SPECIES-SPECIFIC ALLELOPATHIC EFFECTS OF THE DIATOM SKELETONEMA COSTATUM

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1 Thalassas, 27 (1): An International Journal of Marine Sciences SPECIES-SPECIFIC ALLELOPATHIC EFFECTS OF THE DIATOM SKELETONEMA COSTATUM Y. YAMASAKI (1)*, Y. OHMICHI (1), T. SHIKATA (2), M. HIROSE (1), Y. SHIMASAKI (1), Y. OSHIMA (1) & T. HONJO (3) Key words: Allelopathy, growth inhibition, growth promotion, Heterosigma akashiwo, low molecular weight allelochemicals, Skeletonema costatum, species-specific effect ABSTRACT We examined allelopathic effects of Skeletonema costatum on the growth of Heterosigma akashiwo, a major competitor of S. costatum. Growth of H. (1) Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka , Japan. (2) Department of Photoscience, School of Advanced Sciences, Graduate University for Advanced Studies, Shonan Village, Hayama, Kanagawa , Japan. (3) Seto Inland Sea Regional Research Center, Kagawa University, Saiwai, Takamatsu, Kagawa , Japan. *Corresponding author: Y Yamasaki Present address: National Fisheries University, Nagata- Honmachi, Shimonoseki, Yamaguchi , Japan. Tel: Fax: yamasaky@fish-u.ac.jp akashiwo was inhibited when it was exposed to S. costatum filtrates collected in the stationary and decline growth phases. During the decline phase, the filtrate had the strongest inhibitory effect. Deactivation of the allelopathic effect of S. costatum was not observed when the filtrate was preserved at room temperature for 3 days. Additionally, crude extracts greater than 3,5 Da, obtained by dialysis, did not inhibit the growth of H. akashiwo; that is, S. costatum produces low molecular weight allelochemicals. Furthermore, we examined the allelopathic effects of S. costatum on the growth of other species and determined that these effects were both species-specific and dependent upon the cell density of the target species. It is likely that the allelochemicals produced by S. costatum are chemically stable low molecular weight substances, and the allelopathy of S. costatum has the potential to affect the succession of phytoplankton species during dense blooms of S. costatum and/or after a decline in the bloom of S. costatum. 21

2 Y. YAMASAKI, Y. OHMICHI, T. SHIKATA, M. HIROSE, Y. SHIMASAKI, Y. OSHIMA & T. HONJO INTRODUCTION Molisch (1937) introduced the term allelopathy to refer to biochemical interactions between all types of plants including microorganisms. Rice (1974) defined allelopathy as any direct or indirect harmful effect by one plant, including microorganisms, on another through production of chemical compounds that escape into the environment. Later, Rice (1984) recanted his earlier definition because apparently most allelopathic compounds have both inhibitory and stimulatory effects on growth. In general, most allelochemicals, including low-molecular weight (LMW) organic acids, phenolic substances, alkaloids and terpenoids, are known as plant secondary metabolites they exist only in plants and are not directly relevant to the maintenance of life. Until now, both in situ and in vitro studies have suggested that allelopathy plays a key role in the growth dynamics of red-tide blooms through inhibitory effects (Cembella, 23; Legrand et al., 23; Granéli & Hansen, 26; van Rijssel et al., 28; Paul et al., 29). The raphidophyte Heterosigma akashiwo and the diatom Skeletonema costatum form alternating blooms in various coastal waters (Pratt, 1966; Honjo et al., 1978; Honjo & Tabata, 1985; Shikata et al., 28abc). In in situ and in vitro experiments, high concentrations of Olisthodiscus luteus inhibit S. costatum growth, while lower concentrations stimulate S. costatum growth (Pratt 1966). Here, since Honjo (1994) and Smayda (1998) described that most pelagic blooms attributed to O. luteus were considered almost certainly those of H. akashiwo, we will treat O. luteus described by Pratt (1966) and Honjo & Tabata (1985) as H. akashiwo. Similarly, Honjo et al. (1978) reported that H. akashiwo and S. costatum alternated in forming red tide blooms in the Hakozaki Fishing Port. They found that filtrate from dense cultures of H. akashiwo, re-enriched with nutrients, inhibited S. costatum growth. In addition, Honjo & Tabata (1985) reported an apparent and reciprocal codominance between O. luteus and diatoms in a 7 m 3 outdoor tank with Cell density of S. costatum (cells ml -1 ) Growth of H. akashiwo (%) Phase-E Phase-B Phase-C Phase-A Incubation time (days) Phase-D (A) (B) Control Phase-A Phase-B Phase-C Phase-D Phase-E Figure 1: A) Growth of Skeletonema costatum culture showing phases used to produce filtrates. B) Growth of Heterosigma akashiwo exposed to filtrates of S. costatum at different growth phases. Error bars indicate SD. Asterisk (*) indicates significant difference from control treatment (p <.5). flowing coastal water. Furthermore, we investigated growth and interactions between H. akashiwo and S. costatum using bi-algal cultures under axenic conditions, and our findings indicated that the growth of either species could be suppressed by the unidentified allelochemicals of the other, depending on cell densities (Yamasaki et al., 27). Recently, we demonstrated that allelopathic effects of H. akashiwo were both species-specific and dependent upon the cell density of the target species, and allelopathic polysaccharide protein complexes (APPCs) produced by H. akashiwo, which were detected in field samples from the 22

3 SPECIES-SPECIFIC ALLELOPATHIC EFFECTS OF THE DIATOM SKELETONEMA COSTATUM (A) (B) (C) 2 μm to S. costatum and H. akashiwo (Shikata et al., 28ab, 29). Thus, it is also necessary to identify allelochemicals produced by the dominant species S. costatum, and to demonstrate the significance of allelopathy in a multispecies phytoplankton community in the field. In this study, to obtain basic information on properties of allelochemicals produced by S. costatum, we examined the stability and estimated molecularweight of allelochemicals of S. costatum. To confirm allelopathy of S. costatum plays an ecological role, we also investigated allelopathic effects of S. costatum on the growth of S. costatum itself, H. akashiwo and other co-occurring species in the Hakozaki Fishing Port. MATERIALS AND METHODS Algal species and culture conditions Figure 2: Appearance of (A) normal, (B) morphologically-abnormal, and (C) disrupted Heterosigma akashiwo cells, before and after exposure to Skeletonema costatum filtrates. Hakozaki Fishing Port at concentrations exceeding their experimentally-determined action threshold, inhibited the growth of S. costatum (Yamasaki et al., 29). However, chemical nature and speciesspecificity of allelochemicals produced by S. costatum are not well understood. In Hakozaki Fishing Port, with an average water depth of about 3. m, located Fukuoka City, Kyushu, Japan (lat N, long W), there are frequent blooms of several diatoms and flagellates, especially genera Asterionellopsis, Chaetoceros, Thalassiosira, and Prorocentrum in addition Axenic strains of S. costatum (strain NIES-324) and H. akashiwo (strain NIES-1) were obtained from the National Institute for Environmental Studies (NIES) Japan. The two strains were tested for bacterial contamination by the 4',6-diamidino- 2-phenylindole (DAPI) staining method (Porter & Feig, 198), and were verified as axenic. Three diatoms of Asterionellopsis glacialis, Thalassiosira sp. and Chaetoceros sp., and three dinoflagellates of Prorocentrum dentatum, Prorocentrum triestinum and Prorocentrum minimum were isolated from Hakata Bay (Fukuoka, Japan). Of these, only the culture of P. minimum was axenic. Cultures were maintained in 1 ml flasks containing 5 ml autoclaved (121 C, 15 min), buffered (ph ) and modified SWM-3 medium (Yamasaki et al., 27) with a salinity of 25. Cultures were grown at 25 C under 228 (±5) μmol photons m -2 s -1 of cool-white fluorescent illumination on a 12:12 h light:dark cycle. Irradiance in the incubator was measured with a Quantum Scalar Laboratory irradiance sensor (QSL-211; Biospherical Instruments, San Diego, CA, USA). 23

4 Y. YAMASAKI, Y. OHMICHI, T. SHIKATA, M. HIROSE, Y. SHIMASAKI, Y. OSHIMA & T. HONJO Effects of growth phase on the allelopathic effects of S. costatum towards H. akashiwo Skeletonema costatum in the stationary phase ( cells ml -1 ) was inoculated at a concentration of 2 cells ml -1 into five 2 ml glass flasks containing 1 ml modified SWM-3 medium. One hundred milliliter samples from five replicate flasks were withdrawn by glass pipette after 2 d (exponential phase), 4 d (early stationary phase), 9 d (stationary phase), 22 d (late stationary phase) and 24 d (decline phase), and passed through a 5. μm pore size membrane filter on a 47 mm polysulfone holder (Advantec, Tokyo, Japan) under gravity filtration. Nutrients dissolved in deionized water were then added to each 1 ml filtrate to the same final concentration as in the original modified SWM-3 medium, and the filtrate was then passed through a.22 μm pore size syringe filter (Millipore, Billerica, MA, USA). As a control, an equivalent amount of distilled water was added to the modified SWM-3 medium instead of the nutrient solution, and the medium was passed through a.22 μm pore size syringe filter. Filtrates from S. costatum were labeled in sequence as phases A E, corresponding to their phase of growth on days 2, 4, 9, 22 and 24. In addition, the ph of each filtrate was measured (model B-212 ph meter; Horiba, Kyoto, Japan). The allelopathic effects of these filtrates on the growth of H. akashiwo were determined by a bioassay using 48-well plates (see Bioassay using 48-well plates in MATERIALS AND METHODS). Determination of stability of allelochemicals produced by S. costatum Filtrate (Phase E) of S. costatum was prepared by the same method as described in the previous section and stored at 25 C under 228 (±5) μmol photons m -2 s -1 of cool-white fluorescent illumination on a 12:12 h light:dark cycle for 3 d. The allelopathic effects of the filtrate on the growth of H. akashiwo were determined by a bioassay using 48-well plates (see Bioassay using 48-well plates in MATERIALS AND METHODS). Effects of the crude extract prepared from the filtrate of S. costatum Filtrate (Phase E) of S. costatum was prepared by the same method as described in the previous section and then 3 ml of the filtrate was dialyzed against deionized water for 3 d at 4 C using dialysis membranes with a 3,5 Da MW cut-off (MWCO; Spectrum Laboratories, Rancho Dominguez, CA, USA). After dialysis, the inner solution was frozen at -8 C and lyophilized with a Free Zone 2.5 freezedrying apparatus (Labconco, Kansas City, MO, USA). Similarly, 3 ml of the modified SWM-3 medium was dialyzed and lyophilized as a negative control. The allelopathic effects of the crude extract on the growth of H. akashiwo were determined by a bioassay using 48-well plates (see Bioassay using 48-well plates in MATERIALS AND METHODS). Bioassay using 48-well plates Effects of enriched filtrates of S. costatum and the crude extract on H. akashiwo growth were examined in 48-well plates. Heterosigma akashiwo cells from cultures in stationary phase ( cells ml -1 ) were diluted to a density of cells ml -1 with modified SWM-3 culture medium. Then, 1 μl of the cell suspension was inoculated into 99 μl of each sample with six replicate wells for each treatment. After 8 d of incubation at 25 C under 228 (±5) μmol photons m -2 s -1 of cool-white fluorescent illumination on a 12:12 h light:dark cycle, the cells in each of five 1 μl subsamples from each well were counted. Allelopathic effects of S. costatum on phytoplankton growth Skeletonema costatum cells were inoculated at a density of 2 cells ml -1 into 1, ml glass flasks (n = 6) containing 25 ml modified SWM-3 medium. After incubation for 24 d at 25 C under 228 (±5) μmol photons m -2 s -1 of cool-white fluorescent illumination on a 12:12 h light:dark cycle, each 25 ml sample from the six replicate flasks was combined 24

5 SPECIES-SPECIFIC ALLELOPATHIC EFFECTS OF THE DIATOM SKELETONEMA COSTATUM Fluorescence (relative value) H. akashiwo P. dentatum P. triestinum P. minimum (A) 4 (E) 1 (I) (M) % % % Control (B) 4 (F) 1 (J) (N) (C) 4 (G) 1 (K) (O) (D) 4 (H) 1 (L) (P) Incubation time (days) 1 Initial cell density (cells ml -1 ) Figure 3: Growth of four flagellates at different initial cell densities, exposed to Skeletonema costatum filtrates at 1%, 7%, 4%, and % (control) concentrations. and passed through a 5. μm pore-size membrane filter on a 47 mm polysulfone holder under gravity filtration. The 1,5 ml filtrate was aerated to avoid an increase in ph and to supply dissolved inorganic carbon (DIC). After aeration, nutrients dissolved in deionized water were added to 1,5 ml filtrate (ph 8.) to give the same final concentration as in the original modified SWM-3 medium. The filtrate was then passed through a.22 μm pore-size filter. This filtrate, designated 1% filtrate, was diluted to 7% and 4% (v/v) with modified SWM-3 medium. A sample (.5 ml) of each cell suspension of S. costatum, A. glacialis, Thalassiosira sp., Chaetoceros sp., H. akashiwo, P. dentatum, P. triestinum and P. minimum at various inoculum cell densities (initial cell densities, 1,, and cells ml -1 ) was added to 4.5 ml of each test solution (described above) in polystyrene test tubes (Evergreen Scientific, Los Angeles, CA, USA). As a control, each species was grown in the modified SWM-3 medium without the addition of any sample solution. This growth experiment had 16 test conditions, with four replicates per test condition. Following the commencement of incubation, the in vivo fluorescence of each species was measured daily with an in vivo fluorometer (1-AU-5-CE; Turner Designs, Sunnyvale, CA, USA). Growth rate (divisions d -1 ) was calculated for each tube from three consecutive data points, using the method of Brand et al. (1981), and maximum growth rates during the entire incubation period were determined. The lag period before exponential growth 25

6 Y. YAMASAKI, Y. OHMICHI, T. SHIKATA, M. HIROSE, Y. SHIMASAKI, Y. OSHIMA & T. HONJO Fluorescence (relative value) S. costatum A. glacialis T. rotula Chaetoceros sp (A) 3 (E) 2 (I) 3 (M) % % 15 4% Control (B) 3 (F) (J) 3 (N) (C) 3 (G) (K) 3 2 (O) (D) 3 (H) (L) 3 2 (P) Incubation time (days) 1 Initial cell density (cells ml -1 ) Figure 4: Growth of four diatoms at different initial cell densities, exposed to Skeletonema costatum filtrates at 1%, 7%, 4%, and % (control) concentrations. was estimated as the number of days from the start of incubation to the time when fluorescence increased by more than five units (fsu). Statistical analysis The experimental data were checked for assumptions of homogeneity of variance across treatments by using Levene s test. Data were analyzed by one-way analysis of variance (ANOVA) and then were tested by using Dunnett s test. When there was no proof of data homoscedasticity, a Mann-Whitney U-test for nonparametric data was used to compare cell densities between treatments and controls. All analyses were performed using SPSS for Windows (SPSS version 16.; SPSS, Inc., Chicago, IL, USA). A significance level of p <.5 was used for all tests. RESULTS Effects of allelochemicals produced by S. costatum Results of bioassays in 48-well plates showed that enriched filtrate of S. costatum from phase A had no effect, whereas phases B E significantly inhibited growth of H. akashiwo (Fig. 1B). Filtrates from cultures in the late stationary and decline phases had the strongest effects. Heterosigma akashiwo exposed to filtrate from the decline phase showed morphologically abnormal cells within 3 min, prior to disruption of the cells (Fig. 2). Thus, allelochemicals produced by S. costatum proved to have strong lytic activity, as previously reported (Tillmann et al., 27). Although ph of the filtrates from different phases ranged from 7.9 to 8.3, our previous study 26

7 SPECIES-SPECIFIC ALLELOPATHIC EFFECTS OF THE DIATOM SKELETONEMA COSTATUM Table 1: Growth (mean ± SD) of Heterosigma akashiwo exposed to freshly prepared filtrate of Skeletonema costatum cultures in the decline phase, to filtrate after 3 d of storage, or to crude extract of filtrate prepared by dialysis. Asterisk (*) indicates significant difference from control (p <.5). Growth of H. akashiwo (%) Control 1 ± 7 Filtrate of S. costatum * Filtrate of S. costatum stored for 3 days * Crude extract of S. costatum filtrate (MW > 3,5) 97 ± 11 found that this range of ph did not affect growth of H. akashiwo (Yamasaki et al., 27). Storage for 3 days did not affect the allelopathic activity of S. costatum filtrate from the decline phase (Table 1). However, crude extract from the dialysis of the S. costatum filtrate did not affect the growth of H. akashiwo. Allelopathic effects of S. costatum on the growth of co-occurring algal species Allelopathic effects of S. costatum depended on the target species and filtrate concentration. Although all species tested were affected, including S. costatum itself, some effects were inhibitory and others were stimulatory (Figs. 3 and 4, Tables 2 and 3). Among the inhibitory effects, all species had a longer lag period before the onset of exponential growth when grown in S. costatum filtrates than in control cultures; H. akashiwo, P. dentatum, P. triestinum, S. costatum, A. glacialis, Thalassiosira sp., and Chaetoceros sp. exhibited lower maximum growth rates; and S. costatum had lower maximum cell yield. Among the stimulatory effects, all species, except S. costatum, exhibited higher maximum growth rates and cell yields when grown in S. costatum filtrates than in control cultures; and P. minimum and A. glacialis had a shorter lag before the onset of exponential growth. Allelopathic effects of S. costatum also depended on cell density of the target species. For example, when H. akashiwo was inoculated at low cell densities, exposure to S. costatum filtrate was strongly inhibitory (Fig. 3A C). However, when H. akashiwo was inoculated at high cell densities, exposure to filtrate was initially inhibitory, but growth gradually resumed (Fig. 3D). DISCUSSION Mono-specific and multi-species blooms of S. costatum and H. akashiwo also have been observed in various coastal waters (Pratt, 1966; Honjo et al., 1978; Honjo & Tabata, 1985; Shikata et al., 28abc), and this suggests that species interactions including allelopathy may contribute to the formation of these alternating blooms in addition to growth characteristics and environmental factors. In this study, to obtain basic information on properties of allelochemicals produced by S. costatum and confirm allelopathy of S. costatum plays an ecological role, we investigated allelopathic effects of S. costatum on the growth of H. akashiwo and other co-occurring species in the Hakozaki Fishing Port. Growth of H. akashiwo was inhibited by the filtrates of S. costatum depending on culture period of S. costatum (Fig. 1). Yamasaki et al. (29) showed that the APPCs produced by H. akashiwo became deactivated over time (time-scale of days), whereas no deactivation of the allelopathic effect of S. costatum 27

8 Y. YAMASAKI, Y. OHMICHI, T. SHIKATA, M. HIROSE, Y. SHIMASAKI, Y. OSHIMA & T. HONJO Table 2: Growth characteristics of flagellate cultures at different initial cell densities, exposed to Skeletonema costatum filtrate at % (control), 4%, 7%, and 1% concentrations. Downward pointing triangles indicate significant inhibition compared to control. Upward pointing triangles indicate significant stimulation compared to control. Speceis Initial cell density (cells ml -1 ) Filtrate concentration Lag before exponential growth (days) Growth rate (div d -1 ) Maximum cell yield (fsu) H. akashiwo P. dentatum P. triestinum P. minimum Control % ( ) ( ) 91 9 ( ) 7% Nogrowth Nogrowth Nogrowth 1% No growth No growth No growth Control % 8 ( ) ( ) ( ) 7% ( ) ( ) 1% No growth No growth No growth Control % ( ) % ( ) ( ) 1% ( ) ( ) ( ) Control % ( ) 7% 3 ( ) ( ) 1% 4 ( ) ( ) Control % 15 2 ( ) ( ) % 15 ( ) ( ) 1% No growth No growth No growth Control % 13 ( ) % ( ) ( ) 1% No growth No growth No growth Control % ( ) % ( ) % ( ) ( ) Control % ( ) ( ) 7% ( ) ( ) ( ) 1% ( ) ( ) ( ) Control % ( ) ( ) 7% Nogrowth Nogrowth Nogrowth 1% No growth No growth No growth Control % 1.82 ( ) ( ) 7% ( ) ( ) 1% No growth No growth No growth Control % ( ) ( ) 9 5 ( ) 7% ( ) ( ) 99 4 ( ) 1% ( ) ( ) Control % ( ) 7% 3 ( ) ( ) 11 7 ( ) 1% 3 ( ) ( ) ( ) Control % ( ) ( ) ( ) 7% ( ) ( ) ( ) 1% 14 ( ) ( ) ( ) Control % 6 ( ) ( ) 7% ( ) ( ) ( ) 1% ( ) ( ) Control % ( ) ( ) 7% 4 ( ) ( ) 1% ( ) ( ) ( ) Control % ( ) ( ) 7% ( ) ( ) 1% 2 ( ) ( ) 28

9 SPECIES-SPECIFIC ALLELOPATHIC EFFECTS OF THE DIATOM SKELETONEMA COSTATUM was observed when the filtrate was preserved at room temperature for 3 days, and crude extracts (MW > 3,5 Da) obtained from dialysis did not inhibit H. akashiwo growth (Table 1). Unlike the laboratory experiments, note that the allelopathic effects of S. costatum may not continue for long periods in the field because of several environmental factors (e.g. seawater exchange/mixing). In fact, Shikata et al. (28b) reported that H. akashiwo could grow adequately in seawater that originally included dense concentrations of other phytoplankton species, including S. costatum. The difference between field observations and laboratory results could be due to different physical, chemical, and biological conditions in situ, including seawater exchange/ mixing, bacterial degradation, and interactions among multiple allelochemicals produced by co-existing phytoplankton species. Thus, future research should not only focus on purification and identification of the allelochemicals produced by S. costatum, but also on effects of the purified allelochemicals under various culture conditions. Crude dialysis extracts (MW > 3,5 Da) of S. costatum filtrates from the decline phase did not inhibit growth of H. akashiwo, indicating that the allelopathic chemicals are LMW compounds. Imada et al. (1991) reported that S. costatum produces 15S-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid (15-HEPE), an LMW polyunsaturated fatty acid (PUFA), which acts as an autoinhibitor. Recent studies suggested that diatoms produce LMW -, -, -, and -unsaturated aldehydes (PUAs), which have a negative influence on the reproductive success of copepods and other invertebrates (Ianora et al. 24, Pohnert 25, Wichard et al. 25, Ribalet et al., 29). Although further studies are required to identify the allelochemicals produced by S. costatum and to clarify the recognition site on the cell surface of target species, the rapid morphological and lytic effects on H. akashiwo suggest that the allelochemicals may attack cell membranes. To confirm allelopathy of S. costatum which plays an ecological role, we also investigated allelopathic effects of S. costatum on the growth of S. costatum itself, H. akashiwo and other co-occurring species in the Hakozaki Fishing Port. When H. akashiwo was inoculated at low cell densities and exposed to the filtrate from S. costatum culture, its growth was markedly inhibited (Fig. 3A-D). On the other hand, when H. akashiwo was inoculated at high cell densities, H. akashiwo growth was inhibited by the filtrate of S. costatum soon after starting the experiments, but H. akashiwo gradually grew depending on the period of incubation (Fig. 3D). These results supported the finding of a previous study (Yamasaki et al., 27); that is, the species first reaching high cell density limited the cell densities of the other species, but when the initial cell densities of H. akashiwo and S. costatum are high, the growth of each species is only weakly suppressed, and suggested that allelopathy drives the alternation of the two species in the field. Several studies reported that blooms of P. minimum were observed following blooms of S. costatum (Silva, 1985; Bodeanu & Usurelu, 1979; Kondo et al., 199a). By contrast, Kondo et al. (199a) also reported that P. minimum and S. costatum bloomed concurrently. Similarly, Shikata et al. (28b) reported that blooms of P. minimum were observed following blooms of S. costatum, and then blooms of P. dentatum and P. triestinum were observed following P. minimum. These field observations strongly support our laboratory experiments. Especially, P. minimum is the most tolerant species of the three Prorocentrum species against the inhibitory effect of allelopathy of S. costatum, and is also the most susceptible to the promoting effect of allelopathy of S. costatum (Fig. 3E-P). Our results may explain why some of cases are observed as alternating blooms, but other ones are observed as mixed blooms. On the other hand, Kondo et al. (199b) reported that organic substances excreted by S. costatum and low molecular weight fractions collected from the interstitial water of bottom sediments stimulated P. minimum growth. Thus, the previous report and the present study 29

10 Y. YAMASAKI, Y. OHMICHI, T. SHIKATA, M. HIROSE, Y. SHIMASAKI, Y. OSHIMA & T. HONJO Table 3: Growth characteristics of diatom cultures at different initial cell densities, exposed to Skeletonema costatum filtrate at % (control), 4%, 7%, and 1% concentrations. Downward pointing triangles indicate significant inhibition compared to control. Upward pointing triangles indicate significant stimulation compared to control. Speceis Initial cell density (cells ml -1 ) Filtrate concentration Lag before exponential growth (days) Growth rate (div d -1 ) Maximum cell yield (fsu) S. costatum A. glacialis Thalassiosira sp. Chaetoceros sp Control No growth No growth 4% No growth No growth No growth 7% No growth No growth No growth 1% No growth No growth No growth Control No growth No growth 4% No growth No growth No growth 7% No growth No growth No growth 1% No growth No growth No growth Control No growth No growth 4% No growth No growth No growth 7% No growth No growth No growth 1% No growth No growth No growth Control % 2 ( ) ( ) ( ) 7% No growth No growth No growth 1% No growth No growth No growth Control % ( ) ( ) 7% ( ) ( ) ( ) 1% ( ) ( ) Control % 6 ( ) ( ) ( ) 7% 6 ( ) ( ) 29 8 ( ) 1% 6 ( ) ( ) ( ) Control % ( ) 7% ( ) 1% ( ) Control % ( ) 7% ( ) 1% ( ) Control % ( ) ( ) ( ) 7% ( ) 28 7 ( ) 1% ( ) ( ) Control % ( ) ( ) 7% ( ) ( ) 1% 5 ( ) ( ) ( ) Control % ( ) ( ) 7% 3 ( ) ( ) ( ) 1% 3 ( ) ( ) 24 2 ( ) Control % ( ) 7% ( ) 29 7 ( ) 1% ( ) ( ) Control % ( ) ( ) 7% 9 ( ) ( ) 1% ( ) ( ) Control % 6 ( ) ( ) 7% ( ) ( ) 1% 9.58 ( ) ( ) Control % 4 ( ) ( ) ( ) 7% 5 ( ) ( ) ( ) 1% 6 ( ) ( ) ( ) Control % ( ) ( ) 7% ( ) ( ) 1% ( ) 35 5 ( ) 3

11 SPECIES-SPECIFIC ALLELOPATHIC EFFECTS OF THE DIATOM SKELETONEMA COSTATUM suggest that S. costatum produces different kinds of allelochemicals, which were involved in growth inhibition and growth promotion. In the field and laboratory experiments, S. costatum rapidly decline as shown in Fig. 1A. This phenomenon also supports our laboratory experiments. In fact, S. costatum itself is the most sensitive species against the inhibitory effect of allelopathy of S. costatum than any other species used in the present study (Fig. 4A-D). Thus, allelopathy of S. costatum may decrease the abundance of S. costatum itself in the field, but this may have led to preserve biodiversity in phytoplankton community. On the other hand, growth of other diatoms tends to be promoted by allelochemicals produced by S. costatum though Chaetoceros sp. growth was inhibited by the filtrate of S. costatum soon after starting the experiments, but Chaetoceros sp. gradually grew depending on the period of incubation (Fig. 4E-P). These results may explain one of factors on multi-species bloom formation of S. costatum and other diatoms observed by Shikata et al. (29). The allelopathic effect of S. costatum filtrate on the lag period before exponential growth was significantly decreased by increasing target cell concentration (Tables 2 and 3), indicating a saturation effect. Allelochemicals may have been removed from the bioassay system via binding or attachment to the target cells, as has been reported by Tillmann et al. (27). Thus, the negative effects may appear only until all allelochemicals are bound to target cells or, unspecifically, to other particles and/or organic matter. Once the allelochemicals are removed or sufficiently reduced in concentration, the remaining targets can grow unaffected. However, with the exception of S. costatum, the maximum cell yield of all species was significantly enhanced by exposure to filtrate (Tables 2 and 3). These results suggest that S. costatum may produce two different kinds of allelochemicals, growth inhibitor and growth promotor. The growth promotor may affect the target cells after removal or reduction of the growth inhibitor. Results of the present study demonstrated that the allelopathic effects of S. costatum filtrate can be inhibitory or stimulatory, depending on the target species and their cell densities. Thus, it is likely that S. costatum affects phytoplankton communities in the field by inhibiting or stimulating growth of other algal species after the decline of its own density. Effects must also depend on other biological factors, such as microbial degradation, and environmental conditions, such as currents and mixing. Hence, it is necessary to purify and identify allelochemicals of S. costatum that affect the growth of co-occurring species including H. akashiwo, and to verify the role of allelopathy in natural phytoplankton populations. ACKNOWLEDGMENTS We are grateful to anonymous reviewers for thoughtful comments. REFERENCES Bodeanu N, Usurelu M (1979) Dinoflagellate blooms in the Romanian Black Sea coastal waters. In: Taylor DL, Seliger HH, eds., Toxic Dinoflagellate Blooms. Elsevier, New York, Brand LE, Guillard RRL, Murphy LF (1981). A method for the rapid and precise determination of acclimated phytoplankton reproduction rates, Journal of Plankton Research, 3: Cembella AD (23). Chemical ecology of eukaryotic microalgae in marine ecosystems, Phycologia, 42: Granéli E, Hansen PJ (26) Allelopathy in harmful algae: a mechanism to compete for resources? In: Granéli E, Turner TJ, eds., Ecology of Harmful Algae. Springer- Verlag, Berlin, Honjo T, Shimouse T, Hanaoka T (1978). A red tide occurred at the Hakozaki fishing port, Hakata Bay, in The growth process and the chlorophyll content, Bulletin of the Plankton Society of Japan, 25: Honjo T, Tabata K (1985). Growth dynamics of Olisthodiscus luteus in outdoor tanks with flowing coastal water and in small vessels, Limnology and Oceanography, 3: Honjo T (1994). The biology and prediction of representative red tides associated with fish kills in Japan, Review in Fisheries Science, 2: Ianora A, Miralto A, Poulet SA, Carotenuto Y, Buttino I, Romano G, Casotti R, Pohnert G, Wichard T, 31

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