Free polyamine concentrations in coastal seawater during phytoplankton bloom

Transcription

1 FISHERIES SCIENCE 2001; 67: Original Article Free polyamine concentrations in coastal seawater during phytoplankton bloom NAOYOSHI NISHIBORI, 1, *AKIHIKO YUASA, 2,a MOTOSUKE SAKAI, 2,a SHINSUKE FUJIHARA 3 AND SACHIO NISHIO 1 1 Shikoku University Junior College, Shikoku University, Ojin, Tokushima , 2 Naruto Branch, Tokushima Prefectural Fisheries Experimental Station, Naruto, Tokushima, and 3 Laboratory of Natural Resources Management, Shikoku National Agricultural Experiment Station, Zentuzi, Hyogo , Japan SUMMARY: Polyamines are widely distributed in nature and known to have many roles in living organisms. We investigated the concentrations of polyamines together with inorganic nutrients during a summer bloom period in the Seto Inland Sea of Japan. Putrescine and spermidine were the major polyamines in the coastal seawater. The concentrations at 1 m depth varied widely during the sampling period and ranged from 2.0 to 32.6 nm and 1.0 to 14.1 nm. Spermine concentrations were much lower than putrescine and spermidine. In addition, other polyamines (diaminopropane, cadaverine, norspermidine, homospermidine, norspermine) were also detected. Putrescine and spermidine seemed to be significant compounds in dissolved organic nitrogen in coastal seawater. KEY WORDS: bloom, phytoplankton, polyamine, putrescine, seawater, spermidine. INTRODUCTION Harmful and toxic algal blooms (HAB) occur in the coastal water around the world and pose a significant threat to fishery resources and human health through mass mortality of marine animals and/or shellfish poisoning in humans. The Seto Inland Sea of Japan is noted for incessant and extensive fish kills caused by HAB. The growth of bloom-forming phytoplankton is affected essentially by abiotic environmental factors such as light, temperature, salinity and concentrations of organic and inorganic nutrients. Some biologically active organic compounds, namely vitamins, nucleic acids and organic matter such as humic substances and soil extract, also stimulate the growth of bloomforming phytoplankton. 1 Among biologically active organic compounds, polyamines are normal constituents in animals, 2 plants 3 and microorganisms including bloom-forming algae. 4 6 Polyamines have been shown to have some roles in plant and algae as well as animal cell cycle and *Corresponding author: Tel: Fax: n-nishibori@shikoku-u.ac.jp a Present address: Fisheries Division Tokushima Prefectural Government, Bandai, Tokushima , Japan. Received 6 April Accepted 3 August growth Organs such as the apical bud where vigorous cell division occurs revealed high contents of polyamine, and transport and redistribution of exogenous polyamines were observed in plant systems. 11 The growth-stimulating effect of exogenous polyamine in higher plants has been reported and polyamines are considered as one of the plant hormones. 7,12,13 The growth enhancement effect of exogenous polyamine is also reported using natural phytoplankton assemblages and one cyanobacterial culture. 14 Although exogenous polyamines as well as its endogenous levels could be involved in regulation of the growth of microalgae, little is known about the existence and concentration of polyamines in seawater. In this study, we determined the concentration of polyamines in the coastal seawater in the Seto Inland Sea of Japan during bloom. MATERIALS AND METHODS Samples were obtained off the coast of Harima- Nada in the Seto Inland Sea of Japan (where a red tide frequently occurs) from 29 June to 19 August 1996, at 3 to 4 day intervals. Samples for polyamine and nutrient analysis were collected from depths of 1 m, 5 m and 40 m. After collection, the samples

2 80 FISHERIES SCIENCE N Nishibori et al. were immediately filtered through a glass fiber filter (Whatman GF/C, Kent, UK) to remove algae and other macroorganisms and then filtered again with a 0.2 µm nucleopore filter to remove microorganisms. The filtered samples were stored at 20 C until analysis. The vacuum applied was less than 20 cm Hg in both filtrations. Polyamine in seawater was analyzed according to the methods described by Schenkel et al. 15 with slight modifications. After adding 60% PCA to a final concentration of 0.2 M and 500 pmol diaminohexane as an internal standard to 40 ml seawater samples, the samples were mixed and alkalized by adding 6 ml of 7 M NaOH, and then 100 µl of benzoyl chloride was added. After standing for 20 min at room temperature, the polyamine benzoates were extracted with CHCl 3 and NaCl. The extracted polyamine benzoates were treated and analyzed by HPLC as reported by Schenkel et al. 15 with an ODS column ( mm, Spherisorb ODS-2; GL Science, Tokyo, Japan), using 55% MeOH as the eluent, and UV absorption was monitored at 234 nm. Artificial seawater containing 25 nm of each polyamine (acetyl-spermidine, diaminopropane, putrescine, cadaverine, norspermidine, spermidine, norspermine and spermine) was used as a standard and benzoylated as described above. For plankton observation, the water column from the surface to a depth of 5 m was collected using a tube 20 mm in diameter. The cell numbers of phytoplankton in 1 ml samples were counted under a light microscope directly or after 100-fold concentration using an 8 µm membrane filter (Millipore Japan, Tokyo, Japan). Dissolved inorganic nitrogen, phosphate and silicate were analyzed using a TRAACS 8000 auto-analyzer (Norderstedt, Germany). RESULTS Diaminopropane, putrescine, cadaverine, norspermidine, spermidine, homospermidine, norspermine and spermine were detected in seawater samples. Although a rather broad peak which eluted at a position near that of the standard acetylspermidine was observed, this peak was not identified as acetylspermidine due to its broad spectrum (Fig. 1). Average concentrations of the polyamine detected are shown in Fig. 2. Putrescine and spermidine were observed in a relatively high concentration, detected in all of the seawater samples and these two polyamines were the major polyamines in seawater. Spermine was detected in some seawater samples and its concentration was much lower than those of putrescine and sper- Fig. 1 Typical chromatograms of benzoylated seawater samples. (a) Collected from 1 m depth on 19 October, 1996; (b) collected from 5 m depth on 12 October, 1996; and (c)standard polyamine benzoates. 1, Acetylspermidine; 2, putrescine; 3, diaminopropane; 4, cadaverine; 5, diaminohexane (internal standard); 6, norspermidine; 7, spermidine; 8, homospermidine; 9, norspermine; and 10, spermine. midine. Although the average concentrations of norspermine were considerable because of a few high content samples, the concentrations of other polyamines were almost 1 nm. The total polyamine concentration decreased as depth increased. It was 21.6 nm at surface (1 m) and 18.6 nm at 5 m, then at the bottom (40 m), it was 12.4 nm. The distributions of the two major polyamines (putrescine and spermidine) and norspermine are shown in Fig. 3. The concentrations of putrescine and spermidine in surface seawater varied widely during the sampling period and were nm and nm, respectively. Relatively high concentrations of putrescine were observed in the surface layer on 15 July and 19 and 26 August. Spermidine concentrations were higher on July

3 Polyamine concentrations in seawater FISHERIES SCIENCE 81 Fig. 2 Average concentrations of the polyamine detected during investigation at: (a) 1 m, (b) 5 m, and (c) 40 m depth. Bars represent SD (n = 19). Put, putrescine; Dap, diaminopropane; Cad, cadaverine; Nspd, norspermidine; Spd, spermidine; Hspd, homospermidine; Nspm, norspermine; and Spm, spermine. and 8 26 August. High concentrations of norspermine were observed at a depth of 5 m on July 18 (24.3 nm) and in the surface sample on 1 August (16 nm). With the exception of these samples, the norspermine concentrations usually ranged from not detected to 10 nm. In contrast to putrescine and spermidine, although they were found in almost all of the samples, the concentrations of diaminopro-pane and cadaverine were lower and ranged from N.D. 4.5 nm. Norspermidine and homospermidine concentrations varied widely and average concentrations were almost equivalent to those of diaminopropane and cadaverine. Inorganic nutrient concentrations are shown in Fig. 4. The variations of dissolved inorganic nitrogen (DIN) and phosphate concentration at Fig. 3 Distribution of ( ) putrescine, ( ) spermidine and ( ) norspermine. (a) 1 m, (b) 5 m, and (c) 40 m depth. the depths of 1 m and 5 m were similar throughout the sampling period. The concentrations of DIN and phosphate in the surface layer were less than 4.0 µm and N.D µm, respectively. Silicate concentrations were 5 17 µm in the surface water. During the sampling periods, blooms of the dinoflagellate Gymnodinium mikimotoii and two diatom species Chaetoceros sp. and Skeletonema costatum were observed (Fig. 5). The G. mikimotoii cell number showed the maximum of 1616 cells/ml on 8 August. The blooms dominated by Chaetoceros sp. were observed twice during

4 82 FISHERIES SCIENCE N Nishibori et al. Fig. 5 Cell numbers of ( ) Chaetoceros sp., ( ) Skeletonema costatum, and ( ) Gymnodinium mikimotoii observed at 0 5 m depth. Fig. 4 Distribution of inorganic nutrient of (a) DIN, (b) phosphate, and (c) silicate at 1 m ( ) and 5 m ( ) depth. the sampling period. The first bloom was observed during July and a second bloom in which S. costatum was observed as a second dominant species formed during August. DISCUSSION Putrescine and spermidine were the major polyamines in the coastal seawater. Spermine was present at a much lower concentration than putrescine and spermidine. Other than these polyamines we detected diaminopropane, cadaverine, norspermidine, homospermidine and norspermine in the coastal seawater. The concentrations of polyamines varied widely during the sampling period and declined as depth increased (Fig. 2). The average concentrations of total polyamine detected and that of the major two polyamines (putrescine plus spermidine) were 21.6 nm and 13.2 nm at 1 m, 18.6 nm and 11.7 nm at 5 m, and 11.1 nm and 6.0 nm at 40 m, respectively, and the contributions of the major two polyamines were almost one-half of the total polyamine detected. Lorenza et al. 16 reported that concentrations of putrescine and spermidine in seawater were higher than spermine as shown here. In their study, the concentrations of these polyamines were one order of magnitude higher than the results of the present study. This difference seemed to be due to their use of eutrophic water. The concentrations of dissolved organic nitrogen (DON) and dissolved free amino acid (DFAA) in the surface seawater at four stations of Harimanada in July were reported to be µg-atn/l and µg-atn/l, respectively. 17 The nitrogen concentrations calculated from the total polyamine concentrations at 1 m were µg-atn/l. Although we did not determine DON and DFAA concentrations here, from the concentrations reported by Tada et al. 17 the total polyamine concentrations consisted of 0.1% to 1.5% of DON, were almost one-third to one-half of that of DFAA and polyamines seemed one of the significant compounds in DON in coastal seawater. The addition of polyamines, especially putrescine, to natural phytoplankton assemblages gave the increase of in vivo fluorescence. 14 Palenik and Morel 18 and Pantoja et al. 19 reported that certain

5 Polyamine concentrations in seawater FISHERIES SCIENCE 83 marine phytoplankton species oxidize amino acid and amines using cell-surface deaminases, taking up the ammonium across the cell membrane. In this study, the changes in polyamine (putrescine and spermidine) concentrations at 1 m depth (Fig. 3a) as well as DIN and phosphate concentrations (Fig. 4a,b) during the period of investigation resembled the changes in phytoplankton (especially diatoms) cell numbers (Fig. 5). In this study, we detected putrescine and spermidine as major polyamines in seawater and these polyamines seemed one of the significant components in DON in seawater. REFERENCES 1. Iwasaki H. Growth physiology of red-tide microorganisms. Microbiol. Sci. 1984; 1: Matsui-Yuasa I, Obayashi M, Hasumi T, Otani S. Enhancement of spermidine/spermine N1-acetyltransferase activity by treatment with lithium chloride in ehrlich ascites tumor cells. Chem. Biol. Interact. 1992; 81: Flores HE, Galston AW. Analysis of polyamines in higher plants by high performance liquid chromatography. Plant Physiol. 1982; 69: Hamana K, Matsuzaki S. Futher study on polyamines in primitive unicellular eukaryotic algae. J. Biochem. 1985; 97: Hamana K, Matsuzaki S. Polyamines as a chemotaxonomic marker in bacterial systematics. Crit. Rev. Microbiol. 1992; 18: Nishibori N, Nishio S. Occurrence of polyamines in bloom forming toxic dinoflagellate Alexandrium tamarense. Fisheries Sci. 1997; 63: Bagin N, Serafini-Fracassini D, Torrigiani P. Polyamines and cellular growth processes in higher plants. In: Wareing PF (ed.). Plant Growth Substances. Academic Press, London. 1982; Kotzabasis K, Senger H. Free, conjugated and bound polyamines during the cell cycle in synchronized cultures of Scenedesmus obliqus. Z. Naturforsch. 1994; 49: Pfosser M, Köigshoeer H, Kandeler R. Free, conjugated, and bound polyamines during the cell cycle of synchronized cell suspension culture of Nicotiana tubacum. J. Plant Physiol. 1990; 136: Pfosser M. Free and conjugated polyamines during the initiation of the cell cycle of G0-arrested Medicago varia cell cultures. J.Plant Physiol. 1993; 142: Caffaro S, Scaramagli S, Antognoni F, Bagin N. Polyamine content and translocation in soybean plants. J. Plant Physiol. 1993; 141: Oshmarina VI, Shevyakova NI, Shamina ZB. Dynamics of free amino acids and amides in Nicotiana sylverstris cell cultures at different concentrations of putrescine in the medium. Sov. Plant Physiol. 1982; 29: Bradley PM. Plant hormones do have a role in controlling growth and development of algae. J. Phycol. 1991; 27: Maestrini ST, Balode M, Bechemin C, Purina I. Nitrogenous organic substances as potential nitrogen sources, for summer phytoplankton in the gulf of Riga, eastern Baltic sea. Plankton Biol. Ecol. 1999; 46: Schenkel E, Berlaimont V, Dubois J, Cambier MH, Hanoq M. Improved high performance liquid chromatographic method for the determination of polyamines as their benzoylated derivatives: application to P388 cancer cells. J. Chromatogr. B. 1995; 668: Lorenza B, Pistocchi R, Bagni N. Polyamine transport in the seaweed Ulva rigida (Chlorophyta). J. Phycol. 1994; 30: Tada K, Tada M, Maita Y. Dissolved free amino acids in coastal seawater using a modified fluorometric method. J. Oceanogr. 1998; 54: Palenik B, Morel FMM. Amine oxidase of marine phytoplankton. Appl. Environ. Microbiol. 1991; 57: Pantoja S, Lee C, Marecet JF, Palenik BP. Synthesis and use of fluorescent molecular probes for measuring cell-surface enzymatic oxidation of amino acids and amines in seawater. Anal. Biochem. 1993; 211: