First report of the cyanobacterium Aphanizomenon ovalisporum Forti in two Greek lakes and cyanotoxin occurrence

SHORT COMMUNICATION First report of the cyanobacterium Aphanizomenon ovalisporum Forti in two Greek lakes and cyanotoxin occurrence SPYROS GKELIS 1,2, MARIA MOUSTAKA-GOUNI 1, KAARINA SIVONEN 2 AND TOM LANARAS 1 * 1 DEPARTMENT OF BOTANY, SCHOOL OF BIOLOGY, ARISTOTLE UNIVERSITY OF THESSALONIKI, PO BOX 109, GR-541 24 THESSALONIKI, GREECE AND 2 DEPARTMENT OF APPLIED CHEMISTRY AND MICROBIOLOGY, VIIKKI BIOCENTER, HELSINKI UNIVERSITY, PO BOX 56, HELSINKI, FIN-000 14, FINLAND *CORRESPONDING AUTHOR: lanaras@bio.auth.gr Received June 1, 2005; accepted in principle August 25, 2005; accepted for publication October 7, 2005; published online November 22, 2005 Communicating editor: I.R. Jenkinson Aphanizomenon ovalisporum is reported for the first time in Greece, in two warm, monomictic lakes. Aphanizomenon ovalisporum was dominant constituting 99 and 58% of the total cyanobacterial biomass in lakes Lysimachia and Trichonis, respectively. Trichomes were solitary (length 60 700 m), were narrowed slightly at the ends, had a few terminal hyaline cells and had cells containing gas vesicles (length 2.5 6.9, width 2.4 5.1 m). Heterocytes, spherical or ellipsoidal (length 4.4 10.5, width 2.41 5.1 m) and akinetes (length 16.0 27.8, width 6.0 15.9 m) were located in the middle of the trichome. High performance liquid chromatography (HPLC) analysis detected microcystin LR (MC LR) and a putative anabaenopeptin in the L. Lysimachia sample. The sestonic MC LR concentration was 0.9 g L 1. The origin of MC LR in L. Lysimachia is discussed. The other cyanobacteria present were Pseudanabaena sp. and Planktothrix mougeotii (1% of the total cyanobacterial biomass). INTRODUCTION The cyanobacterial genus Aphanizomenon has a worldwide distribution, but the species Aphanizomenon ovalisporum has been reported only rarely. This species was first described in a lake near Istanbul by Forti (Huber-Pestalozzi, 1938). Aphanizomenon ovalisporum belongs to a group of species characterized by solitary trichomes which are narrowed toward the ends and in which elongated terminal cells are absent (Komárek and Kováčik, 1989). Other species in this group are Anabaena bergii, Anabaena aphanizomenoides, Aphanizomenon chinense, Aphanizomenon sphaericum and Aphanizomenon manguini. The position of this group within the Anabaena or Aphanizomenon genus or even as a separate genus is still open (Komárek and Kováčik, 1989). Further, it has been suggested that A. ovalisporum and A. bergii are morphological variants of the same cyanobacterium (Fergusson and Saint, 2000). Aphanizomenon ovalisporum strains isolated from Israel and Australia have been found to produce cylindrospermopsin (Banker et al., 1997; Shaw et al., 1999), a cytotoxic compound which affects several organs, e.g. the liver and the kidneys (see Kuiper-Goodman et al., 1999 for a review). This study constitutes the first report of the occurrence of A. ovalisporum in lakes of the Balkan Peninsula and focuses on the morphological variation of the species in relation to its taxonomic position and its geographical distribution. RESULTS AND DISCUSSION Lakes Lysimachia (38 35 0 N, 21 29 0 E) and Trichonis (38 30 0 N, 21 35 0 E) belong to a group of warm monomictic lakes located in central Greece (for a detailed description of these lakes see Overbeck et al., 1982). A water sample was collected in the summer (22 July 1999) doi:10.1093/plankt/fbi085, available online at www.plankt.oxfordjournals.org Ó The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

from the surface layer (0 1.0 m) of each lake. Water temperature and ph were measured in situ using a portable ph meter (Wissenschlaftlich-Technische Werkstatten portable instruments, model 196T, Germany). The water samples (1.5 L) were kept in 1.5-L airtight plastic bottles. Bottles were placed in insulated boxes in the dark, transported to the laboratory and filtered through Whatman GF/C filters within 12 h of collection. The filtrates were used for the determination of dissolved inorganic nutrient concentrations. Filters and filtrates were stored at 20 C immediately following filtration and were analysed within 6 months. Soluble reactive phosphorous (SRP), nitrate (NO 3 N) and nitrite (NO 2 N) nitrogen were determined using the methods of the American Public Health Association (American Public Health Association, 1976). Ammonia nitrogen (NH 4 N) was determined according to Liddicoat et al. (Liddicoat et al., 1976). Phytoplankton samples (200 ml) were preserved in situ in acidic Lugol s solution. The preserved samples were examined using a light microscope and phytoplankton species were identified in accordance with Huber-Pestalozzi (Huber-Pestalozzi, 1938), Komárek and Kováčik (Komárek and Kováčik, 1989), Pollingher et al. (Pollingher et al., 1998) and Komárek (Komárek, 2003). The following parameters were selected to describe the morphometry of the species studied: the length (l) and width (w) of vegetative cells, heterocytes and akinetes; the corresponding l : w ratio; the shape of terminal cells; the presence or absence of terminal heterocytes and gas vesicles; the shape and length of filaments. The abundance of cyanobacterial filaments and cells was determined in accordance with Utermöhl (Utermöhl, 1958). Cell (vegetative, heterocyte and akinete) or filament dimensions were used for biovolume calculation. Cell or filament volumes were converted to biomass (wet weight) by assuming a density of 1 g ml 1. The extraction of microcystins (MCs) from filters and high performance liquid chromatography (HPLC) analysis were carried out according to Gkelis et al. (Gkelis et al., 2005). Purified MCs, MC LR, and its demethylated variants [D-Asp 3 ] MC LR, [Dha 7 ] MC LR, [D-Asp 3,Dha 7 ] MC LR, MC-RR and its demethylated variants [D-Asp 3 ] MC-RR, [Dha 7 ] MC-RR, [D-Asp 3, Dha 7 ] MC-RR, MC-LA and MC-YR (Sivonen et al., 1995) and anabaenopeptins A and B and anabaenopeptilide 90A (Fujii et al., 1996) were used as standards for HPLC. Nodularin (5 ml of 100 ng ml 1 ) was used as an internal standard for the quantification of MCs. The water temperature and ph at the time of sampling, the dissolved inorganic nutrient concentrations, the abundance and biomass of A. ovalisporum and other cyanobacterial species present in the water samples from L. Lysimachia and L. Trichonis are given in Table I. Table I: Temperature, ph, dissolved inorganic nutrient concentrations of the water, Aphanizomenon ovalisporum abundance and biomass and the other cyanobacterial taxa present in water samples from lakes Lysimachia and Trichonis Lake Lysimachia Trichonis Water temperature ( C) 30.8 29.2 ph 7.2 7.5 NO 3 N(mM) 3.54 3.77 NO 2 N(mM) 0.02 0.01 NH 4 N(mM) Not detected Not detected Soluble reactive phosphorus (mm) 0.03 0.03 A. ovalisporum Abundance (trichomes L 1 ) 199 682 Biomass (mg L 1 ) 3.2 1.5 Percent of total biomass 99.4 58.2 Other cyanobacteria present Pseudanabaena sp., Planktothrix mougeotii Anabaena sp. In both lakes, total phytoplankton abundance was low (<500 individuals L 1 ). Cyanobacteria constituted more than 90% of the total phytoplankton abundance. Aphanizomenon ovalisporum was the dominant species in both lakes constituting 99 and 58% of the total cyanobacterial biomass in L. Lysimachia and L. Trichonis, respectively (Table I). Anabaena sp. constituted 42% of the total cyanobacterial biomass in L. Trichonis. In L. Lysimachia Pseudanabaena sp. and Planktothrix mougeotii constituted <1% of the total cyanobacterial biomass, i.e. <3 mg L 1 (Table I). The morphometric characteristics of A. ovalisporum from L. Lysimachia and L. Trichonis are given in Table II. In both cases A. ovalisporum trichomes were solitary (Fig. 1a) (l = 60 700 mm) with a low incidence of terminal hyaline cells (Fig. 1c), were narrowed slightly toward the ends and had gas vesicle containing cells (l = 2.5 6.9 mm, w = 2.4 5.1 mm) (Table II). Heterocytes (Fig. 1b and d) were spherical or ellipsoidal (l =4.4 10.5mm, w = 4.8 8.2 mm). Heterocytes were usually located in the middle of the trichome (Fig. 1d), but were also observed near the tip, and were connected to neighboring cells by small bridges. No terminal heterocytes were observed. Akinetes (l = 16.0 27.8 mm, w = 6.0 15.9 mm) (Table II) were located in the middle of the trichomes (Fig. 1a and b). 1296

1297 Table II: Morphometric measurements of Aphanizomenon ovalisporum from lakes Lysimachia and Trichonis, Greece, and those given by Forti (Forti, 1911; original description), Pollingher et al. (Pollingher et al., 1998) and Shaw et al. (Shaw et al., 1999) Length (mm) Lake Lysimachia Trichonis Forti, 1911 a Pollingher et al., 1998 Shaw et al., 1999 Mean SD Median Minimum Maximum n Mean SD Median Minimum Maximum n Minimum Maximum Minimum Maximum Minimum Maximum Trichome l 303 191 224 126 703 50 280 142 263 60 609 50 500 1000 25 400 Vegetative l cell 4.6 0.8 4.4 3.1 6.9 126 b 3.7 0.6 3.7 2.5 5.0 132 b 4 12 5 10 5 13.7 w 4.1 0.4 4.2 3.3 5.1 3.1 0.5 3.0 2.4 4.3 4 5 3 4 5 6.2 l : w c 1.2 1.1 0.7 1.7 1.2 1.2 0.8 1.6 Heterocyte l 8.7 0.9 8.8 7.6 10.5 21 6.7 1.1 6.9 4.4 9.2 27 12 12 5 11.5 w 7.2 0.7 7.2 5.7 8.2 5.9 0.9 5.8 4.8 8.2 5 d 7 d or 8 3 5 5 7.5 l : w 1.2 1.2 1.1 1.3 1.1 1.1 0.9 1.5 Akinete l 20.2 3.1 20.9 16.0 24.2 18 20.5 3.3 19.8 17.3 27.8 20 18 20 7 15 10 12.5 w 10.2 3.5 10.9 6.0 15.9 10.6 3.1 10.5 9.3 13.4 12 14 5 10 10 12.5 l : w 2.1 2.1 1.1 2.9 1.9 2.0 1.6 3.0 l, length; w, width. a Given in Huber-Pestalozzi (Huber-Pestalozzi, 1938). b Measured from 25 filaments. c l : w is a nondimensional size. d When spherical. Data S. GKELIS ETAL. j APHANIZOMENON OVALISPORUM AND CYANOTOXIN OCCURRENCE

Fig. 1. Photomicrographs (a, b, c and d) of Aphanizomenon ovalisporum trichomes (preserved samples) showing akinetes (A), terminal hyaline cells (black arrowheads) and heterocytes (H). Bar, 20 mm. A wide variation in the morphological characteristics of A. ovalisporum in natural populations has been reported in the literature (Bazzichelli and Abdelahad, 1994; Pollingher et al., 1998; Shaw et al., 1999). However, the morphometric and morphological characteristics of A. ovalisporum from L. Lysimachia and L. Trichonis conform to the species description of Forti (Forti, 1911; cited in Huber-Pestallozi, 1938) (Table II) with wide variations in the lengths of trichomes and the dimensions of vegetative cells and akinetes. The akinetes (see l : w ratio, Table II) were from nearly cylindrical (characteristic of Anabaena minderi; Hindák, 2000) to nearly spherical (characteristic of A. ovalisporum and A. bergii; Hindák, 2000). Akinete length of A. ovalisporum from L. Lysimachia resembles the akinete length of A. bergii described in Turkey (Cirik-Altindag et al., 1992). Furthermore, many of the A. ovalisporum morphological characters studied here overlap with the description of A. bergii by Hindák (Hindák, 2000). It appears that all the populations described as A. ovalisporum to date (Bazzichelli and Abdelahad, 1994; Pollingher et al., 1998; Shaw et al., 1999; this study) exhibit a morphological variability which ranges from typical A. bergii to A. ovalisporum to A. minderi. It is of particular interest that populations of A. ovalisporum were encountered in Greek lakes since this cyanobacterium has been reported rarely elsewhere in the world. To date, A. ovalisporum has been reported in lakes in Israel (Banker et al., 1997; Pollingher et al., 1998), Greece (this study) and Italy (Cannicci, 1954; Bazzichelli and Abdelahad, 1994) (latitudes between 32 N and 41 N) and in Australian freshwaters (Shaw et al., 1999) (latitude 25 S) with Mediterranean or subtropical climates, respectively. There is one report of the presence of A. ovalisporum f. brevicellum in the Caucasian area (Bazzichelli and Abdelahad, 1994). The closely related species A. bergii has been found at latitudes greater than 41 N (Hindák, 2000). HPLC analysis of the L. Lysimachia sample (filter extract) for cyanotoxins resolved two peaks (peaks 2 and 3; Fig. 2a). Nodularin was used as the internal standard (peak 1). The retention time of peak 2 did not correspond to any of the MC or anabaenopeptin standards used. However, the UV-absorption spectrum of peak 2 had a maximum at 273 nm (Fig. 2b) which is typical of anabaenopeptins (Harada et al., 2004). This peak was considered to be a putative anabaenopeptin but was not further characterized. Peak 3 was identified as MC LR by comparison to the retention time of the standard and had a UV-absorption spectrum profile (Fig. 2c) typical of MCs (Lawton et al., 1994). The sestonic MC LR concentration in the L. Lysimachia water was 0.9 mg L 1. Cyanotoxins were not detected in the L. Trichonis sample by HPLC analysis. In L. Lysimachia in which A. ovalisporum was dominant, and Pseudanabaena sp. and P. mougeotii had a minor contribution to the total cyanobacterial biomass (<1%), MC LR and a putative anabaenopeptin were detected. To date, A. ovalisporum has been reported to produce only cylindrospermopsin (Banker et al., 1997; Shaw et al., 1999). Although P. mougeotii is known to produce MCs (Sivonen and Jones, 1999), its biomass in L. Lysimachia was very low (<50 filaments L 1 ). In addition, a P. mougeotii strain has been described which does not produce MCs (Welker et al., 2004). Aphanizomenon species have not been reported to produce MCs (Sivonen and Jones, 1999; Lyra et al., 2001). Anabaenopeptins A and B have previously been detected in water samples from lakes Kastoria, Pamvotis and Zazari, in Greece (Gkelis et al., 2005). Planktothrix spp. are known to produce mainly demethylated MC variants (Henriksen, 1996; Fastner et al., 1999; Christiansen et al., 2003; Welker et al., 2004). 1298

S. GKELIS ETAL. j APHANIZOMENON OVALISPORUM AND CYANOTOXIN OCCURRENCE Fig. 2. High performance liquid chromatography (HPLC) profile (a) indicating three peaks. Peak 1, internal standard (nodularin); peak 2, a putative anabaenopeptin, based on the UV-absorption spectrum (b); peak 3, microcystin LR (MC LR), identified by comparison of the retention time with that of a MC LR standard and the UV-absorption spectrum (c). Aphanizomenon ovalisporum was also dominant in L. Trichonis, and Anabaena sp. was codominant. Anabaena species are known to produce MCs (Sivonen and Jones, 1999) and anabaenopeptins (Fujii et al., 1996). However, these compounds were not detected in L. Trichonis. The accumulation of cyanobacterial cells in waterbodies presents waterborne hazards to humans and animals which range from mild to fatal depending on the exposure (Codd et al., 2005). In Greece, inland waterbodies are managed in terms of water usage but are not yet managed with respect to their ecological quality and cyanotoxin concentrations (Cook et al., 2005). The MC LR concentration of 0.9 mg L 1 detected in L. Lysimachia is very close to the provisional guideline value of 1 mg L 1 in drinking water set by the World Health Organisation (Falconer et al., 1999). Thus, the absence of cyanotoxin management policies for the lake may present a potential hazard for human health and wildlife concerning the consumption of L. Lysimachia products and water. In conclusion, this is the first report of the presence of the rare cyanobacterium A. ovalisporum in Greek lakes. The occurrence of A. ovalisporum and the presence of MC LR and a putative anabaenopeptin in L. Lysimachia are indications that potential problems andhazardsmayariseinthefuturethroughtheuse of the lake water. The identification of the MC and anabaenopeptin producing species in L. Lysimachia remains unresolved. ACKNOWLEDGEMENTS This study was supported by the EU project CYANO- TOX (IC18-CT98-0293), the Academy of Finland to K.S. and the General Secretariat of Research and Technology, Greece, (Herakleitos) to T.L. and S.G. We thank Dr. C. M. Cook, National Agricultural Research Foundation (NAGREF), Greece, for making critical suggestions to the manuscript. REFERENCES American Public Health Association. (1976) Standard Methods for the Examination of Water and Waste Water, 14th edn. American Public Health Association, Washington. Banker, R., Carmeli, S., Hadas, O. et al. (1997) Identification of cylindrospermopsin in Aphanizomenon ovalisporum (Cyanophyceae) isolated from Lake Kinneret, Israel. J. Phycol., 33, 613 616. Bazzichelli, G. and Abdelahad, N. (1994) Caractérisation morphométrique et statistique de deux populations d Aphanizomenon du groupe Aphanizomenon ovalisporum Forti des lacs de Nemi et Albano (Italie). Algol. Stud., 73, 1 21. Cannicci, G. (1954) Su una eccezionale fioritura del lago di Albano (con notizie sul fitoplankton e i rotiferi). Boll. Pesca E Idrobiol. (N.S.), 8, 221 233. Christiansen, G., Fastner, J., Erhard, M. et al. (2003) Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation. J. Bacteriol., 185, 564 572. Cirik-Altindag, S., Couté, A. and Cirik, S. (1992) Quelques cyanophycées rares du lac de Bafa (Turquie). Cryptogam. Algol., 13, 235 246. 1299

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