Open Life Sci. 2015; 10: 52–60

Research Article Open Access

Spyros Gkelis*, Pablo Fernández Tussy, Nikos Zaoutsos Isolation and preliminary characterization of strains from freshwaters of

Abstract: Cyanobacterial harmful algal blooms (or 1 Introduction CyanoHABs) represent one of the most conspicuous waterborne microbial hazards. The characterization of Cyanobacteria are photosynthetic, prokaryotic organisms the bloom communities remains problematic because which occur primarily in freshwater and saline the cyanobacterial of certain genera has not environments, but also in terrestrial ecosystems. Their yet been resolved. In this study, 29 planktic and benthic presence in lakes with high nutrient levels can to a cyanobacterial strains were isolated from freshwaters mass increase in cyanobacterial cell numbers, with the located in Greece. The strains were assigned to the genera formation of blooms, which results in a depreciation of Chroococcus, Microcystis, Synechococcus, Jaaginema, water quality [1]. Cyanobacterial harmful algal blooms Limnothrix, Pseudanabaena, Anabaena, and Calothrix (or CyanoHABs) represent one of the most conspicuous and screened for the production of the cyanotoxins waterborne microbial hazards to human and agricultural microcystins (MCs), cylindrospermopsins (CYNs), and water supplies, fishery production, and freshwater and saxitoxins (STXs) using molecular (PCR amplification of marine ecosystems [2]. This hazard results from the seven genes implicated in cyanotoxin biosynthesis) and production of cyanotoxins, harmful secondary metabolites, immunological (ELISA) methods. This study presents, which can have deleterious effects within reservoirs and in for the first time, a cyanobacteria culture collection downstream receiving water systems during releases [3]. from Greece, thus providing missing study material for In Greece, common bloom-forming cyanobacteria the understanding of bloom formation and cyanotoxin mainly belong to the genera Microcystis and Anabaena, production in the Mediterranean and for the polyphasic followed by Cylindrospermopsis and Aphanizomenon [1, characterization of important components of the 4, 5]. In addition to the bloom-forming cyanobacteria, phytoplankton. The combined use of molecular and a wide range of less abundant and lesser-known immunochemical methods allowed the identification of cyanobacteria, such as, filamentous (e.g. Pseudanabaena) MC producing strains, but further data are needed for CYN- or colonial (e.g. Aphanocapsa, Chroococcus, Cyanodictyon) and STX-producing cyanobacteria. The high percentage nanoplanktonic (2-20 μm) species [4] and Synechococcus- of MC-producing Microcystis strains in the urban Lakes type picocyanobacteria (<2 μm) [6] are present in blooms Kastoria and Pamvotis, frequently used for agriculture that rarely become dominant, but can represent an irrigation, fishing and recreation, highlights the potential important part of the total cyanobacterial biomass. risk for human health. Occasionally, benthic and/or periphytic cyanobacteria can be observed in phytoplankton. Keywords: Microcystis, Anabaena, Limnothrix, Calothrix, The characterization of the bloom communities’ cyanotoxins, molecular detection, lakes, ELISA structure remains problematic because the cyanobacterial taxonomy of certain genera has not yet been resolved [7]. Today, cyanobacterial diversity is examined using a DOI 10.1515/biol-2015-0006 polyphasic approach by assessing morphological and Received January 28, 2014; accepted August 21, 2014 molecular data (e.g. 8, 9), often combined with toxicological characters [10, 11]. The traditional cyanobacterial *Corresponding author: Spyros Gkelis: Department of Botany, classification [12-15] and the bacteriological classification School of Biology, Aristotle University of , GR-541 24 [16] are based on morphological and genotypic (partial Thessaloniki, Macedonia, Greece, E-mail: [email protected] 16S rRNA gene sequences) data [9]. The comparison of Pablo Fernández Tussy, Nikos Zaoutsos: Department of Botany, morphological and genetic data is sometimes hindered School of Biology, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Macedonia, Greece by the lack of cultures of several cyanobacterial

© 2015 Spyros Gkelis et al., licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Isolation and characterization of cyanobacteria strains 53 morphospecies and inadequate morphological data of Thessaloniki (AUTH) microalgae collection (Department of sequenced strains [8]. Furthermore, in order to evaluate the Botany, School of Biology) and can be accessed in http:// phenotypic plasticity within defined taxa, the variability cyanobacteria.myspecies.info/. observed in cultures has to be compared to the range in natural variation [17]. 2.3 Light microscopy The taxonomy of some of the potentially toxic cyanobacteria remains challenging [18], especially due to A Zeiss Axio imager z2 (Carl Zeiss, Germany) microscope the co-occurrence of several different morphotypes [7]. using bright field and differential interference contrast In Greece, cyanobacteria diversity and toxicity is mainly (EC Plan-Neofluar 5x/0,16,EC Plan-Neofluar 10x/0.3, Plan- known by field (e.g. [4, 5, 19]) and culture-independent Apochromat 20x/0.8, Plan-Neofluar 40x/0.75 DIC, Plan- 16S rRNA gene studies (e.g. [7]); only one publication [18] Neofluar 63x/1.25 Oil DIC, Plan-Neofluar 100x/1.30 Oil DIC) refers to Limnothrix cyanobacteria isolates. The objective was used. Microphotographs were taken with an Axio Cam of this paper is to isolate and characterize cyanobacteria MRc5 digital camera (Carl Zeiss, Germany). from freshwaters of Greece, with respect to their ability to produce cyanotoxins. 2.4 Identification

The strains were identified to the species or genus level 2 Experimental Procedures according to Anagnostidis & Komárek [12, 13), Komárek & Anagnostidis (14, 15, 23, 24), Castenholz [16], taking into 2.1 Growth media and growth conditions consideration current taxonomic status [17].

Solid growth medium: agar plates 53 and 90 mm in 2.5 DNA extraction and PCR analyses diameter containing BG-11 media [20] with or without (for the nitrogen-fixing strains) nitrogen, 1.2% w/v [21] agar In order to identify toxic strains, different primer pairs, (Sigma-Aldrich, Germany). Liquid growth medium: BG-11 previously described in the literature, were used to with or without nitrogen in 100, 250 and 500 mL culture detect different gene targets known to be involved in the flasks. biosynthesis of either MC, CYN or STX. DNA was extracted Cultures were grown as liquid batch cultures at 20±2oC using the protocol described in Atashpaz et al. [25] for Gram or 25±1oC (for Microcystis) at a photosynthetic photon negative . PCR was carried out on the DNA extracts flux density of 20 μmol m-2 s-1 provided by cool white light using the primer pairs shown in Table 2 and PCR conditions fluorescent lamps (Sylvania Standard F36W/154-T8, SLI) in described in detail by Gkelis & Zaoutsos [5]. Thermal a 16:8 h light:dark cycle. cycling was carried out using an Eppendorf MasterCycler Pro (Eppendorf). PCR products were separated by 1.5% 2.2 Sampling Sites and Strain isolation (w/v) agarose gel in 1X TAE buffer. The gels were stained with ethidium bromide and photographed under UV Strains were isolated from surface water samples collected transillumination. from freshwaters of Greece between 1999 and 2010 (Table DNA extracted from Microcystis aeruginosa M6 strain 1); for a description of the Kerkini Reservoir and Lakes was used as positive control for the amplification of mcyA, Amvrakia, Doirani, Kastoria, Mikri Prespa, Pamvotis, mcyB and mcyE gene targets; DNA from Cylindrospermopsis Paralimni, Volvi, see [1 and 4]. Lake Pikrolimni is located raciborskii Aqs strain was used as positive control for in the basin of plain, near Thessaloniki (23 km), in the amplification of the ps (peptide syntethase) and pks northern Greece. It is a small, shallow lake which usually (polyketide synthase) genetic determinants; DNA from dries out during summer. It has an average depth of Aphanizomenon gracile A040 strain was used as positive about 0.5–0.7 m and covers an area about 4.5 km2 when control for the detection of sxtI target gene (see [26]). All it is flooded [22]. Strains were isolated on solid growth positive controls we used produced an amplification media using classical microbiological techniques and product under the tested conditions. grown as batch clonal unialgal cultures. The strains were purified unialgal by repeated transfer of single colonies or 2.6 Cyanotoxin analyses trichomes of cyanobacteria on BG-11 medium agar plates; all strains were derived from a single colony or trichome. The Abraxis Microcystin (520011), Saxitoxin (52255B), and The isolates were deposited in Aristotle University of Cylindrospermopsin (522011) Microtiter Plate Kits were 54 S. Gkelis et al. used to determine the presence of Microcystins (MCs), percentage of >15% were not accepted. Strains were Saxitoxins (STXs), and Cylindrospermopsins (CYNs), considered positive for a cyanotoxin when concentration respectively. Eight-to-ten mL from each culture were was higher of the lowest concentration of the standards centrifuged and the pellet was freeze-dried. provided for each cyanotoxin. MC, STX and CYN from each strain were extracted by placing up to 1200mg of freeze-dried material in eight mL of water in glass tubes, immersed in ice and sonicated 3 Results for 10 min. After sonication, the mixture was stirred for Twenty-nine strains were isolated from surface water 30 min at room temperature, centrifuged for 10 min at samples collected from nine lakes (Amvrakia, Cheimaditis, 13,000 g and the supernatant was collected. The pellet Doirani, Kastoria, Mikri Prespa, Pamvotis, Paralimni, was resuspended in eight mL of water and re-extracted. Pikrolimni and Volvi) and one reservoir (Kerkini) located The resulting solutions were then applied to the above in Greece (Table 1). Isolation procedures resulted in 25 mentioned ELISA kits following the manufacturer’s planktic, and four benthic isolates (Table 1) representing instructions. eight genera (Figure 1). The isolated cyanobacterial The microtiter plates were read at 450 and 630 nm, for strains were assigned to the following genera: strain MCs and at 450 nm for CYNs and STXs, and B values (%) 0 0599, Chroococcus; strains 0410, 0610, 0710, 1410, 1510, were calculated. Samples with a coefficient of variation

Table 1: Cyanobacteria strains isolated from freshwaters of Greece and their origin. The isolates are deposited in Aristotle University of Thes- saloniki (AUTH) microalgae collection (Department of Botany, School of Biology) Strain Origin Geographic coordinates Habitat Collection date (Lake or Reservoir) (N) (E) Chroococcus minutus AUTH 0599 Mikri Prespa 40o45΄ 21o07΄ planktic 5/8/1999 Microcystis flos-aquae AUTH 0410 Pamvotis 39o40΄ 20o51΄ planktic 21/8/2010 Microcystis aeruginosa AUTH 0610 Kastoria 40o31΄ 21o18΄ planktic 24/8/2010 Microcystis sp. AUTH 0710 Kastoria 40o31΄ 21o18΄ planktic 24/8/2010 Microcystis flos-aquae AUTH 1410 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Microcystis flos-aquae AUTH 1510 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Microcystis sp. AUTH 1610 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Microcystis sp. AUTH 1710 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Microcystis viridis AUTH 1810 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Microcystis sp. AUTH 2010 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Microcystis sp. AUTH 2110 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Microcystis sp. AUTH 2310 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Microcystis flos-aquae AUTH 2410 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Synechococcus sp. AUTH 0499 Cheimaditis 40o36΄ 21o33΄ planktic 5/8/1999 Synechococcus sp. AUTH 3010 Pamvotis 39o40΄ 20o51΄ planktic 1/11/2010 Limnothrix redekei AUTH 0310 Doirani 41o11΄ 22o45΄ planktic 21/8/2010 Jaaginema sp. AUTH 0110 Volvi 40o41΄ 23o25΄ planktic 21/8/2010 Jaaginema sp. AUTH 0210 Doirani 41o11΄ 22o45΄ planktic 21/8/2010 Jaaginema sp. AUTH 2210 Kerkini 41o08΄ 23o13΄ planktic 21/8/2010 Pseudanabaena sp. AUTH 0104 Pikrolimni 40o83΄ 22o82΄ benthic 27/9/2004 Anabaena cf. oscillarioides AUTH 0199 Paralimni 38o27΄ 23o21΄ benthic 19/7/1999 Anabaena sp. AUTH 0299 Paralimni 38o27΄ 23o21΄ benthic 19/7/1999 Anabaena cf. cylindrica AUTH 0699 Amvrakia 38o45΄ 21o11΄ planktic 19/8/1999 Anabaena sp. AUTH 0799 Kerkini 41o08΄ 23o13΄ planktic 26/8/1999 Anabaena sp. AUTH 0899 Kerkini 41o08΄ 23o13΄ planktic 26/8/1999 Anabaena sp. AUTH 2510 Doirani 41o11΄ 22o45΄ planktic 21/8/2010 Anabaena sp. AUTH 2610 Doirani 41o11΄ 22o45΄ planktic 21/8/2010 Anabaena sp. AUTH 2710 Doirani 41o11΄ 22o45΄ planktic 21/8/2010 Calothrix sp. AUTH 0399 Pamvotis 39o40΄ 20o51΄ benthic 22/7/1999 Isolation and characterization of cyanobacteria strains 55

1610, 1710, 1810, 2010, 2110, 2310 and 2410, Microcystis; for MCs (Table 3). We also obtained fragments targeting strains 0499 and 3010, Synechococcus; strains 0110, the mcyB, mcyE, and mcyE/ndaF genes in 10, 10 and 9 0210 and 2210, Jaaginema; strain 0310, Limnothrix; Microcystis isolates, respectively (Table 3). All three primer strain 0104, Pseudanabaena; strains 0199, 0299, 0699, pairs were co-amplified, except for one strain (Microcystis 0799, 0899, 2510, 2610 and 2710 Anabaena; and strain sp. AUTH 0710) where mcyE/ndaF gene fragment was not 0399, Calothrix (Table 1). Morphological features (data amplified (Table 3). Only the mcyB gene fragment was not shown) allowed us to identify 10 strains to the amplified in strains Synechococcus sp. AUTH 0499 and species level: strain 0599 Chroococcus minutus; strains Anabaena sp. AUTH 0299; only mcyE/ndaF gene fragment 0410, 1410 and 1510, Microcystis flos-aquae; strain 0610, was amplified in strains Pseudanabaena sp. AUTH 0104 Microcystis aeruginosa; strain 1810, Microcystis viridis; and Anabaena sp. AUTH 0799; only mcyE gene fragment strain 0310, Limnothrix redekei; strain 0199, Anabaena cf. was amplified in strain Anabaena sp. AUTH 2710; none oscillarioides; strain0699, Anabaena cf. cylindrica (Table of the strains where only one MC gene fragment was 1). amplified gave positive ELISA results for MCs (Table 3). A PCR product of about 300 bp was obtained using None of the assayed isolates gave positive PCR results the mcyA-Cd 1F/mcyA-Cd 1R primer pair, indicating the using the psM13/PSM14 primer pair, thus suggesting the presence of mcyA gene, in eight out of the 12 Microcystis absence of the ps gene in the strains. However, the 422 bp isolates; six of those strains gave positive ELISA results fragment of the pks gene was amplified using the primer

Figure 1: Microphotographs of strains representing eight genera of cyanobacteria isolated from freshwaters of Greece.[a] Chroococcus minutus AUTH 0599; [b] Microcystis flos-aquae AUTH 1410; [c] Synechococcus sp. AUTH 3010; [d] Limnothrix redekei AUTH 0310; [e] Jaagi- nema sp. AUTH 0110; [f] Pseudanabaena sp. AUTH 0104; [g] Anabaena sp. AUTH 0899; [h] Calothrix sp. AUTH 0399. Bars, 20 μm. 56 S. Gkelis et al.

Table 2: PCR primers used in the analyses of cyanobacteria strains isolated in this study for the detection of genes involved in microcystin, cylindrospermopsin and saxitoxin production

Primer Target-gene Sequence (5’–3’) Size (bp) Reference mcyA-Cd1F mcyA AAAATTAAAAGCCGTATCAAA 297 [46] mcyA-Cd1R AAAAGTGTTTTATTAGCGGCTCAT mcyB2959F mcyB TGGGAAGATGTTCTTCAGGTATCCAA 320 [29] mcyB3278R AGAGTGGAAACAATATGATAAGCTAC

PKEF1 mcyE CGCAAACCCGATTTACAG 755 [31] PKER1 CCCCTACCATCTTCATCTTC

HEPF mcyE/ndaF TTTGGGGTTAACTTTTTTGGGCATAGTC 472 [33] HEPR AATTCTTGAGGCTGTAAATCGGGTTT psM13 ps GGCAAATTGTGATAGCCACGAGC 597 [42] psM14 GATGGAACATCGCTCACTGGTG pksK18 pks CCTCGCACATAGCCATTTGC 422 [47] pksM4 GAAGCTCTGGAATCCGGTAA

Sxt1-F sxtI GCTTACTACCACGATAGTGCTGCCG 1669 [44] Sxt1-R GGTTCGCCGCGGACATTAAA pair pksM4/PKSK18 in strains Microcystis sp. AUTH 2310 management of toxic cyanobacteria, awareness of benthic and Anabaena sp. AUTH 0899; both strains were found strains is important because toxic benthic cyanobacteria positive for CYN in ELISA (Table 3). have caused animal deaths in Scotland and in Switzerland None of the isolates gave positive PCR results for the [28]. presence of the sxtI gene indicating the absence of STX Microcystins were found in 90% of the Microcystis producing strains (Table 3). However, nine strains (belonging strains examined in this study thus providing further to Microcystis, Synechococcus, Jaaginema, and Anabaena) evidence that in Greece cyanobacteria blooms, often were found positive for STX using ELISA (Table 3). dominated by Microcystis spp., are highly likely to contain microcystins [5, 19]. ELISA detection of microcystins was in accordance with PCR amplification of the mcy regions, 4 Discussion especially the mcyA gene region. This is in accordance with Vasconcelos et al. [26] that detected MCs using ELISA only The strains isolated in this study were assigned to species in samples where the mcyA gene region was amplified. and genera known to occur and/or form blooms in Greek However, Gkelis & Zaoutsos [5] recently reported that in lakes. Chroococcus minutus, Microcystis flos-aquae, M. environmental samples the mcyA region was amplified aeruginosa, and M. viridis have been previously recorded only where Microcystis formed blooms and where MC in the phytoplankton of Lakes Mikri Prespa [27], Pamvotis, concentrations where >40 μg L-1, indicating that mcyA and Kastoria [4] where there were isolated from. This is the gene region is not always amplified or is amplified when first report of Limnothrix redekei occurring in Lake Doirani. Microcystis reaches high biovolumes. Thus, we suggest At the collection dates, water blooms formed mainly that mcyA gene is a suitable molecular marker for MC by Microcystis spp., Anabaena spp., Aphanizomenon production in the presence of bloom-forming Microcystis issatschenkoi, and Cylindrospermopsis raciborskii were populations. observed in Kerkini Reservoir and Lakes Cheimaditis, In some Synechococcus, Pseuadanabaena, and Doirani, Mikri Prespa, Kastoria, and Pamvotis [1, 5, 19]. All Anabaena strains of this study mcyB, mcyE and mcyE/ of these genera, with the exception of the benthic Calothrix, ndaF regions were amplified, but no MCs were found are known to occur in Greek lakes [4, 6]. To the best of our using ELISA. The mcyB2959F/mcyB3278R primer targets knowledge, this is the first report of Calothrix occurring mcyB gene [29], which encodes a peptide synthetase in Greek freshwaters. It is known that, occasionally, containing two modules each possessing adenylation, benthic cyanobacteria can be observed in phytoplankton thiolation, and condensation domains. The mcyB may [28], especially when surface samples are collected from be involved with the activation of “variable L-amino littoral stations, like in this study. For monitoring and acids” [30]. The PKEF1/PKER1 primer pair targets the Isolation and characterization of cyanobacteria strains 57

Table 3: Cyanobacteria strains evaluated for cyanotoxin production by ELISA and genetic properties. Bold letter mark strains where toxicity was confirmed by both PCR and ELISA for a cyanotoxin; *indicates strains where toxicity was confirmed only by ELISA; MC: microcystin; CYN: cylindrospermopsin; STX: saxitoxin; n.a.: not analysed. Cyanotoxin genes are explained in Table 2.

Strain Detection of gene fragments ELISA detection

mcyA mcyB mcyE mcyE/ndaF PKS PS sxtI MC CYN STX

Chroococcus minutus AUTH 0599 ------

Microcystis flos-aquae AUTH 0410 + + + + - - - + + -

Microcystis aeruginosa AUTH 0610 ------

Microcystis AUTH 0710 - + + - - - - + - -

Microcystis flos-aquae AUTH 1410 + + + + - - - + + -

Microcystis flos-aquae AUTH 1510 + + + + - - - + - +

Microcystis AUTH 1610 + + + + - - - + - +

Microcystis sp. AUTH 1710 + + + + - - - n.a. n.a. n.a.

Microcystis viridis AUTH 1810* ------+ - -

Microcystis sp. AUTH 2010 + + + + - - - n.a. n.a. n.a.

Microcystis sp. AUTH 2110 - + + + - - - + - +

Microcystis sp. AUTH 2310 + + + + + - - + + -

Microcystis flos-aquae AUTH 2410 + + + + - - - + - -

Synechococcus sp. AUTH 0499* - + ------+

Synechococcus sp. AUTH 3010 ------

Limnothrix redekei AUTH 0310 ------n.a. n.a. n.a.

Jaaginema sp. AUTH 0110 ------

Jaaginema sp. AUTH 0210* ------+

Jaaginema sp. AUTH 2210 ------

Pseudanabaena sp. AUTH 0104 - - - + ------

Anabaena cf. oscillarioides AUTH 0199 ------

Anabaena sp. AUTH 0299* - + ------+

Anabaena cf. cylindrica AUTH 0699 ------

Anabaena sp. AUTH 0799 - - - + ------

Anabaena sp. AUTH 0899* - - - - + - - + + +

Anabaena sp. AUTH 2510 ------

Anabaena sp. AUTH 2610 ------+

Anabaena sp. AUTH 2710 - - + ------+

Calothrix sp. AUTH 0399 ------

mcyE gene in all the MC-producing strains [31]. The production of MC or nodularin, respectively [33]. Species mcyE gene codes for the glutamate-1-semialdehyde of the genus Anabaena are well known for microcystin aminotransferase (GSA-AMT) domain, whose role in MC production [34] and are very often one of the dominant biosynthesis is to supply the glutamate group to Adda bloom-forming species in Greece [4, 5, 19]. However, [32]. In addition, the HEPF/HEPR primer pair targets the planktic and benthic strains we isolated from Lakes the AMT domain of either mcyE or ndaF, involved in the Doirani, Kerkini, and Paralimni seem to be incapable of 58 S. Gkelis et al. producing MCs, with the exception of strain Anabaena 5 Conclusions sp. AUTH 0899 which needs further investigation to clarify the discrepancy of the ELISA positive results This study presents, for the first time, a cyanobacteria and the absence of amplification of mcy regions. No culture collection from Greece, thus providing missing Synechococcus strain has been reported to produce MCs. study material for the understanding of bloom formation Nonetheless, recent findings [35, 36] associate marine and cyanotoxin production in the Mediterranean and for Synechococcus with MCs. Pseudanabaena spp. are also the polyphasic characterization of important components not considered microcystin-producing; however, a new of the phytoplankton. The data conclude that Microcystis Pseudanabaena species of low microcystin toxicity was are the main MC producing strains, but further data are recently found [11]. needed to assess CYN and STX producing cyanobacteria Two strains (Microcystis sp. AUTH 2310 and Anabaena strains. The high percentage of MC-producing Microcystis sp. AUTH 0899) were found positive for CYN, but only strains in the urban Lakes Kastoria and Pamvotis, one region (ps) targeting CYN-implicated genes was frequently used for agriculture irrigation, fishing and amplified. The ps (aoaB/cyrB) gene spans 8.7 kb and recreation, highlights the potential risk for human health. catalyzes a step in the CYN synthesis. It encodes a PKS/ NRPS hybrid [37]. The pks (aoaC/cyrC) gene spans 5.0 Acknowledgments: Part of this work was supported kb and encodes a PKS [36]. Other studies have shown by Aristotle University Research Committee Program the presence of parts of aoaC/cyrC, aoaB/cyrB (and also “Enhancing Young Investigators-Lecturers: CYaNobacteria aoaA/cyrA) genes associated with the biosynthesis of CYN diversity Assessment throUgh sTrain pHylogenetic in non-CYN-producing cyanobacterial strains [38, 39]. Up analyses) [CYN-AUTH]” (Contract No 87903). We thank to now, no Microcystis species has been found to produce Vitor Vasconcelos for providing freeze-dried material CYN or contain any gene found in the cluster responsible of cyanobacteria strains used as positive control in PCR for CYN biosynthesis [40]. However, cylindrospermopsin studies and E. Panteris for valuable help with microscopy. genes have been reported from Oscillatoria sp. PCC6506 Microphotographs presented here are part of Konstantina [41]. Genetic analyses have shown that the CYN genes may Siampouli’s final-year project supervised by SG. We thank have been horizontally transferred [40]. CYN was also all students who helped maintenance of the culture detected in three Microcystis-dominated blooms in Greece collection during the course of this work. (in two of them, fragments targeting the pks (AoaC/cyrC) gene were also obtained) [5]. In contrast, CYN production Conflicts of Interest: The authors declare that there are has been reported, although rarely, for Anabaena strains no conflicts of interest. [42, 43]. In view of the absence of cyrB genes, further studies are needed to assess whether strains Microcystis sp. AUTH 2310 and Anabaena sp. AUTH 0899 are capable References of producing CYN. [1] Cook C.M., Vardaka E., Lanaras T., Toxic cyanobacteria in Greek In all of the strains found positive for STX in this freshwaters, 1987-2000: Occurrence, toxicity and impacts in study the sxtI gene region was not amplified. The sxtI the Mediterranean region., Acta Hyrdoch. Hydrob. 2004, 32, gene product is a putative carbamoyltransferase of the 107-124 STX biosynthetic cluster that catalyzes the transfer of a [2] Paerl H.W., Hall N.S., Calandrino E.S., Controlling harmful cyanobacterial blooms in a world experiencing anthropogenic carbamoyl group in STX biosynthesis [44]. The sxtI gene and climatic-induced change, Sci. Total Environ., 2011, 409, is exclusively present in PSP toxin producing strains 1739-1745 of Anabaena, Aphanizomenon, Cylindrospermopsis, [3] Paerl H.W., Otten G.T., Harmful Cyanobacterial Blooms: Causes, and Lyngbya [44, 45]. Up to now, no Microcystis, Consequences, and Controls, Microb. Ecol., 2013, 65, 995-1010 Synechococcus or Jaaginema strains has been reported [4] Vardaka E., Moustaka-Gouni M., Cook C.M., Lanaras T., to produce STX; furthermore, no cyanobacterium has Cyanobacterial blooms and water quality in Greek waterbodies. J. Appl. Phycol., 2005, 17, 391-401 been reported to produce STX in the absence of sxtI [5] Gkelis S., Zaoutsos N., Cyanotoxin occurrence and potentially gene. Thus, the possibility of ELISA false-positive results toxin producing cyanobacteria in freshwaters of Greece: a has to be considered and further studies are needed to multi-disciplinary approach, Toxicon, 2014, 78, 1-9 investigate the strains found positive for STX, although [6] Moustaka-Gouni M., Vardaka E., Michaloudi E., Kormas K.A., no false-positive results have been reported for STX [10] Tryfon E., Mihalatou H., Gkelis S., Lanaras T. , Plankton food web structure in a eutrophic polymictic lake with a history in when analyzed with both chromatographic methods and ELISA. Isolation and characterization of cyanobacteria strains 59

toxic cyanobacterial blooms, Limnol. Oceanogr., 2006, 51, Greece? Geochemical evidence, J. Geochem. Explor., 2009, 715-727 103, 133-143 [7] Moustaka-Gouni M., Kormas K.A., Vardaka E., Katsiapi M., [23] Komárek J., Anagnostidis K., Cyanoprokaryota. 1. Teil: Gkelis S., Raphidiopsis mediterranea Skuja represents . Süßwasserflora von Mitteleuropa Band 19/1, p. non-heterocytous life-cycle stages of Cylindrospermopsis 270-323, Spektrum Akademischer Verlag, 1999 raciborskii (Woloszynska) Seenayya et Subba Raju in Lake [24] Komárek J., Anagnostidis K., Cyanoprokaryota. 2. Teil: Oscilla- Kastoria (Greece), its type locality: Evidence by morphological toriales. Süßwasserflora von Mitteleuropa Band 19/2, p. 1-759, and phylogenetic analysis, Harmful Algae, 2009, 8, 864-872 Spektrum Akademischer Verlag, 2005 [8] Rajaniemi P., Hrouzek P., Kaštovská K., Willame R., Rantala A., [25] Atashpaz S., Khani S., Barzegari A., Barar J., Vahed S.Z., Hoffmann L., Komárek J., Sivonen K., Phylogenetic and morpho- Azarbaijani, R., Omidi, Y., A robust universal method logical evaluation of the genera Anabaena, Aphanizomenon, for extraction of genomic DNA from bacterial species. Trichormus and Nostoc (Nostocales, Cyanobacteria), Int. J. Syst. Microbiology, 2010, 79, 538-542 Evol. Micr., 2005, 55, 11-26 [26] Vasconcelos V., Martins A., Vale M., Antunes A., Azevedo [9] Willame R., Boutte C., Grubisic S., Wilmotte A., Komárek J., J., Welker M., Lopez O., Montejano G., First report on the Hoffmann L., Morphological and molecular characterization occurrence of microcystins in planktonic cyanobacteria from of planktonic cyanobacteria from Belgium and Luxemburg., J. Central Mexico, Toxicon, 2010, 56, 425-431 Phycol., 2006, 42, 1312-1332 [27] Tryfon E., Moustaka-Gouni M., Nikolaidis G., Planktic [10] Ballot A., Fastner J., Wiedner C., Paralytic shellfish poisoning cyanophytes and their ecology in the shallow Lake Mikri toxin-producing cyanobacterium Aphanizomenon gracile Prespa, Greece, Nord. J. Bot., 1997, 17, 439-448 in northeast Germany, Appl. Environ. Microbiol., 2010, 76, [28] Mur L.R., Skulberg O.M., Utkilen H., Cyanobacteria in 1173-1180. the environment, In: I Chorus and J Bartram (Eds) Toxic [11] Kling H.J., Laughinghouse H.D., Šmarda J., Komárek J., Cyanobacteria in Water. E & FN Spon, London, UK, pp 41-112, Acreman J., Bruun K., Watson S.B., Chen F., A new red colonial 1999 Pseudanabaena (Cyanoprokaryota, Oscillatoriales) from North [29] Nonneman D., Zimba P., A PCR-based test to assess the American large lakes, Fottea, 2012, 12, 327-339 potential for microcystin occurrence in channel catfish [12] Anagnostidis K., Komárek J., Modern approach to the production ponds, J. Phycol., 2002, 38, 230-234 classification system of cyanophytes. 1- Introduction, Arch. [30] Tillett D., Dittmann E., Erhard M., Döhren H.V., Börner T., Neilan Hydrobiol., 1985, 71, 291-302 B.A., Structural organization of microcystin biosynthesis [13] Anagnostidis K. & Komárek J., Modern approach to the in Microcystis aeruginosa PCC7806: an integrated peptide classification system of cyanophytes. 3- Oscillatoriales, Arch. polyketide synthetase system, Chem Biol, 2000. 7. 753-764 Hydrobiol., 1988, 80, 327-472 [31] Ouahid Y., Perez-Silva G., Del Campo F.F., Identification of [14] Komárek J., Anagnostidis K., Modern approach to the classi- potentially toxic environmental Microcystis by individual and fication system of cyanophytes. 2- Chroococales. Arch. multiple PCR amplification of specific microcystin synthetase Hydrobiol., 1986, 73, 157-226 gene regions, Environ. Toxicol., 2005, 20, 235-242 [15] Komárek J., Anagnostidis K., Modern approach to the [32] Tillett D., Parker D.L., Neilan B.A., Detection of toxigenicity by classification-system of cyanophytes 4 - Nostocales. Arch. a probe for the microcystin synthetase A gene (mcyA) of the Hydrobiol., 1989, 82: 247-345 cyanobacterial genus Microcystis: comparison of toxicities with [16] Castenholz R.W., Oxygenic photosynthetic bacteria. In: Boone 16S rRNA and phycocyanin operon (Phycocyanin Intergenic D.R. & R. W. Castenholz (Eds), Bergey’s Manual of Systematic Spacer) phylogenies, Appl. Environ. Microbiol., 2001, 67, Bacteriology, vol. 1, p. 474-600, Springer-Verlag, New York, 2810-2818 2001 [33] Jungblut A.D., Neilan B.A., Molecular identification and [17] Komárek J., Mareš J., An update to modern taxonomy (2011) of evolution of the cyclic peptide hepatotoxins, microcystin and freshwater planktic heterocytous cyanobacteria, Hydrobiologia, nodularin, synthetase genes in three orders of cyanobacteria, 2012, 698, 327-351 Arch. Microbiol., 2006, 185, 107-114 [18] Gkelis S., Rajaniemi P., Vardaka E., Moustaka-Gouni M., [34] Sivonen K, Jones G., Cyanobacterial toxins. In: I Chorus and Lanaras T., Sivonen K., Limnothrix redekei (Van Goor) Meffert J Bartram (Eds) Toxic Cyanobacteria in Water. E & FN Spon, (cyanobacteria) strains from Lake Kastoria form a separate London, UK, pp 113-153, 1999 phylogenetic group, Microb. Ecol., 2005, 49, 176-182 [35] Frazão B., Martins R., Vasconcelos V., Are Known Cyanotoxins [19] Gkelis S., Harjunpää V., Lanaras T., Sivonen K., Diversity of Involved in the Toxicity of Picoplanktonic and Filamentous hepatotoxic microcystins and bioactive anabaenopeptins North Atlantic Marine Cyanobacteria? Mar. Drugs, 2010, 8, in cyanobacterial blooms from Greek freshwaters, Environ. 1908-1919 Toxicol., 2005, 20, 249-256 [36] Vareli K., Zarali E., Zacharioudakis G.S.A., Vagenas G., Varelis [20] Rippka R., Isolation and purification of cyanobacteria, Method. V., Pilidis G., Briasoulis E., Sainis I., Microcystin producing Enzymol., 1988, 167, 3-27 cyanobacterial communities in Amvrakikos Gulf (Mediterranean [21] de Chazal N.M., Smaglinski S., Smith G.D., Methods involving Sea, NW Greece) and toxin accumulation in mussels (Mytilus light variation of cyanobacteria: Characterization of isolates galloprovincialis), Harmful Algae, 2012, 15, 109-118 from central Australia, Appl. Environ. Microb., 1992, 58, [37] Mihali, T.K., Kellmann, R., Muenchhoff, J., Barrow K.D., Neilan 3561-3566 B.A., Characterization of the gene cluster responsible for [22] Dotsika E., Poutoukis D., Tzavidopoulosa I., Maniatis Y., cylindrospermopsin biosynthesis, Appl. Environ. Microbiol., Ignatiadou D., Raco B., A source at Pikrolimni Lake in 2008, 74, 716-722 60 S. Gkelis et al.

[38] Wood S.A., Rasmussen J.P., Holland P.T., Campbell R., Crowe [43] Spoof L., Berg K.A., Rapala J., Laht, K., Lepisto L., Metcalf A.L.M., First report of the cyanotoxin anatoxin-a from Aphani- J.S., Codd G.A., Meriluoto J., First observation of cylindro- zomenon issatschenkoi (Cyanobacteria), J. Phycol., 2007, 43, spermopsin in Anabaena lapponica isolated from the Boreal 356-365 Environment (Finland), Environ. Toxicol., 2006, 21, 552-560 [39] Ballot, A., Ramm, J., Rundberget, T., Kaplan-Levy, R.N., Hadas, [44] Kellmann R., Mihali T.K., Jeon Y.J., Pickford R., Pomati F., O., Sukenik, A., Wiedner C., Occurrence of non-cylindro- Neilan B.A., Biosynthetic intermediate analysis and functional spermopsin-producing Aphanizomenon ovalisporum and homology reveal a saxitoxin gene cluster in cyanobacteria, Anabaena bergii in Lake Kinneret (Israel), J. Plankton Res., Appl. Environ. Microb., 2008, 74, 4044-4053 2011, 33, 1736-1746 [45] Dittmann E., Fewer D.P., Neilan B.A, Cyanobacterial toxins: [40] Stücken, A., Jakobsen, K.S., The cylindrospermopsin gene biosynthetic routes and evolutionary routes, FEMS Microbiol. cluster of Aphanizomenon sp. strain 10E6: organization and Rev., 2013, 37, 23-43 recombination, Microbiology+, 2010, 156, 2438–2451 [46] Hisbergues M., Christiansen G., Rouhiainen L., Sivonen K., [41] Mazmouz R., Chapuis-Hugon F., Mann S., Pichon V., Méjean A., Borner T., PCR-based identification of microcystin-producing Ploux O., Biosynthesis of cylindrospermopsin and 7-epicylind- genotypes of different cyanobacterial genera, Arch. Microbiol., rospermopsin in Oscillatoria sp. strain PCC 6506: identification 2003, 180, 402-410 of the cyr gene cluster and toxin analysis, Appl. Environ. [47] Fergusson K.M., Saint C.P, Multiplex PCR assay for Cylindro- Microbiol., 2010, 67, 4943–4949 spermopsis raciborskii and cylindrospermopsin-producing [42] Schembri M., Neilan B., Saint C., Identification of genes cyanobacteria, Environ. Toxicol., 2003, 18, 120-125 implicated in toxin production in the cyanobacterium Cylindro- spermopsis raciborskii, Environ.Toxicol., 2001, 16, 413-421