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Monitoring cYanobacteria and cYanotoxins in Alqueva dam Amely Zavattieri; Susana Nunes; Alexandra Penha; Maria Manuela Queiroz Martins Mantero Morais Corresponding author: A. Zavattieri, Email: [email protected]. Rua da Barba Rala, n.º 1, Parque Industrial e Tecnológico de Évora, 7005-345 Évora, Portugal. Tel/Fax: (+ 351) 266 768 060 INTRODUCTION Alqueva region is now recognized as one of the most productive and recreational area of Alentejo, in Portugal. For these reason ALEX project (Alqueva Hidro Meteorological Experiment) included complementary physical and biological studies to asses the ecological situation of Alqueva dam. A good knowledge of the whole system is mandatory to propose adequate management strategies. Several biological quality indicators of reservoirs are widely recognized as the phytoplankton composition, specially cyanobacteria. Polymerase chain reaction (PCR) was selected to corroborate microscopic identification of cyanobacteria found during ALEX campaign in the summer of 2014 , and also to verify the presence of toxic genes in the samples. This technique allows the identification of the major toxin biosynthesis genes not requiring axenic cultures, since such cultures are difficult to obtain (Choit al., 2008). This molecular approach subsequently facilitates an analysis of the distribution of genotypes based on the presence or absence of a combination of target genes in the samples. In the current study, an initial screening of samples was completed by PCR using a combination of primer sets (Table 1). MATERIAL AND METHODS PRELIMINARY RESULTS Study area and sampling The field campaign in Alqueva reservoir took place from June to September 2014. In situ measurements, water samples and biological elements were monthly collected from three fixed platforms placed in the lacustrine zone and from selected sites in the margins. An additional campaign for cyanobacteria was performed in October due to a bloom situation. In each campaign, vertical profiles of temperature, dissolved, pH, oxidation-reduction potential and turbidity were taken. Samples were collected with a plankton net from platforms and margins and separate in two sub-samples (100 ml each) one for microscopic identification and isolation for in vitro culture and the other for molecular studies (the latest were kept on ice until transported to the laboratory). Microscopic identification of collected samples indicate the presence of serveral species of cyanobacteria in Alqueva dam during the summer of 2014 (Fig. 2). Isolation for in vitro culture was no easy particularly for the Genus Microcystis. apparently the growth of this cyanobacterium is inhibited in agar medium (Allen and Gorham, 1981). Filamentous cyanobacteria grew well in BG-11 medium (solid and liquid) with or without the addition of antibiotics (quais usamos) (Fig. 3) a e Fig. 1 Meteorological platform in which cynobacterial samples were taken with plankton net. DNA extraction directly from local samples Cells used to extract DNA for PCR analysis were collected by filtering 100 ml of the sample collected as described above onto 0,2 µm pore size polycarbonate membrane filters (Millipore), and immediately frozen in Eppendorfs (-20ºC) until processing. Extraction of DNA was latter perform using a modification of the protocol described by Rinta-Kanto et al., 2005. Conventional PCR was performed for cyanobacterial reference strains and bloom samples to check the presence of toxin genes; and second, to check the specificity of the PCR primers to amplify single genes. The set of primers and primers targets used for PCR analysis are summarized in table 1. Each single reaction, with a final volume of 25 µL, contained 1 µL of template??, 0.2 µM of each primer (final concentration), 0.2 µM dNTPs (final concentration, Invitrogen, California ), 5X GoTaq® Flexi buffer (Promega, Madison, WI), 3 mM MgCl2 (final concentration) and 0.025 U/µL GoTaq® G2 Flexi DNA Polymerase (Promega, Madison, WI). The PCR protocol consisted of an initial denaturation step at 94 °C for 3 min, 35 cycles at 94 °C for 45 s, primer annealing at 50 °C for 30 s, primer elongation at 72 °C for 1 min, and a final single step at 72 °C for 7 min. The PCR reactions were performed in a thermocycler (Qual?)and the specificity of the reaction was verified by a 1% agarose-gel electrophoresis. b f c g d h Fig. 2 Microscopic images of the cyanobacteria identify in the summer of 2014 for Alqueva Dam . Anabaena catenula (a); Cylindrospermopsis sp. (b) ; Lyngbya sp. (c); Oscillatoria sp. (d); Microcystis aeruginosa (e); Aphanozomenon flos-aquae (f); Woronichinia naegeliana (g); Microcystis aeruginosa (h) Fig. 3 Different phases of in vitro culture. Isolated cyanobacteria on solid G-11 medium (a) ; liquid medium without antibiotic (b). Table 1. PCR primers used GENE SIZE (bp) REFERENCE 780 Neilan et al. 1997; Jungblut et al. 2005 754 Neilan et al. 1997 220 Neilan et al. 1997 GGCATTCCTAGTTATATTGCCATACTA GCCCGTTTTTGTCCCTTTGCTGC 300 Wilson et al., 2000 mcyA-Cd1F mcyA-Cd1R AAAATTAAAAGCCGTATCAAA AAAAGTGTTTTATTAGCGGCTCAT 297 Hisbergues et al. 2003 mcyB2959F mcyB3278R TGGGAAGATGTTCTTCAGGTATCCAA AGAGTGGAAACAATATGATAAGCTAC 350 Nonneman & Zimba 2002 PKEF1 PKER1 CGCAAACCCGATTTACAG CCCCTACCATCTTCATCTTC 755 Ouahid et al. 2005). HEPF HEPR TTTGGGGTTAACTTTTTTGGCCATAGTC AATTCTTGAGGCTGTAAATCGGGTTT 472 Jungblut e Neilan 2006 PKSM4 PKMS5 M13 M14 GAAGCTCTGGAATCCGGTAA AATCCTTACGGGATCCGGTGC GGCAAATTGTGATAGCCACGAGC GATGGAACATCGCTCACTGGTG 650 Schembri et al. 2001 597 Schembri et al. 2001 PRIMER SEQUENCE 5’-3’ 27F 809R 740F 1494R Micr184F Micr431R AGAGTTTGATCCTGGCTCAG GCTTCGGCACGGCTCGGGTCGATA GGCYRWAWCTGACACTSAGGGA TACGGTTACCTTGTTACGAC GCCGCRAGGTGAAAMCTAA AATCCAAARACCTTCCTCCC GYL2 GYL4 Microcystin production mcyB, gene present in Microcystis that produce 16sRNA specific fragment for cyanobacteria 16s specific for Microcystis Specific for Cilindrospermopsis raciborskii microcistin Gene mcyE, from Genera Microcystis mcyE & ndaF, microcystin and nodularin sinthetase Cylindrospermopsin polyketide synthetase (pks) Cylindrospermopsin peptide synthetase (ps) REFERENCES Choi GG., Bae MS., Ahn CY, Oh HM. 2008 Induction of axenic culture of Arthrospira (Spirulina) platensis based on antibiotic sensitivity of contaminating bacteria. Biotechnology Letters 30, 87–92. Allen EAD., Gorham PR. 1981. Culture of planktonic cyanophytes on agar, In W. W. Carmichael (ed.), The water environment: algal toxins and health. Plenum Publishing. Corp., New York. p. 185-192. Hisbergues M, Christiansen G, Rouhiainen L, Sivonen K, Borner T. 2003. PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera. Archives of Microbiology 180(6):402-410. Jungblut A-D, Neilan BA. 2006. Molecular identification and evolution of the cyclic peptide hepatotoxins, microcystin and nodularin, synthetase genes in three orders of cyanobacteria. Archives of Microbiology 185(2):107-114. Neilan BA., Jacobs D., DelDot T., Blackall LL.,Hawkins PR., Cox PT., Goodman AE. 1997. rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis. Int.ernational Journal of Syst.ematic Bacteriol.ogy 47:693-697. Nonneman D, Zimba PV. 2002. A PCR-based test to assess the potential for microcystin occurrence in channel catfish production ponds. Journal of Phycology 38(1):230-233. Ouahid Y, Perez-Silva G, del Campo F. 2005. Identification of potentially toxic environmental Microcystis by individual and multiple PCR amplification of specific microcystin synthetase gene regions. Environmental Toxicology 20(3):235-242. Rinta-Kanto JM, Ouellette AJA., Twiss MR., Bridgeman TB., Wilhelm SW. 2005. Quantification of toxic Microcystis spp. during the 2003 and 2004 blooms in western Lake Erie using quantitative real-time PCR. Environmental Science and Technolology 39:4198–4205. Schembri MA, Neilan BA, Saint CP. 2001. Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii. Environmental Toxicology 16(5):413-421. Wilson KM, Schembri MA, Baker PD, Saint CP. 2000. Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR. Applied and Environmental Microbiology 66(1):332-338. Fig. 4 Electrophoretic gel with the results of the 10 primers used PCR procedure confirmed the presence of cyanobacteria in the Alqueva by way of the conservative region of the cyanobacterial genome in 16SrRNA with product of 780bp (1) and 754bp (2) Fig 4. The presence of Microcystis was also confirmed by the sequences 220bp (3). It was also possible to observed specific for ........and (Fig. 4). On the other hand, it was no possible to identify specific genes for Cylindrospermopsis raciborskii. being necessary to identify the specie of Cylindrospermopsis found by microscopical observation. The results associated with the production of the toxin Cylindrospermopsin like cylindrospermpsin polyketide synthetase (pks) and polipeptide synthetase were also no conclusive being necessarily to adjust the PCR reaction conditions for the fragments 597bp (9) and 650bp (10). CONCLUSIONS The combination of microscopical identification and PCR confirmation with specif primers was very usefull for a . In the future we will introduce pRT-PCR for a rapid identification of cyano and toxins as well as quantification of.........