The genetic diversity and the potential toxicity of bloom-forming cyanobacteria were studied in four lagoons located in the state of Sao Paulo (Campinas, Limeira and Piracicaba cities). Bloom samples were collected on the water surface and cyanobacterial communities were evaluated using DGGE fingerprinting and 16S rDNA clone library. The amplification of genes encoding secondary metabolites such as microcystin ( mcy ), anatoxin ( ana ), cylindrospermopsin ( cyr ), saxitoxin ( sxt ), cyanopeptolin ( mcn ) and aeruginosin ( aer ) was performed and their production analyzed by LC-MS. The comparison of DGGE banding pattern among the different water samples suggested that some operational taxonomic units (OTUs) in these locations were predominant over others. The 16S rDNA clone libraries sequences matched with nine different known cyanobacterial genera available in NCBI, identified as Anabaena , Brasilonema , Cylindrospermopsis , Limnococcus , Microcystis , Nostoc , Pseudanabaena , Synechococcus and Woronichinia . The lagoons ESALQ2, Taquaral and Limeira had more than 80% of the cyanobacterial community assigned to the genus Microcystis . Genes encoding aeruginosin, cyanopeptolin and microcystin synthetases and saxitoxin synthase were amplified, and LC-MS/MS confirmed the production of aeruginosin, cyanopeptolin and microcystin. Rapid and sensitive methods for the detection of these secondary metabolites, especially toxins, using chemical and molecular tools together, can be used for a faster diagnostic of toxic cyanobacterial blooms.
Siqueira, D.B. and Oliveira-Filho, E.C. (2005) Cianobacterias de agua doce e saude publica: Uma revisao. Universitas Ciencias da Saude, 3, 109-127.
Paerl, H.W. (2008) Nutrient and Other Environmental Controls of Harmful Cyanobacterial Blooms along the Freshwater-Marine Continuum. Advances in Experimental Medicine and Biology, 619, 217-237. http://dx.doi.org/10.1007/978-0-387-75865-7_10
Sivonen, K. and Jones, G. (1999) Cyanobacterial Toxins. In: Chorus, I. and Bartram, J., Eds., Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring, and Management, E & FN Spon, London, 41-111.
Carmichael, W.W., Azevedo, S.M., An, J.S., Molica, R.J., Jochimsen, E.M., Lau, S., Rinehart, K.L., Shaw, G.R. and Eaglesham, G.K. (2001) Human Fatalities from Cyanobacteria: Chemical and Biological Evidence for Cyanotoxins. Environmental Health Perspective, 109, 663-668.
Merel, S., Walker, D., Chicana, R., Snyder, S., Baures, E. and Thomas, O. (2013) State of Knowledge and Concerns on Cyanobacterial Blooms and Cyanotoxins. Environmental International, 59, 303-327. http://dx.doi.org/10.1016/j.envint.2013.06.013
Lawton, L.A., Edwards, C. and Codd, G.A. (1994) Extraction and High Performance Liquid Chromatographic Method for the Determination of Microcystins in Raw and Treated Water. Analyst, 119, 1525-1530. http://dx.doi.org/10.1039/an9941901525
Watanabe, M.F., Ohishi, S., Harada, K.I., Matsuura, K., Kawai, H. and Suzuki, M. (1988) Toxins Contained in Microcystis Species of Cyanobacteria (Blue-Green Algae). Toxicon, 26, 1017-1025. http://dx.doi.org/10.1016/0041-0101(88)90200-0
Oehrle, S.A., Southwell, B. and Westrick, J. (2010) Detection of Various Freshwater Cyanobacterial Toxins Using Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry. Toxicon, 55, 965-972. http://dx.doi.org/10.1016/j.toxicon.2009.10.001
An, J. and Carmichael, W.W. (1994) Use of a Colorimetric Protein Phosphatase Inhibition Assay and Enzyme Linked Immunosorbent Assay for the Study of Microcystins and Nodularins. Toxicon, 32, 1495-1507. http://dx.doi.org/10.1016/0041-0101(94)90308-5
Al-Tebrineh, J., Gehringer, M.M., Akcaalan, R. and Neilan, B.A. (2011) A New Quantitative PCR Assay for the Detection of Hepatotoxigenic Cyanobacteria. Toxicon, 57, 546-556. http://dx.doi.org/10.1016/j.toxicon.2010.12.018
Molica, R.J.R. and Azevedo, S.M.F.O. (2009) Ecofisiologia de cianobacterias produtoras de cianotoxinas. Oecologia Brasiliensis, 13, 229-246.
Neilan, B.A., Jacobs, D., Del Dot, T., Blackall, L.L., Hawkins, P.R., Cox, P.T. and Goodman, A.E. (1997) rRNA Sequences and Evolutionary Relationships among Toxic and Nontoxic Cyanobacteria of the Genus Microcystis. International Journal of Systematic Bacteriology, 47, 693-697. http://dx.doi.org/10.1099/00207713-47-3-693
Nübel, U., Garcia-Pichel, F. and Muyzer, G. (1997) PCR Primers to Amplify 16S rRNA Genes from Cyanobacteria. Applied and Environmental Microbiology, 63, 3327-3332.
Rigonato, J., Alvarenga, D.O., Andreote, F.D., Dias, A.C.F., Melo, I.S., Kent, A. and Fiore, M.F. (2012) Cyanobacterial Diversity in the Phyllosphere of a Mangrove Forest. FEMS Microbiology Ecology, 80, 312-322. http://dx.doi.org/10.1111/j.1574-6941.2012.01299.x
Blum, H., Beier, H. and Gross, H. (1987) Improved Silver Staining of Plant Proteins, RNA and DNA in Polyacrylamide Gels. Electrophoresis, 8, 93-99. http://dx.doi.org/10.1002/elps.1150080203
Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: A Laboratory Manual. 3rd Edition, Cold Spring Harbor Laboratory, New York.
Drummond, A.J., Ashton, B., Cheung, M., Heled, J., Kearse, M., Moir, R., Scones-Havas, S., Thierer, T. and Wilson, A. (2009) Geneious v4.7. http://www.geneious.com/
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL-W: Improving the Sensitivity of Progressive Multiple Sequence Alignment through Sequence Weighting, Position-Specific Gap Penalties and Weigh Matrix Choice. Nucleic Acids Research, 22, 4673-4680. http://dx.doi.org/10.1093/nar/22.22.4673
Felsenstein, J. (1985) Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evolution, 39, 783-791. http://dx.doi.org/10.2307/2408678
Schloss, P.D. and Handelsman, J. (2005) Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness. Applied and Environmental Microbiology, 71, 1501-1506. http://dx.doi.org/10.1128/AEM.71.3.1501-1506.2005
Komarek, J. (2010) Recent Changes (2008) in Cyanobacteria Taxonomy Based on a Combination of Molecular Background with Phenotype and Ecological Consequences (Genus and Species Concept). Hydrobiologia, 639, 245-259. http://dx.doi.org/10.1007/s10750-009-0031-3
Schloss, P.D., Larget, B.R. and Handelsman, J. (2004) Integration of Microbial Ecology and Statistics: A Test to Compare Gene Libraries. Applied and Environmental Microbiology, 70, 5485-5492. http://dx.doi.org/10.1128/AEM.70.9.5485-5492.2004
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731-2739. http://dx.doi.org/10.1093/molbev/msr121
Mejean, A., Mann, S., Maldiney, T., Vassiliadis, G., Lequin, O. and Loux, O. (2009) Evidence That Biosynthesis of the Neurotoxic Alkaloids Anatoxin-a and Homoanatoxin-a in the Cyanobacterium Oscillatoria PCC 6506 Occurs on a Modular Polyketide Synthase Initiated by L-Proline. Journal of the American Chemical Society, 131, 7512-7513. http://dx.doi.org/10.1021/ja9024353
Kellmann, R., Mills, T. and Neilan, B.A. (2006) Functional Modeling and Phylogenetic Distribution of Putative Cylindrospermopsin Biosynthesis Enzymes. Journal of Molecular Evolution, 62, 267-280. http://dx.doi.org/10.1007/s00239-005-0030-6
Mihali, T.K., Kellmann, R., Muenchhoff, J., Barrow, K.D. and Neilan, B.A. (2008) Characterization of the Gene Cluster Responsible for Cylindrospermopsin Biosynthesis. Applied and Environmental Microbiology, 74, 716-722. http://dx.doi.org/10.1128/AEM.01988-07
Rantala, A., Fewer, D.P., Hisbergues, M., Rouhiainen, L., Valtomaa, J., Borner, T. and Sivonen, K. (2004) Phylogenetic Evidence for the Early Evolution of Microcystin Synthesis. Proceedings of the National Academy of Sciences of the United States, 101, 568-573. http://dx.doi.org/10.1073/pnas.0304489101
Fiore, M.F., Crespim, E., Silva-Stenico, M.E., Genuario, D.B. and Silva, C.S.P. (2010) Putative Saxitoxin Gene in Terrestrial Heterocytous Cyanobacteria Isolated from Brazilian Rainforests. The 8th International Conference on Toxic Cyanobacteria, Istambul.
Cadel-Six, S., Dauga, C., Castets, A.M., Rippka, R., Bouchier, C., Marsac, N.T. and Welker, M. (2008) Halogenase Genes in Nonribosomal Peptide Synthetase Gene Clusters of Microcystis (Cyanobacteria): Sporadic Distribution and Evolution. Molecular Biology and Evolution, 25, 2031-2041. http://dx.doi.org/10.1093/molbev/msn150
Silva-Stenico, M.E., Silva, C.S.P., Lorenzi, A.S., Shishido, T.K., Etchegaray, A., Lira, S.P., Moraes, L.A.B. and Fiore, M.F. (2011) Non-Ribosomal Peptides Produced by Brazilian Cyanobacterial Isolates with Antimicrobial Activity. Microbiological Research, 166, 161-175. http://dx.doi.org/10.1016/j.micres.2010.04.002
Welker, M., Fastner, J., Erhard, M. and von Dohren, H. (2002) Applications of MALDI-TOF MS Analysis in Cyanotoxin Research. Environmental Toxicology, 17, 367-374. http://dx.doi.org/10.1002/tox.10073
Welker, M. and Erhard, M. (2007) Consistency between Chemotyping of Single Filaments of Planktothrix rubescens (Cyanobacteria) by MALDI-TOF and the Peptide Patterns of Strains Determined by HPLC-MS. Journal of Mass Spectrometry, 48, 1062-1068. http://dx.doi.org/10.1002/jms.1237
Welker, M. and von Dohren, H. (2006) Cyanobacterial Peptides—Nature’s Own Combinatorial Biosynthesis. FEMS Microbiology Reviews, 30, 530-563. http://dx.doi.org/10.1111/j.1574-6976.2006.00022.x
Czarnecki, O., Henning, M., Lippert, I. and Welker, M. (2006) Identification of Peptide Metabolites of Microcystis (Cyanobacteria) That Inhibit Trypsin-Like Activity in Planktonic Herbivorous Daphnia (Cladocera). Environmental Microbiology, 8, 77-87. http://dx.doi.org/10.1111/j.1462-2920.2005.00870.x
Welker, M., Brunke, M., Preussel, K., Lippert, I. and von Dohren, H. (2004) Diversity and Distribution of Microcystis (Cyanobacteria) Oligopeptide Chemotypes from Natural Communities Studied by Single Colony Mass Spectrometry. Microbiology, 150, 1785-1796. http://dx.doi.org/10.1099/mic.0.26947-0
Okino, T., Matsuda, H., Murakami, M. and Yamaguchi, K. (1993) Microginin, an Angiotensin-Converting Enzyme Inhibitor from the Blue-Green Alga Microcystis aeruginosa. Tetrahedron Letters, 34, 501-504. http://dx.doi.org/10.1016/0040-4039(93)85112-A
Molica, R.J.R., Onodera, H., Garcia, C., Rivas, M., Andrinolo, D., Nascimento, S., Meguro, H., Oshima, Y., Azevedo, S.M.F.O. and Lagos, N. (2002) Toxins in the Freshwater Cyanobacterium Cylindrospermopsis raciborskii (Cyanophyceae) Isolated from Tabocas Reservoir in Caruaru, Brazil, Including Demonstration of a New Saxitoxin Analogue. Phycologia, 41, 606-611. http://dx.doi.org/10.2216/i0031-8884-41-6-606.1
Sant’Anna, C.L., Melcher, S.S., Carvalho, M.C., Gemelgo, M.P. and Azevedo, M.T.P. (2007) Planktic Cyanobacteria from Upper Tiete Basin Reservoirs, SP, Brazil. Revista Brasileira de Botanica, 30, 1-17.
Sotero-Santos, R.B., Carvalho, E.G., Dellamano-Oliveira, M.J. and Rocha, O. (2008) Occurrence and Toxicity of an Anabaena Bloom in a Tropical Reservoir (Southeast Brazil). Harmful Algae, 7, 590-598. http://dx.doi.org/10.1016/j.hal.2007.12.017
Rosembaum, V. and Riesner, D. (1987) Temperature-Gradient Gel Electrophoresis: Thermodynamic Analysis of Nucleic Acids and Proteins in Purified Form and in Cellular Extracts. Biophysical Chemistry, 26, 235-246. http://dx.doi.org/10.1016/0301-4622(87)80026-1
Vetriani, C., Jannasch, H.W., MacGregor, B.J., Stahl, D.A. and Reysenbach, A.L. (1999) Population Structure and Phylogenetic Characterization of Marine Benthic Archaea in Deep-Sea Sediments. Applied and Environmental Microbiology, 65, 4375-4384.
Muyzer, G. (1999) DGGE/TGGE a Method for Identifying Genes from Natural Ecosystems. Current Opinion in Microbiology, 2, 317-322. http://dx.doi.org/10.1016/S1369-5274(99)80055-1
Sant’Anna, C.L., Carvalho, L.R., Fiore, M.F., Silva-Stenico, M.E., Lorenzi, A.S., Rios, F.R., Konno, K., Garcia, C. and Lagos, N. (2011) Highly Toxic Microcystis aeruginosa Strain, Isolated from Sao Paulo—Brazil, Produce Hepatotoxins and Paralytic Shellfish Poison Neurotoxins. Neurotoxicity Research, 19, 389-402. http://dx.doi.org/10.1007/s12640-010-9177-z
Funari, E. and Testai, E. (2008) Human Health Risk Assessment Related to Cyanotoxins Exposure. Critical Reviews in Toxicology, 38, 97-125. http://dx.doi.org/10.1080/10408440701749454
Jaiswal, P., Singh, R.K. and Prasanna, R. (2008) Cyanobacterial Bioactive Molecules—An Overview of Their Toxic Properties. Canadian Journal of Microbiology, 54, 701-717. http://dx.doi.org/10.1139/W08-034
Hoff-Risseti, C., Dorr, F.A., Schaker, P.D.C., Pinto, E., Werner, V.R. and Fiore, M.F. (2013) Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater. PLOS One, 8, e74238. http://dx.doi.org/10.1371/journal.pone.0074238
Molica, R.J.R., Oliveira, E.J.A., Carvalho, P.V.V.C., Costa, A.P.N.S.F., Cunha, M.C.C., Melo, G.L. and Azevedo, S.M.F.O. (2005) Occurrence of Saxitoxins and an Anatoxin-a(s)-Like Anticholinesterase in a Brazilian Drinking Water Supply. Harmful Algae, 4, 743-753. http://dx.doi.org/10.1016/j.hal.2004.11.001
Genuario, D.B., Silva-Stenico, M.E., Welker, M., Moraes, L.A.B. and Fiore, M.F. (2010) Characterization of a Microcystin and Detection of Microcystin Synthetase Genes from a Brazilian Isolate of Nostoc. Toxicon, 55, 846-854. http://dx.doi.org/10.1016/j.toxicon.2009.12.001
Willame, R., Jurczak, T., Iffly, J., Kull, T., Meriluoto, J. and Hoffman, L. (2005) Distribution of Hepatotoxic Cyanobacterial Blooms in Belgium and Luxembourg. Hydrobiologia, 551, 99-117. http://dx.doi.org/10.1007/s10750-005-4453-2
Matsumura-Tundisi, T., Hino, K. and Rocha, O. (1986) Caracteristicas limnologicas da Lagoa do Taquaral (Campinas S.P.)-um ambiente hipereutrofico. Ciencia e Cultura, 38, 420-425.
Borges, J.T. (1998) Avaliacao do estado trofico e sanitario e a adsorcao de fosforo no sedimento da Lagoa do Taquaral-Campinas-SP. Dissertation, Universidade Estadual de Campinas, Campinas.
Sant’Anna, C.L., Azevedo, M.T.P., Werner, V.R., Dogo, C.R., Rios, F.R. and Carvalho, L.R. (2008) Review of Toxic Species of Cyanobacteria in Brazil. Algological Studies, 126, 251-265. http://dx.doi.org/10.1127/1864-1318/2008/0126-0251
Crossetti, L.O. and Bicudo, C.E.M. (2008) Adaptations in Phytoplankton Life Strategies to Composed Change in a Shallow Urban Tropical Eutrophic Reservoir, Garcas Reservoir, over 8 Years. Hydrobiologia, 614, 91-105. http://dx.doi.org/10.1007/s10750-008-9539-1
Tucci, A. and Sant’Anna, C.L. (2003) Cylindrospermopsis raciborskii (Woloszynska) Seenayya & Subba Raju (Cyanobacteria): Variacao semanal e relacoes com fatores ambientais em um reservatorio eutrofico, Sao Paulo, SP, Brasil. Revista Brasileira de Botanica, 26, 97-112.
Paerl, H.W. and Huisman, J. (2008) Blooms Like It Hot. Science, 320, 57-58. http://dx.doi.org/10.1126/science.1155398
Namikoshi, M. and Rinehart, K.L. (1996) Bioactive Compounds Produced by Cyanobacteria. Journal of Industrial Microbiology and Biotechnology, 17, 373-384. http://dx.doi.org/10.1007/BF01574768
Schwarzenberger, A., Zitt, A., Kroth, P., Mueller, S. and Elert, E.V. (2010) Gene Expression and Activity of Digestive Proteases in Daphnia: Effects of Cyanobacterial Protease Inhibitors. BMC Physiology, 10, 6. http://dx.doi.org/10.1186/1472-6793-10-6
Welker, M., Marsalek, B., Sejnohova, L. and von Dohren, H. (2006) Detection and Identification of Oligopeptides in Microcystis (Cyanobacteria) Colonies: Toward an Understanding of Metabolic Diversity. Peptides, 27, 2090-2103. http://dx.doi.org/10.1016/j.peptides.2006.03.014
Lagos, N., Onodera, H., Zagatto, P.A., Andrinolo, D., Azevedo, S.M.F.O. and Oshima, Y. (1999) The First Evidence of Paralytic Shellfish Toxins in the Freshwater Cyanobacterium Cylindrospermopsis raciborskii, Isolated from Brazil. Toxicon, 37, 1359-1373. http://dx.doi.org/10.1016/S0041-0101(99)00080-X
Ferrao-Filho, A.S., Cunha, R., Magalhaes, V.F., Soares, M.C.S. and Baptista, D.F. (2007) Evaluation of Sub-Lethal Toxicity of Cyanobacteria on the Swimming Activity of Aquatic Organisms by Image Analysis. Journal of the Brazilian Society of Ecotoxicology, 2, 93-100. http://dx.doi.org/10.5132/jbse.2007.02.001
Hawkins, P.R., Runnegar, M.T.C., Jackson, A.R.B. and Falconer, I.R. (1985) Severe Hepatotoxicity Caused by the Tropical Cyanobacterium (Blue-Green Alga) Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju Isolated from a Domestic Water Supply Reservoir. Applied and Environmental Microbiology, 50, 1292-1295.
Saker, M.L. and Eaglesham, G.K. (1999) The Accumulation of Cylindrospermopsin from the Cyanobacterium Cylindrospermopsis raciborskii in Tissues of the Redclaw Crayfish Cherax quadricarinatus. Toxicon, 37, 1065-1077. http://dx.doi.org/10.1016/S0041-0101(98)00240-2
Li, R., Carmichael, W.W., Brittain, S., Eaglesham, G.K., Shaw, G.R., Mahakhant, A., Noparatnaraporn, N., Yongmanitchai, W., Kaya, K. and Watanabe, M.M. (2001) Isolation and Identification of the Cyanotoxin Cylindrospermopsin and Deoxy-Cylindrospermposin from a Thailand Strain of Cylindrospermopsis raciborskii. Toxicon, 39, 973-980. http://dx.doi.org/10.1016/S0041-0101(00)00236-1
Bernard, C., Harvey, M., Briand, J.F., Bire, R., Krys, S. and Fontaine, J.J. (2003) Toxicological Comparison of Diverse Cylindrospermopsis raciborskii Strains: Evidence of Liver Damage Caused by a French C. raciborskii Strain. Environmental Toxicology, 18, 176-186. http://dx.doi.org/10.1002/tox.10112
Wood, S.A. and Stirling, D.J. (2003) First Identification of the Cylindrospermopsin-Producing Cyanobacterium Cylindrospermopsis raciborskii in New Zealand. New Zealand Journal of Marine and Freshwater Research, 37, 821-828. http://dx.doi.org/10.1080/00288330.2003.9517211
Chonudomkul, D., Yongmanitchai, W., Theeragool, G., Kawachi, M., Kasai, F., Kaya, K. and Watanabe, M.M. (2004) Morphology, Genetic Diversity, Temperature Tolerance and Toxicity of Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) Strains from Thailand and Japan. FEMS Microbiology Ecology, 48, 345-355. http://dx.doi.org/10.1016/j.femsec.2004.02.014
Everson, S., Fabbro, L., Kinnear, S. and Wright, P. (2011) Extreme Differences in Akinete, Heterocyte and Cylindrospermopsin Concentrations with Depth in a Successive Bloom Involving Aphanizomenon ovalisporum (Forti) and Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju. Harmful Algae, 10, 265-276. http://dx.doi.org/10.1016/j.hal.2010.10.006
Silva-Stenico, M.E., Rigonato, J., Leal, M.G., Vaz, M.G.M.V., Andreote, A.P.D. and Fiore, M.F. (2012) Non-Ribosomal Halogenated Protease Inhibitors from Cyanobacterial Isolates as Attractive Drug Targets. Current Medicinal Chemistry, 19, 5205-5213. http://dx.doi.org/10.2174/092986712803530539