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Application of real-time PCR in the assessment of the toxic cyanobacterium Cylindrospermopsis raciborskii abundance and toxicological potential

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Cyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.

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  1. Abrantes N, Pereira R, Gonçalves F (2006) First step for an ecological risk assessment to evaluate the impact of diffuse pollution in Lake Vela (Portugal). Environ Monit Assess 117:411–431

  2. Banker R, Carmeli S, Hadas O, Teltsch B, Porat R, Sukenik A (1997) Identification of cylindrospermopsin in the cyanobacterium Aphanizomenon ovalisporum (Cyanophyceae) isolated from Lake Kinneret. J Phycol 33:613–616

  3. Berry JP, Lind O (2010) First evidence of “paralytic shellfish toxins” and cylindrospermopsin in a Mexican freshwater system, Lago Catemaco, and apparent bioaccumulation of the toxins in “tegogolo” snails (Pomacea patula catemacensis). Toxicon 55(5):930–938

  4. Bláhová L, Oravec M, Maršálek B, Šejnohová L, Šimek Z, Bláha L (2009) The first occurrence of the cyanobacterial alkaloid toxin cylindrospermopsin in the Czech Republic as determined by immunochemical and LC/MS methods. Toxicon 53:519–524

  5. Briand JF, Robillot C, Quiblier-Llobéras C, Humbert JF, Couté A, Bernard C (2002) Environmental context of Cylindrospermopsis raciborskii (Cyanobacteria) blooms in a shallow pond in France. Water Res 36:3183–3192

  6. Brient L, Lengronne M, Bormans M, Fastner J (2009) First occurrence of cylindrospermopsin in freshwater in France. Environ Toxicol 24(4):415–420

  7. Bourke ATC, Hawes RB, Neilson A, Stallman ND (1983) An outbreak of hepato-enteritis (the Palm Island mystery disease) possibly caused by algal intoxication. Toxicon 21(Supplement 3):45–48

  8. Chiswell R, Smith M, Norris R, Eaglesham G, Shaw G, Seawright A, Moore M (1997) The cyanobacterium, Cylindrospermopsis raciborskii, and its related toxin, cylindrospermopsin. Australas J Ecotoxicol 3:7–23

  9. de Figueiredo DR, Reboleira ASSP, Antunes SC, Abrantes N, Azeiteiro U, Gonçalves F, Pereira MJ (2006) The effect of environmental parameters and cyanobacterial blooms on phytoplankton dynamics of a Portuguese temperate lake. Hydrobiologia 568:145–157

  10. de Figueiredo DR, Pereira MJ, Moura A, Silva L, Bárrios S, Fonseca F, Henriques I, Correia A (2007) Bacterial community composition over a dry winter in meso- and eutrophic Portuguese water bodies. FEMS Microbiol Ecol 59:638–650

  11. Falconer IR (2005) Cyanobacterial toxins of drinking water supplies. CRC, Boca Raton

  12. Falconer IR, Humpage AR (2001) Preliminary evidence for in vivo tumour initiation by oral administration of extracts of the bluegreen alga Cylindrospermopsis raciborskii containing the toxin cylindrospermopsin. Environ Toxicol 16:192–195

  13. Fastner J, Heinze R, Humpage AR, Mischke U, Eaglesham GK, Chorus I (2003) Cylindrospermopsin occurrence in two German lakes and preliminary assessment of toxicity and toxin production of Cylindrospermopsis raciborskii (Cyanobacteria) isolates. Toxicon 42:313–321

  14. Fergusson KM, Saint PC (2003) Multiplex PCR assay for Cylindrospermopsis raciborskii and cylindrospermopsin-producing cyanobacteria. Environ Toxicol 18(2):120–125

  15. Freitas M (2009) Monitorização de cianobactérias e cianotoxinas nas lagoas Portuguesas de Mira e Vela comparando métodos moleculares, imunológicos e volumes de amostragem. Dissertation, University of Porto

  16. Hamilton PB, Ley LM, Dean S, Pick FR (2005) The occurrence of the cyanobacterium Cylindrospermopsis raciborskii in Constance Lake, an exotic cyanoprokaryote new to Canada. Phycologia 44:17–25

  17. Harada K, Ohtani I, Iwamoto K, Suzuki M, Wantanabe MF, Wantanabe M, Terao K (1994) Isolation of cylindrospermopsin from a cyanobacterium Umezakia natans and its screening method. Toxicon 32:73–84

  18. Hawkins PR, Runnegar MTC, Jackson ARB, Falconer IR (1985) Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju isolated from a domestic supply reservoir. Appl Environ Microbiol 50:1292–1295

  19. Humpage AR, Falconer IR (2003) Oral toxicity of the cyanobacterial toxin cylindrospermopsin in male Swiss albino mice: determination of no observed adverse effect level for deriving a drinking water guideline value. Environ Toxicol 18(2):94–103

  20. Humpage AR, Fenech M, Thomas P, Falconer IR (2000) Micronucleus induction and chromosome loss in transformed human white cells indicate clastogenic and aneugenic action of the cyanobacterial toxin, cylindrospermopsin. Mutat Res 472:155–161

  21. Kurmayer R, Kutzenberger T (2003) Application of real-time PCR for quantification of microcystin genotypes in a population of the toxic cyanobacterium Microcystis sp. Appl Environ Microbiol 69(11):6723–6730

  22. Lagos N, Onodera H, Zagatto PA, Andrinolo D, Azevedo SMFO, Oshima Y (1999) The first evidence of paralytic shellfish toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil. Toxicon 37:1359–1373

  23. Li R, Carmichael WW, Brittain S, Eaglesham GK, Shaw GR, Liu Y, Watanabe MM (2001) First report of the cyanotoxins cylindrospermopsin and deoxycylindrospermopsin from Raphidiopsis curvata (Cyanobacteria). J Phycol 37:1121–1126

  24. Manti G, Mattei D, Messineo V, Melchiorre S, Bogialli S, Sechi N, Casiddu P, Luglié L, Di Brizio M, Bruno M (2005) First report of Cylindrospermopsis raciborskii in Italy. Harmful Algal News 28:8–9

  25. Neilan BA, Saker ML, Fastner J, Torokne A, Burnes BP (2003) Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii. Mol Ecol 12:113–140

  26. Nübel U, Garcia-Pichel F, Muyzer G (1997) PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl Environ Microbiol 63(8):3327–3332

  27. Padisák J (1997) Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju, an expanding, highly adaptive cyanobacterium: worldwide distribution and review of its ecology. Arch Hydrobiol/Suppl 107:563–593

  28. Pearson LA, Neilan B (2008) The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk. Curr Opin Biotechnol 19(3):281–288

  29. Preußel K, Stüken A, Wiedner C, Chorus I, Fastner J (2006) First report on cylindrospermopsin producing Aphanizomenon flos-aquae (Cyanobacteria) isolated from two German lakes. Toxicon 47:156–162

  30. Quesada A, Moreno E, Carrasco D, Paniagua T, Wormer L, De Hoyos C, Sukenik A (2006) Toxicity of Aphanizomenon ovalisporum (Cyanobacteria) in a Spanish water reservoir. Eur J Phycol 41:39–45

  31. Rasmussen JP, Giglio S, Monis PT, Campbell RJ, Saint SP (2007) Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria. J Appl Microbiol 104(5):1503–1515

  32. Romo S, Miracle MR (1994) Population dynamics and ecology of subdominant phytoplankton species in a shallow hypertrophic lake (Albufera of Valencia, Spain). Hydrobiologia 273:37–56

  33. Rinta-Kanto JM, Ouellette AJA, Boyer GL, 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. Environ Sci Technol 39:4198–4205

  34. Runnegar MT, Kong SM, Zhong YZ, Ge JL, Lu SC (1994) The role of glutathione in the toxicity of a novel cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes. Biochem Biophys Res Commun 201:235–241

  35. Saker ML, Thomas AD, Norton JH (1999) Cattle mortality attributed to the toxic cyanobacterium Cylindrospermopsis raciborskii in an outback region of the North Queensland. Environ Toxicol 14(1):179–182

  36. Saker ML, Nogueira ICG, Vasconcelos VM, Neilan BA, Eaglesham GK, Pereira P (2003) First report and toxicological assessment of the cyanobacterium Cylindrospermopsis raciborskii from Portuguese freshwaters. Ecotox Environ Safe 55:243–250

  37. Saker ML, Vale M, Kramer D, Vasconcelos VM (2006) Molecular techniques for the early warning of toxic cyanobacteria blooms in freshwater lakes and rivers. Appl Microbiol Biotechnol 75(2):441–449

  38. Schembri MA, Neilan BA, Saint CP (2001) Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii. Environ Toxicol 16:413–421

  39. Seifert M, McGregor G, Eaglesham G, Wickramasinghe W, Shaw G (2007) First evidence for the production of cylindrospermopsin and deoxy-cylindrospermopsin by the freshwater benthic cyanobacterium, Lyngbya wollei (Farlowex Gomont) Speziale and Dyck. Harmful Algae 6:73–80

  40. Spoof L, Berg KA, Rapala J, Lahti K, Lepistö L, Metcalf JS, Codd GA, Meriluoto J (2006) First observation of cylindrospermopsin in Anabaena lapponica isolated from the boreal environment (Finland). Environ Toxicol 21:552–560

  41. Thomas AD, Saker ML, Norton JH, Olsen RD (1998) Cyanobacterium Cylindrospermopsis raciborskii as a probable cause of death in cattle in northern Queensland. Aust Vet J 76(9):592–594

  42. Valério E, Pereira P, Saker ML, Franca S, Tenreiro R (2005) Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters. Harmful Algae 4:1044–1052

  43. Vasconcelos VM, Campos T, Amorim A, Soares AMVM (1993) Toxicidade de estirpes de cianobactérias isoladas a partir das lagoas das Braças, Vela e Mira. Boletim UCA, UCTRA, Univ Algarve 1:193–201

  44. Welker M, Bickel H, Fastner J (2002) HPLC-PDA detection of cylindrospermopsin—opportunities and limits. Water Res 36:4659–4663

  45. Wilson KM, Schembri MA, Baker PD, Saint CP (2000) Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR. Appl Environ Microbiol 66(1):332–338

  46. Yilmaz M, Philips ED, Szabo NJ, Badylak S (2008) A comparative study of Florida strains of Cylindrospermopsis and Aphanizomenon for cylindrospermopsin production. Toxicon 51:130–139

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We would like to thank Paula Ferreira, from the Chemistry Laboratory of CIIMAR, for the LC/MS analysis. This research was funded in part by the projects from Fundação para a Ciência e a Tecnologia PTDC/AMB/67075/2006 and PTDC/AAC-AMB/104983/2008. Cristiana Moreira was supported by the PhD fellowship SFRH/BD/47164/2008.

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Correspondence to Vitor Vasconcelos.

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Moreira, C., Martins, A., Azevedo, J. et al. Application of real-time PCR in the assessment of the toxic cyanobacterium Cylindrospermopsis raciborskii abundance and toxicological potential. Appl Microbiol Biotechnol 92, 189–197 (2011).

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  • C. raciborskii
  • Monitoring
  • Real-time PCR
  • Cylindrospermopsin genes