Multiple cyanotoxin congeners produced by sub-dominant cyanobacterial taxa in riverine cyanobacterial and algal mats

Autoři: Laura T. Kelly aff001;  Keith Bouma-Gregson aff003;  Jonathan Puddick aff002;  Rich Fadness aff004;  Ken G. Ryan aff001;  Timothy W. Davis aff005;  Susanna A. Wood aff002
Působiště autorů: School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand aff001;  Cawthron Institute, The Wood, Nelson, New Zealand aff002;  Office of Information Management and Analysis, California State Water Resources Control Board, Sacramento, California, United States of America aff003;  North Coast Regional Water Quality Control Board, Santa Rosa, California, United States of America aff004;  Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, United States of America aff005
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0220422


Benthic cyanobacterial proliferations in rivers are have been reported with increasing frequency worldwide. In the Eel and Russian rivers of California, more than a dozen dog deaths have been attributed to cyanotoxin toxicosis since 2000. Periphyton proliferations in these rivers comprise multiple cyanobacterial taxa capable of cyanotoxin production, hence there is uncertainty regarding which taxa are producing toxins. In this study, periphyton samples dominated by the cyanobacterial genera Anabaena spp. and Microcoleus spp. and the green alga Cladophora glomerata were collected from four sites in the Eel River catchment and one site in the Russian River. Samples were analysed for potential cyanotoxin producers using polymerase chain reaction (PCR) in concert with Sanger sequencing. Cyanotoxin concentrations were measured using liquid chromatography tandem-mass spectrometry, and anatoxin quota (the amount of cyanobacterial anatoxins per toxigenic cell) determined using droplet digital PCR. Sequencing indicated Microcoleus sp. and Nodularia sp. were the putative producers of cyanobacterial anatoxins and nodularins, respectively, regardless of the dominant taxa in the mat. Anatoxin concentrations in the mat samples varied from 0.1 to 18.6 μg g-1 and were significantly different among sites (p < 0.01, Wilcoxon test); however, anatoxin quotas were less variable (< 5-fold). Dihydroanatoxin-a was generally the most abundant variant in samples comprising 38% to 71% of the total anatoxins measured. Mats dominated by the green alga C. glomerata contained both anatoxins and nodularin-R at concentrations similar to those of cyanobacteria-dominated mats. This highlights that even when cyanobacteria are not the dominant taxa in periphyton, these mats may still pose a serious health risk and indicates that more widespread monitoring of all mats in a river are necessary.

Klíčová slova:

Algae – Cyanobacteria – Eels – Polymerase chain reaction – Rivers – Russian people – Toxins – Anabaena


1. Edwards C, Beattie KA, Scrimgeour CM, Codd GA. Identification of anatoxin-a in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland. Toxicon. 1992;30(10): 1165–75. doi: 10.1016/0041-0101(92)90432-5 1440622

2. Mez K, Beattie KA, Codd GA, Hanselmann K, Hauser B, Naegeli H, et al. Identification of a microcystin in benthic cyanobacteria linked to cattle deaths on alpine pastures in Switzerland. Eur J Phycol. 1997;32(2): 111–7.

3. Hamill KD. Toxicity in benthic freshwater cyanobacteria (blue‐green algae): First observations in New Zealand. New Zealand Journal of Marine and Freshwater Research. 2001;35(5).

4. Hudon C, De Sève M, Cattaneo A. Increasing occurrence of the benthic filamentous cyanobacterium Lyngbya wollei: a symptom of freshwater ecosystem degradation. Freshwater Science. 2014;33(2): 606–18.

5. Gaget V, Humpage AR, Huang Q, Monis P, Brookes JD. Benthic cyanobacteria: A source of cylindrospermopsin and microcystin in Australian drinking water reservoirs. Water Research. 2017;124: 454–64. doi: 10.1016/j.watres.2017.07.073 28787682

6. Belykh OI, Tikhonova IV, Kuzmin AV, Sorokovikova EG, Fedorova GA, Khanaev IV, et al. First detection of benthic cyanobacteria in Lake Baikal producing paralytic shellfish toxins. Toxicon. 2016;121: 36–40. doi: 10.1016/j.toxicon.2016.08.015 27569199

7. Quiblier C, Wood S, Echenique-Subiabre I, Heath M, Villeneuve A, Humbert J-F. A review of current knowledge on toxic benthic freshwater cyanobacteria–ecology, toxin production and risk management. Water Research. 2013;47(15): 5464–79. doi: 10.1016/j.watres.2013.06.042 23891539

8. McAllister TG, Wood SA, Hawes I. The rise of toxic benthic Phormidium proliferations: A review of their taxonomy, distribution, toxin content and factors regulating prevalence and increased severity. Harmful Algae. 2016;55: 282–94. doi: 10.1016/j.hal.2016.04.002 28073542

9. Cantoral Uriza E, Asencio A, Aboal M. Are we underestimating benthic cyanotoxins? Extensive sampling results from Spain. Toxins. 2017;9(12): 385. doi: 10.3390/toxins9120385 29182536

10. Furey PC, Lowe RL, Power ME, Campbell-Craven AM. Midges, Cladophora, and epiphytes: shifting interactions through succession. Freshwater Science. 2012;31(1): 93–107.

11. Zulkifly S, Hanshew A, Young EB, Lee P, Graham ME, Graham ME, et al. The epiphytic microbiota of the globally widespread macroalga Cladophora glomerata (Chlorophyta, Cladophorales). American Journal of Botany. 2012;99(9): 1541–52. doi: 10.3732/ajb.1200161 22947483

12. Merel S, Villarín MC, Chung K, Snyder S. Spatial and thematic distribution of research on cyanotoxins. Toxicon. 2013;76: 118–31. doi: 10.1016/j.toxicon.2013.09.008 24055553

13. Kaebernick M, Neilan BA. Ecological and molecular investigations of cyanotoxin production. FEMS Microbiol Ecol. 2001;35(1): 1–9. doi: 10.1111/j.1574-6941.2001.tb00782.x 11248384

14. Pearson LA, Neilan BA. The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk. Current Opinion in Biotechnology. 2008;19(3): 281–8. doi: 10.1016/j.copbio.2008.03.002 18439816

15. Wood S, Puddick J. The abundance of toxic genotypes is a key contributor to anatoxin variability in Phormidium-dominated benthic mats. Marine Drugs. 2017;15(10): 307. doi: 10.3390/md15100307 29019928

16. Bouma-Gregson K, Kudela RM, Power ME. Widespread anatoxin-a detection in benthic cyanobacterial mats throughout a river network. PLOS ONE. 2018;13(5): e0197669. doi: 10.1371/journal.pone.0197669 29775481

17. Fetscher AE, Howard MD, Stancheva R, Kudela RM, Stein ED, Sutula MA, et al. Wadeable streams as widespread sources of benthic cyanotoxins in California, USA. Harmful Algae. 2015;49: 105–16.

18. Bouma-Gregson K, Olm MR, Probst AJ, Anantharaman K, Power ME, Banfield JF. Impacts of microbial assemblage and environmental conditions on the distribution of anatoxin-a producing cyanobacteria within a river network. The ISME Journal. 2019. doi: 10.1038/s41396-019-0374-3 30809011

19. Anderson B, Voorhees J, Phillips B, Fadness R, Stancheva R, Nichols J, et al. Extracts from benthic anatoxin‐producing Phormidium are toxic to 3 macroinvertebrate taxa at environmentally relevant concentrations. Environmental Toxicology and Chemistry. 2018;37(11): 2851–9. doi: 10.1002/etc.4243 30066467

20. Kelly L, Wood S, McAllister T, Ryan K. Development and application of a quantitative PCR assay to assess genotype dynamics and anatoxin content in Microcoleus autumnalis-dominated mats. Toxins. 2018;10(11): 431. doi: 10.3390/toxins10110431 30373141

21. Rogers S, Puddick J, Wood SA, Dietrich DR, Hamilton DP, Prinsep MR. The effect of cyanobacterial biomass enrichment by centrifugation and GF/C filtration on subsequent microcystin measurement. Toxins. 2015;7(3): 821–34. doi: 10.3390/toxins7030821 25763766

22. Wood SA, Puddick J, Fleming R, Heussner AH. Detection of anatoxin-producing Phormidium in a New Zealand farm pond and an associated dog death. New Zealand Journal of Botany. 2017;55(1): 36–46.

23. Wood SA, Kuhajek JM, de Winton M, Phillips NR. Species composition and cyanotoxin production in periphyton mats from three lakes of varying trophic status. FEMS Microbiol Ecol. 2012;79(2): 312–26. doi: 10.1111/j.1574-6941.2011.01217.x 22092304

24. Jungblut A-D, Neilan BA. Molecular identification and evolution of the cyclic peptide hepatotoxins, microcystin and nodularin, synthetase genes in three orders of cyanobacteria. Arch Microbiol. 2006;185(2): 107–14. doi: 10.1007/s00203-005-0073-5 16402223

25. Ballot A, Fastner J, Wiedner C. Paralytic shellfish poisoning toxin-producing cyanobacterium Aphanizomenon gracile in Northeast Germany. Applied and Environmental Microbiology. 2010;76(4): 1173–80. doi: 10.1128/AEM.02285-09 20048055

26. Rantala-Ylinen A, Känä S, Wang H, Rouhiainen L, Wahlsten M, Rizzi E, et al. Anatoxin-a synthetase gene cluster of the cyanobacterium Anabaena sp. strain 37 and molecular methods to detect potential producers. Applied and Environmental Microbiology. 2011;77(20): 7271–8. doi: 10.1128/AEM.06022-11 21873484

27. Mihali TK, Kellmann R, Muenchhoff J, Barrow KD, Neilan BA. Characterization of the gene cluster responsible for cylindrospermopsin biosynthesis. Applied and Environmental Microbiology. 2008;74(3): 716–22. doi: 10.1128/AEM.01988-07 18065631

28. Akcaalan R, Mazur-Marzec H, Zalewska A, Albay M. Phenotypic and toxicological characterization of toxic Nodularia spumigena from a freshwater lake in Turkey. Harmful Algae. 2009;8(2): 273–8.

29. McGregor GB, Sendall BC. Iningainema pulvinus gen nov., sp nov.(Cyanobacteria, Scytonemataceae) a new nodularin producer from Edgbaston Reserve, north-eastern Australia. Harmful Algae. 2017;62: 10–9. doi: 10.1016/j.hal.2016.11.021 28118884

30. Gehringer MM, Adler L, Roberts AA, Moffitt MC, Mihali TK, Mills TJ, et al. Nodularin, a cyanobacterial toxin, is synthesized in planta by symbiotic Nostoc sp. The ISME Journal. 2012;6(10): 1834. doi: 10.1038/ismej.2012.25 22456448

31. Bouma-Gregson K, Power ME, Bormans M. Rise and fall of toxic benthic freshwater cyanobacteria (Anabaena spp.) in the Eel river: Buoyancy and dispersal. Harmful Algae. 2017;66: 79–87. doi: 10.1016/j.hal.2017.05.007 28602256

32. Foss AJ, Butt J, Aubel MT. Benthic periphyton from Pennsylvania, USA is a source for both hepatotoxins (microcystins/nodularin) and neurotoxins (anatoxin-a/homoanatoxin-a). Toxicon. 2018;150: 13–6. doi: 10.1016/j.toxicon.2018.05.002 29746979

33. Echenique-Subiabre I, Tenon M, Humbert J-F, Quiblier C. Spatial and temporal variability in the development and potential toxicity of Phormidium biofilms in the Tarn River, France. Toxins. 2018;10(10): 418. doi: 10.3390/toxins10100418 30336603

34. Mann S, Cohen M, Chapuis-Hugon F, Pichon V, Mazmouz R, Méjean A, et al. Synthesis, configuration assignment, and simultaneous quantification by liquid chromatography coupled to tandem mass spectrometry, of dihydroanatoxin-a and dihydrohomoanatoxin-a together with the parent toxins, in axenic cyanobacterial strains and in environmental samples. Toxicon. 2012;60(8): 1404–14. doi: 10.1016/j.toxicon.2012.10.006 23085422

35. Faassen EJ, Harkema L, Begeman L, Lurling M. First report of (homo) anatoxin-a and dog neurotoxicosis after ingestion of benthic cyanobacteria in the Netherlands. Toxicon. 2012;60(3): 378–84. doi: 10.1016/j.toxicon.2012.04.335 22534073

36. Skulberg OM, Skulberg R, Carmichael WW, Andersen RA, Matsunaga S, Moore RE. Investigations of a neurotoxic oscillatorialean strain (Cyanophyceae) and its toxin. Isolation and characterization of homoanatoxin‐a. Environmental Toxicology and Chemistry: An International Journal. 1992;11(3): 321–9.

37. Stevens D, Krieger R. Effect of route of exposure and repeated doses on the acute toxicity in mice of the cyanobacterial nicotinic alkaloid anatoxin-a. Toxicon. 1991;29(1): 134–8. doi: 10.1016/0041-0101(91)90047-u 1903000

38. Valentine WM, Schaeffer DJ, Beasley VR. Electromyographic assessment of the neuromuscular blockade produced in vivo by anatoxin-a in the rat. Toxicon. 1991;29(3): 347–57. doi: 10.1016/0041-0101(91)90288-3 1904660

39. Wood SA, Smith FM, Heath MW, Palfroy T, Gaw S, Young RG, et al. Within-mat variability in anatoxin-a and homoanatoxin-a production among benthic Phormidium (cyanobacteria) strains. Toxins. 2012;4(10): 900–12. doi: 10.3390/toxins4100900 23162704

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