Development of a quantitative PCR assay for the detection and enumeration of a potentially ciguatoxin-producing dinoflagellate, Gambierdiscus lapillus (Gonyaulacales, Dinophyceae)

Autoři: Anna Liza Kretzschmar aff001;  Arjun Verma aff001;  Gurjeet Kohli aff001;  Shauna Murray aff001
Působiště autorů: Climate Change Cluster (C3), University of Technology Sydney, Ultimo, New South Wales, Australia aff001;  ithree institute (i3), University of Technology Sydney, Ultimo, New South Wales, Australia aff002;  Alfred Wegener-Institut Helmholtz-Zentrum fr Polar- und Meeresforschung, Bremerhaven, Germany aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224664


Ciguatera fish poisoning (CFP) is an illness contracted through the ingestion of seafood containing ciguatoxins. It is prevalent in tropical regions worldwide, including in Australia. Ciguatoxins are produced by some species of Gambierdiscus. Therefore, screening of Gambierdiscus species identification through quantitative PCR (qPCR), along with the determination of species toxicity, can be useful in monitoring potential ciguatera risk in these regions. In Australia, CFP is prevalent in tropical Queensland and increasingly in sub-tropical regions of Australia, but has a report rate of approximately 10%. Yet the identity, distribution and abundance of ciguatoxin producing Gambierdiscus spp. is largely unknown. In this study, we developed a rapid qPCR assay to quantify the presence and abundance of Gambierdiscus lapillus, a likely ciguatoxic species first described from Australia. We assessed the specificity and efficiency of the qPCR assay. The assay was tested on 25 environmental samples from the Heron Island reef in the southern Great Barrier Reef, a ciguatera endemic region, to determine the presence and patchiness of this species across samples from Chnoospora sp., Padina sp. and Sargassum sp. macroalgal hosts.

Klíčová slova:

Algae – Australia – DNA – DNA extraction – Islands – Polymerase chain reaction – Ribosomal RNA – Toxins


1. Woerner J. Padina (Brown Algae)); 2011. Integration and Application Network, University of Maryland Center for Environmental Science.

2. Kraeer K, van Essen-Fishman L. Pseudocaranx dentex (White Trevally); 2010. Integration and Application Network, University of Maryland Center for Environmental Science.

3. Saxby T. Scomberomorus munroi (Australian Spotted Mackerel); 2004. Integration and Application Network, University of Maryland Center for Environmental Science.

4. Baumann F, Bourrat MB, Pauillac S. Prevalence, symptoms and chronicity of ciguatera in New Caledonia: Results from an adult population survey conducted in Noumea during 2005. Toxicon. 2010;56(5):662–7. doi: 10.1016/j.toxicon.2009.10.015 19835903

5. Sims JK. A theoretical discourse on the pharmacology of toxic marine ingestions. Annals of emergency medicine. 1987;16(9):1006–1015. doi: 10.1016/s0196-0644(87)80750-3 3307551

6. Friedman M, Fleming L, Fernandez M, Bienfang P, Schrank K, Dickey R, Bottein MY, Backer L, Ayyar R, Weisman R, Watkins S. Ciguatera fish poisoning: treatment, prevention and management. Marine drugs. 2008;6(3):456–79. doi: 10.3390/md20080022 19005579

7. Chinain M, Darius HT, Ung A, Fouc MT, Revel T, Cruchet P, et al. Ciguatera risk management in French Polynesia: the case study of Raivavae Island (Australes Archipelago). Toxicon. 2010;56(5):674–690. doi: 10.1016/j.toxicon.2009.05.032 19505491

8. Chinain M, Germain M, Sako Y, Pauillac S, Legrand AM. Intraspecific variation in the dinoflagellate Gambierdiscus toxicus (Dinophyceae). I. Isozyme Analysis. Journal of Phycology. 1997;33(1):36–43.

9. Holmes MJ. Gambierdiscus yasumotoi sp. nov.(Dinophyceae), a toxic benthic dinoflagellate from southeastern Asia. Journal of Phycology. 1998;34(4):661–668.

10. Lewis RJ, Holmes MJ. Origin and transfer of toxins involved in ciguatera. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology. 1993 Nov 1;106(3):615–28.

11. Adachi R, Fukuyo Y. The thecal structure of a marine toxic dinoflagellate Gambierdiscus toxicus gen. et sp. nov. collected in a ciguatera-endemic area. Bulletin of the Japanese Society of Scientific Fisheries (Japan). 1979;45:67–71.

12. Faust MA. Observation of sand-dwelling toxic dinoflagellates (Dinophyceae) from widely differing sites, including two new species. Journal of Phycology. 1995;31(6):996–1003.

13. Smith KF, Rhodes L, Verma A, Curley BG, Harwood DT, Kohli GS, et al. A new Gambierdiscus species (Dinophyceae) from Rarotonga, Cook Islands: Gambierdiscus cheloniae sp. nov. Harmful Algae. 2016;60:45–56. doi: 10.1016/j.hal.2016.10.006 28073562

14. Fraga S, Rodríguez F, Riobó P, Bravo I. Gambierdiscus balechii sp. nov (Dinophyceae), a new benthic toxic dinoflagellate from the Celebes Sea (SW Pacific Ocean). Harmful Algae. 2016;.

15. Litaker RW, Vandersea MW, Faust MA, Kibler SR, Nau AW, Holland WC, et al. Global distribution of ciguatera causing dinoflagellates in the genus Gambierdiscus. Toxicon. 2010;56(5):711–730. doi: 10.1016/j.toxicon.2010.05.017 20561539

16. Chinain M, Faust MA, Pauillac S. Morphology and molecular analyses of three toxic species of Gambierdiscus (Dinophyceae): G. pacificus, sp. nov., G. australes, sp. nov., and G. polynesiensis, sp. nov. Journal of Phycology. 1999;35(6):1282–1296.

17. Litaker RW, Vandersea MW, Faust MA, Kibler SR, Chinain M, Holmes MJ, et al. Taxonomy of Gambierdiscus including four new species, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ruetzleri (Gonyaulacales, Dinophyceae). Phycologia. 2009;48(5).

18. Dai X, Mak YL, Lu CK, Mei HH, Wu JJ, Lee WH, et al. Taxonomic assignment of the benthic toxigenic dinoflagellate Gambierdiscus sp. type 6 as Gambierdiscus balechii (Dinophyceae), including its distribution and ciguatoxicity. Harmful Algae. 2017;67:107–118. doi: 10.1016/j.hal.2017.07.002 28755713

19. Nishimura T, Sato S, Tawong W, Sakanari H, Yamaguchi H, Adachi M. Morphology of Gambierdiscus scabrosus sp. nov.(Gonyaulacales): a new epiphytic toxic dinoflagellate from coastal areas of Japan. Journal of Phycology. 2014;50(3):506–514. doi: 10.1111/jpy.12175 26988323

20. Rhodes L, Smith KF, Verma A, Curley BG, Harwood DT, Murray S, et al. A new species of Gambierdiscus (Dinophyceae) from the south-west Pacific: Gambierdiscus honu sp. nov. Harmful Algae. 2017;65:61–70. doi: 10.1016/j.hal.2017.04.010 28526120

21. Kretzschmar AL, Verma A, Harwood T, Hoppenrath M, Murray S. Characterization of Gambierdiscus lapillus sp. nov.(Gonyaulacales, Dinophyceae): A new toxic dinoflagellate from the Great Barrier Reef (Australia). Journal of Phycology. 2017;53(2):283–297. doi: 10.1111/jpy.12496 27885668

22. Fraga S, Rodríguez F, Caillaud A, Diogène J, Raho N, Zapata M. Gambierdiscus excentricus sp. nov.(Dinophyceae), a benthic toxic dinoflagellate from the Canary Islands (NE Atlantic Ocean). Harmful Algae. 2011;11:10–22.

23. Xu Y, Richlen ML, Morton SL, Mak YL, Chan LL, Tekiau A, et al. Distribution, abundance and diversity of Gambierdiscus spp. from a ciguatera-endemic area in Marakei, Republic of Kiribati. Harmful Algae. 2014;34:56–68.

24. Fraga S, Rodríguez F. Genus Gambierdiscus in the Canary Islands (NE Atlantic Ocean) with description of Gambierdiscus silvae sp. nov., a new potentially toxic epiphytic benthic dinoflagellate. Protist. 2014;165(6):839–853. doi: 10.1016/j.protis.2014.09.003 25460234

25. Kretzschmar AL, Larsson ME, Hoppenrath M, Doblin MA, Murray S. Characterisation of two toxic Gambierdiscus spp. (Gonyaulacales, Dinophyceae) from the Great Barrier Reef (Australia): G. lewisii sp. nov. and G. holmesii sp. nov. Protist. 2019 Forthcoming.

26. Holmes MJ, Lewis RJ, Gillespie NC. Toxicity of Australian and French Polynesian strains of Gambierdiscus toxicus (Dinophyceae) grown in culture: Characterization of a new type of maitotoxin. Toxicon. 1990;28(10):1159–1172. doi: 10.1016/0041-0101(90)90116-o 2264067

27. Holmes MJ, Lewis RJ, Poli MA, Gillespie NC. Strain dependent production of ciguatoxin precursors (gambiertoxins) by Gambierdiscus toxicus (Dinophyceae) in culture. Toxicon. 1991;29(6):761–775. doi: 10.1016/0041-0101(91)90068-3 1926177

28. Holmes MJ, Lewis RJ. Purification and characterisation of large and small maitotoxins from cultured Gambierdiscus toxicus. Natural toxins. 1994;2(2):64–72. doi: 10.1002/nt.2620020204 8075895

29. Kohli GS, Murray SA, Neilan BA, Rhodes LL, Harwood DT, Smith KF, et al. High abundance of the potentially maitotoxic dinoflagellate Gambierdiscus carpenteri in temperate waters of New South Wales, Australia. Harmful Algae. 2014;39:134–145.

30. Bravo I, Figueroa RI, Fraga S. Cellular and nuclear morphological variability within a single species of the toxigenic dinoflagellate genus Gambierdiscus: Relationship to life-cycle processes. Harmful Algae. 2014;40:1–8.

31. Satake M, Murata M, Yasumoto T. Gambierol: a new toxic polyether compound isolated from the marine dinoflagellate Gambierdiscus toxicus. Journal of the American Chemical Society. 1993;115(1):361–362.

32. Nagai H, Murata M, Torigoe K, Satake M, Yasumoto T. Gambieric acids, new potent antifungal substances with unprecedented polyether structures from a marine dinoflagellate Gambierdiscus toxicus. The Journal of Organic Chemistry. 1992;57(20):5448–5453.

33. Rodriguez I, Genta-Jouve G, Alfonso C, Calabro K, Alonso E, Sanchez JA, et al. Gambierone, a ladder-shaped polyether from the dinoflagellate Gambierdiscus belizeanus. Organic letters. 2015;17(10):2392–2395. doi: 10.1021/acs.orglett.5b00902 25950201

34. Murata M, Naoki H, Iwashita T, Matsunaga S, Sasaki M, Yokoyama A, et al. Structure of maitotoxin. Journal of the American Chemical Society. 1993;115(5):2060–2062.

35. Murata M, Legrand AM, Ishibashi Y, Yasumoto T. Structures of ciguatoxin and its congener. Journal of the American Chemical Society. 1989;111(24):8929–8931.

36. Intergovernmental Oceanographic Commission of UNESCO. Global Ciguatera Strategy; 2015 (accessed January 7, 2016).

37. Diogène J. The Chemistry of Ciguatoxins: From the First Records to Current Challenges of Monitoring Programs. Toxins and Biologically Active Compounds from Microalgae. 2014;1:176.

38. Chinain M, Darius HT, Ung A, Cruchet P, Wang Z, Ponton D, et al. Growth and toxin production in the ciguatera-causing dinoflagellate Gambierdiscus polynesiensis (Dinophyceae) in culture. Toxicon. 2010;56(5):739–750. doi: 10.1016/j.toxicon.2009.06.013 19540257

39. Rhodes L, Harwood T, Smith K, Argyle P, Munday R. Production of ciguatoxin and maitotoxin by strains of Gambierdiscus australes, G. pacificus and G. polynesiensis (Dinophyceae) isolated from Rarotonga, Cook Islands. Harmful Algae. 2014;39:185–190.

40. Rhodes LL, Smith KF, Murray S, Harwood DT, Trnski T, Munday R. The epiphytic genus Gambierdiscus (Dinophyceae) in the Kermadec Islands and Zealandia regions of the southwestern Pacific and the associated risk of ciguatera fish poisoning. Marine Drugs. 2017;15(7):219.

41. Larsson ME, Laczka OF, Harwood DT, Lewis RJ, Himaya S, Murray SA, et al. Toxicology of Gambierdiscus spp.(Dinophyceae) from Tropical and Temperate Australian Waters. Marine Drugs. 2018;16(1):7.

42. Smith KF, Biessy L, Argyle PA, Trnski T, Halafihi T, Rhodes LL. Molecular Identification of Gambierdiscus and Fukuyoa (Dinophyceae) from Environmental Samples. Marine Drugs. 2017;15(8):243.

43. Darius HT, Roué M, Sibat M, Viallon J, Vandersea MW, Tester PA, et al. Tectus niloticus (Tegulidae, Gastropod) as a novel vector of ciguatera poisoning: detection of Pacific ciguatoxins in toxic samples from Nuku Hiva Island (French Polynesia). Toxins. 2017;10(1):2.

44. Nishimura T, Hariganeya N, Tawong W, Sakanari H, Yamaguchi H, Adachi M. Quantitative PCR assay for detection and enumeration of ciguatera-causing dinoflagellate Gambierdiscus spp.(Gonyaulacales) in coastal areas of Japan. Harmful Algae. 2016;52:11–22. doi: 10.1016/j.hal.2015.11.018 28073467

45. Vandersea MW, Kibler SR, Holland WC, Tester PA, Schultz TF, Faust MA, et al. Development of semi-quantitative PCR assays for the detection and enumeration of Gambierdiscus species (Gonyaulacales, Dinophyceae) 1. Journal of Phycology. 2012;48(4):902–915. doi: 10.1111/j.1529-8817.2012.01146.x 27009001

46. Conte J, Potoczniak MJ, Tobe SS. Using synthetic oligonucleotides as standards in probe-based qPCR. Biotechniques. 2018;64(4):177–9. doi: 10.2144/btn-2018-2000 29661012

47. Kon NF, Teng ST, Hii KS, Yek LH, Mujahid A, Lim HC, et al. Spatial distribution of toxic Alexandrium tamiyavanichii (Dinophyceae) in the southeastern South China Sea-Sulu Sea: A molecular-based assessment using real-time quantitative PCR (qPCR) assay. Harmful Algae. 2015;50:8–20.

48. Gómez F, Qiu D, Lopes RM, Lin S. Fukuyoa paulensis gen. et sp. nov., a New Genus for the Globular Species of the Dinoflagellate Gambierdiscus (Dinophyceae). Journal of Molecular Evolution. 2015;10(4):e0119676.

49. Kohli GS, Papiol GG, Rhodes LL, Harwood DT, Selwood A, Jerrett A, et al. A feeding study to probe the uptake of Maitotoxin by snapper (Pagrus auratus). Harmful Algae. 2014;37:125–132.

50. Queensland Government, Queensland Health. Notifiable conditions annual reporting; 2016 (accessed December 30, 2016).

51. Lewis RJ. Ciguatera: Australian perspectives on a global problem. Toxicon. 2006;48(7):799–809. doi: 10.1016/j.toxicon.2006.07.019 16930661

52. Tonge J, Battey Y, Forbes J, Grant E, et al. Ciguatera poisoning: a report of two out-breaks and a probable fatal case in Queensland. Medical Journal of Australia. 1967;2(24):1088–90. 6074504

53. Farrell H, Zammit A, Harwood DT, McNabb P, Shadbolt C, Manning J, et al. Clinical diagnosis and chemical confirmation of ciguatera fish poisoning in New South Wales, Australia. Communicable Diseases Intelligence. 2016;40(1).

54. Farrell H, Murray SA, Zammit A, Edwards AW. Management of Ciguatoxin Risk in Eastern Australia. Toxins. 2017;9(11):367.

55. Murray S, Momigliano P, Heimann K, Blair D. Molecular phylogenetics and morphology of Gambierdiscus yasumotoi from tropical eastern Australia. Harmful Algae. 2014;39:242–252.

56. Sparrow L, Momigliano P, Russ GR, Heimann K. Effects of temperature, salinity and composition of the dinoflagellate assemblage on the growth of Gambierdiscus carpenteri isolated from the Great Barrier Reef. Harmful Algae. 2017;65:52–60. doi: 10.1016/j.hal.2017.04.006 28526119

57. Richlen ML, Morton SL, Barber PH, Lobel PS. Phylogeography, morphological variation and taxonomy of the toxic dinoflagellate Gambierdiscus toxicus (Dinophyceae). Harmful Algae. 2008;7(5):614–629.

58. Hallegraeff GM, Bolch C, Hill D, Jameson I, LeRoi J, McMinn A, et al. Algae of Australia: Phytoplankton of Temperate Coastal Waters.; 2010.

59. Kohli GS, Neilan BA, Brown MV, Hoppenrath M, Murray SA. Cob gene pyrosequencing enables characterization of benthic dinoflagellate diversity and biogeography. Environmental Microbiology. 2014;16(2):467–485. doi: 10.1111/1462-2920.12275 24147781

60. Verma A, Hoppenrath M, Dorantes-Aranda JJ, Harwood DT, Murray SA. Molecular and phylogenetic characterization of Ostreopsis (Dinophyceae) and the description of a new species, Ostreopsis rhodesae sp. nov., from a subtropical Australian lagoon. Harmful algae. 2016;60:116–130. doi: 10.1016/j.hal.2016.11.004 28073555

61. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research. 2004;32(5):1792–1797. doi: 10.1093/nar/gkh340 15034147

62. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647–1649. doi: 10.1093/bioinformatics/bts199 22543367

63. Zhou Z, Miwa M, Hogetsu T. Analysis of genetic structure of a Suillus grevillei population in a Larix kaempferi stand by polymorphism of inter-simple sequence repeat (ISSR). New Phytologist. 1999;144(1):55–63.

64. R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria; 2013.

65. RStudio Team. RStudio: Integrated Development Environment for R. Boston, MA; 2015.

66. Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York; 2009.

67. Berdalet E, Bravo I, Evans J, Fraga S, Kibler S, Kudela M, et al. Global ecology and oceanography of harmful algal blooms, GEOHAB Core Research Project: HABs in benthic systems. GEOHAB report. 2012;.

68. Hariganeya N, Tanimoto Y, Yamaguchi H, Nishimura T, Tawong W, Sakanari H, et al. Quantitative PCR method for enumeration of cells of cryptic species of the toxic marine dinoflagellate Ostreopsis spp. in coastal waters of Japan. PLoS ONE. 2013;8(3):e57627. doi: 10.1371/journal.pone.0057627 23593102

69. Galluzzi L, Bertozzini E, Penna A, Perini F, Garcés E, Magnani M. Analysis of rRNA gene content in the Mediterranean dinoflagellate Alexandrium catenella and Alexandrium taylori: implications for the quantitative real-time PCR-based monitoring methods. Journal of Applied Phycology. 2010;22(1):1–9.

70. Brosnahan ML, Kulis DM, Solow AR, Erdner DL, Percy L, Lewis J, Anderson DM. Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: predominance of heterotypic encystment and implications for mating interactions and biogeography. Deep Sea Research Part II: Topical Studies in Oceanography. 2010;57(3–4):175–89.

71. Yasumoto T, Nakajima I, Bagnis R, Adachi R. Finding of a dinoflagellate as a likely culprit of ciguatera. Bulletin of the Japanese Society of Scientific Fisheries (Japan). 1977;43(8):1021–1026.

72. Bomber JW. Ecology, genetic variability and physiology of the ciguatera-causing dinoflagellate Gambierdiscus toxicus Adachi & Fukuyo; PhD thesis, Florida Institute of Technology 1987.

73. Parsons ML, Settlemier CJ, Ballauer JM. An examination of the epiphytic nature of Gambierdiscus toxicus, a dinoflagellate involved in ciguatera fish poisoning. Harmful algae. 2011;10(6):598–605. doi: 10.1016/j.hal.2011.04.011 21966283

74. Taylor F, Gustavson M. An underwater survey of the organism chiefly responsible for ciguatera fish poisoning in the eastern Caribbean region: the benthic dinoflagellate Gambierdiscus toxicus. In: Proceedings of the 7th. International Diving Science Symposium. CMAS, University of Padua; 1986. p. 95–111.

75. Bomber JW, Rubio MG, Norris DR. Epiphytism of dinoflagellates associated with the disease ciguatera: substrate specificity and nutrition. Phycologia. 1989;28(3):360–368.

76. Tester PA, Kibler SR, Holland WC, Usup G, Vandersea MW, Leaw CP, et al. Sampling harmful benthic dinoflagellates: Comparison of artificial and natural substrate methods. Harmful Algae. 2014;39:8–25.

77. Cruz-Rivera E, Villareal TA. Macroalgal palatability and the flux of ciguatera toxins through marine food webs. Harmful Algae. 2006;5(5):497–525.

78. Lobel PS, Anderson DM, Durand-Clement M. Assessment of ciguatera dinoflagellate populations: sample variability and algal substrate selection. The Biological Bulletin. 1988;175(1):94–101.

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