Skip to main content

Development of real-time PCR for quantification of Cylindrospermopsis raciborskii cells and potential cylindrospermopsin-producing genotypes in subtropicalreservoirs of southern China

Journal of Applied Phycology volume 31, pages 3749–3758 (2019)Cite this article

Abstract

Cylindrospermopsin (CYN) is of great concern to human and animal health due to its potential toxicity. The cyanobacterium Cylindrospermopsis raciborskii is considered the most common cyanobacterial species that produces CYN. The discovery of the cyr gene cluster responsible for CYN biosynthesis allows us to develop molecular methods that detect and quantify potentially CYN-producing C. raciborskii. This paper describes the development of real-time PCR (qPCR) assays capable of quantifying the total and CYN-producing C. raciborskii in subtropical reservoirs of southern China. We designed primers and probes specifically targeting the rpoC1 and cyrJ genes of C. raciborskii, and enabling quantification of its total cell numbers and potentially toxic genotypes, respectively. The qPCR showed strong linearity between 102 and 106 copies per reaction for both genes. This molecular method was validated against microscopic counting and a high correlation was found between them for quantifying C. raciborskii cell numbers in cyanobacterial cultures and water samples. Using qPCR, we detected potentially CYN-producing C. raciborskii in 34 of the 46 subtropical reservoirs of southern China, with the cyrJ/rpoC1 proportion ranging from 0.3 to 34.7%. CYN concentrations significantly correlated with both the copy numbers of cyrJ gene and the proportion of toxic C. raciborskii. Thus, the present real-time PCR method provides a reliable and faster method for estimating the potential toxicity of C. raciborskii blooms in this region. The wide distribution of potential CYN-producing C. raciborskii in the investigated subtropical reservoirs highlights the need for further monitoring.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Aguilera A, Gómez EB, Kaštovský J, Echenique RO, Salerno GL (2018) The polyphasic analysis of two native Raphidiopsis isolates supports the unification of the genera Raphidiopsis and Cylindrospermopsis (Nostocales, cyanobacteria). Phycologia 57:130–146

    CAS  Google Scholar 

  • Al-Tebrineh J, Merrick C, Ryan D, Humpage A, Bowling L, Neilan BA (2012) Community composition, toxigenicity, and environmental conditions during a cyanobacterial bloom occurring along 1,100 kilometers of the Murray River. Appl Environ Microbiol 78:263–272

    CAS  PubMed  PubMed Central  Google Scholar 

  • Antunes JT, Leão PN, Vasconcelos VM (2015) Cylindrospermopsis raciborskii: review of the distribution, phylogeography, and ecophysiology of a global invasive species. Front Microbiol 6:473–485

    PubMed  PubMed Central  Google Scholar 

  • Ballot A, Ramm J, Rundberget T, Kaplan-Levy RN, Hadas O, Sukenik A, Wiedner C (2011) Occurrence of non-cylindrospermopsin-producing Aphanizomenon ovalisporum and Anabaena bergii in Lake Kinneret (Israel). J Plankton Res 33:1736–1746

    CAS  Google Scholar 

  • Bar-Yosef Y, Sukenik A, Hadas O, Viner-Mozzini Y, Kaplan A (2010) Enslavement in the water body by toxic Aphanizomenon ovalisporum, inducing alkaline phosphatase in phytoplankton. Curr Biol 20:1557–1561

    CAS  PubMed  Google Scholar 

  • Burford MA, Davis TW, Orr PT, Sinha R, Willis A, Neilan BA (2014) Nutrient-related changes in the toxicity of field blooms of the cyanobacterium, Cylindrospermopsis raciborskii. FEMS Microbiol Ecol 89:135–148

    CAS  PubMed  Google Scholar 

  • Burford M, Beardall J, Willis A, Orr P, Magalhaes VF, Rangel LM, Azevedo SMFOE, Neilan BA (2016) Understanding the winning strategies used by the bloom-forming cyanobacterium Cylindrospermopsis raciborskii. Harmful Algae 54:44–53

    PubMed  Google Scholar 

  • Campo E, Lezcano M-Á, Agha R, Cirés S, Quesada A, El-Shehawy R (2013) First TaqMan assay to identify and quantify the cylindrospermopsin-producing cyanobacterium Aphanizomenon ovalisporum in water. Adv Microbiol 3:430–437

    CAS  Google Scholar 

  • Davis TW, Orr PT, Boyer GL, Burford MA (2014) Investigating the production and release of cylindrospermopsin and deoxy-cylindrospermopsin by Cylindrospermopsis raciborskii over a natural growth cycle. Harmful Algae 31:18–25

    CAS  PubMed  Google Scholar 

  • Dyble J, Tester PA, Litaker RW (2006) Effects of light intensity on cylindrospermopsin production in the cyanobacterial HAB species Cylindrospermopsis raciborskii. Afr J Mar Sci 28:309–312

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Ha JH, Hidaka T, Tsuno H (2009) Quantification of toxic-Microcystis and evaluation of its dominance ratio in blooms using real-time PCR. Environ Sci Technol 43:812–818

    CAS  PubMed  Google Scholar 

  • 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 18:94–103

    CAS  PubMed  Google Scholar 

  • Jiang Y, Xiao P, Yu G, Sano T, Pan Q, Li R (2012) Molecular basis and phylogenetic implications of deoxycylindrospermopsin biosynthesis in the cyanobacterium Raphidiopsis curvata. Appl Environ Microbiol 78:2256–2263

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang Y, Xiao P, Yu G, Shao J, Liu D, Azevedo SM, Li R (2014) Sporadic distribution and distinctive variations of cylindrospermopsin genes in cyanobacterial strains and environmental samples from Chinese freshwater bodies. Appl Environ Microbiol 80:5219–5230

    PubMed  PubMed Central  Google Scholar 

  • Jungblut AD, Hawes I, Mountfort D, Hitzfeld B, Dietrich DR, Burns BP, Neilan BA (2005) Diversity within cyanobacterial mat communities in variable salinity meltwater ponds of McMurdo ice shelf, Antarctica. Environ Microbiol 7:519–529

    CAS  PubMed  Google Scholar 

  • Kardinaal WEA, Janse I, Agterveld MPK, Meima M, Snoek J, Mur LR, Huisman J, Zwart G, Visser PM (2007) Microcystis genotype succession in relation to microcystin concentrations in freshwater lakes. Aquat Microb Ecol 48:1–12

    Google Scholar 

  • Kellmann R, Mills T, Neilan BA (2006) Functional modeling and phylogenetic distribution of putative cylindrospermopsin biosynthesis enzymes. J Mol Evol 62:267–280

    CAS  PubMed  Google Scholar 

  • Komárek J, Komárková J (2003) Phenotype diversity of the cyanoprokaryotic genus Cylindrospermopsis (Nostocales); review 2002. Czech Phycol 3:1–30

    Google Scholar 

  • Lei L, Peng L, Huang X, Han BP (2014) Occurrence and dominance of Cylindrospermopsis raciborskii and dissolved cylindrospermopsin in urban reservoirs used for drinking water supply, South China. Environ Monit Assess 186:3079–3090

    CAS  PubMed  Google Scholar 

  • Lei L, Peng L, Yang Y, Han BP (2018a) Development of time-resolved fluoroimmunoassay for detection of cylindrospermopsin using its novel monoclonal antibodies. Toxins (Basel) 10:255

    Google Scholar 

  • Lei M, Peng L, Han BP, Lei L (2018b) Distribution and affecting factors of Cylindrospermopsis raciborskii in Guangdong reservoirs. Environ Sci 39:5523–5531

    Google Scholar 

  • 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

    CAS  Google Scholar 

  • Manali KM, Arunraj R, Kumar T, Ramya M (2017) Detection of microcystin producing cyanobacteria in Spirulina dietary supplements using multiplex HRM quantitative PCR. J Appl Phycol 29:1279–1286

    CAS  Google Scholar 

  • Mankiewicz-Boczek J, Kokociński M, Gagała I, Pawełczyk J, Jurczak T, Dziadek J (2012) Preliminary molecular identification of cylindrospermopsin-producing cyanobacteria in two polish lakes (Central Europe). FEMS Microbiol Lett 326:173–179

    CAS  PubMed  Google Scholar 

  • Mazmouz R, Chapuis-Hugon F, Mann S, Pichon V, Méjean A, Ploux O (2010) Biosynthesis of cylindrospermopsin and 7-epicylindrospermopsin in Oscillatoria sp. strain PCC 6506: identification of the cyr gene cluster and toxin analysis. Appl Environ Microbiol 76:4943–4949

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mihali TK, Kellmann R, Muenchhoff J, Barrow KD, Neilan BA (2008) Characterization of the gene cluster responsible for cylindrospermopsin biosynthesis. Appl Environ Microbiol 74:716–722

    CAS  PubMed  Google Scholar 

  • Moreira C, Martins A, Azevedo J, Freitas M, Regueiras A, Vale M, Antunes A, Vasconcelos V (2011) Application of real-time PCR in the assessment of the toxic cyanobacterium Cylindrospermopsis raciborskii abundance and toxicological potential. Appl Microbiol Biotechnol 92:189–197

    CAS  PubMed  Google Scholar 

  • Moreira C, Azevedo J, Antunes A, Vasconcelos V (2013) Cylindrospermopsin: occurrence, methods of detection and toxicology. Appl Microbiol 114:605–620

    CAS  Google Scholar 

  • Moreira C, Ramos V, Azevedo J, Vasconcelos V (2014) Methods to detect cyanobacteria and their toxins in the environment. Appl Microbiol Biotechnol 98:8073–8082

    CAS  PubMed  Google Scholar 

  • Moustaka-Gouni M, Kormas KA, Vardaka E, Katsiapi M, Gkelis S (2009) Raphidiopsis mediterranea Skuja represents non-heterocytous life-cycle stages of Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju in Lake Kastoria (Greece), its type locality: evidence by morphological and phylogenetic analysis. Harmful Algae 8:864–872

    Google Scholar 

  • Neilan BA, Jacobs D, Therese DD, Blackall LL, Hawkins PR, Cox PT, Goodman AE (1997) rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis. Int J Syst Bacteriol 47:693–697

    CAS  PubMed  Google Scholar 

  • Orr PT, Rasmussen JP, Burford MA, Eaglesham GK, Lennox SM (2010) Evaluation of quantitative real-time PCR to characterise spatial and temporal variations in cyanobacteria, Cylindrospermopsis raciborskii (Woloszynska) Seenaya et Subba Raju and cylindrospermopsin concentrations in three subtropical Australian reservoirs. Harmful Algae 9:243–254

    CAS  Google Scholar 

  • Pacheco AB, Guedes IA, Azevedo SM (2016) Is qPCR a reliable indicator of cyanotoxin risk in freshwater? Toxins (Basel) 8:172

    Google Scholar 

  • Pichardo S, Cameán AM, Jos A (2017) In vitro toxicological assessment of cylindrospermopsin: a review. Toxins (Basel) 9:402

    Google Scholar 

  • Pierangelini M, Sinha R, Willis A, Burford MA, Orr PT, Beardall J, Neilan BA (2015) Constitutive cylindrospermopsin pool size in Cylindrospermopsis raciborskii under different light and CO2 partial pressure conditions. Appl Environ Microbiol 81:3069–3076

    CAS  PubMed  PubMed Central  Google Scholar 

  • Poniedziałek B, Rzymski P, Kokociński M (2012) Cylindrospermopsin: water-linked potential threat to human health in Europe. Environ Toxicol Pharmacol 34:651–660

    PubMed  Google Scholar 

  • Rasmussen JP, Giglio S, Monis PT, Campbell RJ, Saint CP (2008) Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria. Appl Microbiol 104:1503–1515

    CAS  Google Scholar 

  • Rzymski P, Poniedziałek B (2014) In search of environmental role of cylindrospermopsin: a review on global distribution and ecology of its producers. Water Res 66:320–337

    CAS  PubMed  Google Scholar 

  • Saker ML, Neilan BA (2001) Varied diazotrophies, morphologies, and toxicities of genetically similar isolates of Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) from northern Australia. Appl Environ Microbiol 67:1839–1845

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Scherer PI, Millard AD, Miller A, Schoen R, Raeder U, Geist J, Zwirglmaier K (2017) Temporal dynamics of the microbial community composition with a focus on toxic cyanobacteria and toxin presence during harmful algal blooms in two South German lakes. Front Microbiol 8:2387

    PubMed  PubMed Central  Google Scholar 

  • Sinha R, Pearson LA, Davis TW, Muenchhoff J, Pratama R, Jex A, Burford MA, Neilan BA (2014) Comparative genomics of Cylindrospermopsis raciborskii strains with differential toxicities. BMC Genomics 15:83–96

    PubMed  PubMed Central  Google Scholar 

  • Stucken K, John U, Cembella A, Murillo AA, Soto-Liebe K, Fuentes-Valdés JJ, Friedel M, Plominsky AM, Vásquez M, Glöckner G (2010) The smallest known genomes of multicellular and toxic cyanobacteria: comparison, minimal gene sets for linked traits and the evolutionary implications. PLoS One 5:9235

    Google Scholar 

  • Te SH, Chen EY, Gin KY (2015) Comparison of quantitative PCR and droplet digital PCR multiplex assays for two genera of bloom-forming cyanobacteria, Cylindrospermopsis and Microcystis. Appl Environ Microbiol 81:5203–5211

    CAS  PubMed  PubMed Central  Google Scholar 

  • Willis A, Adams MP, Chuang AW, Orr PT, O’Brien KR, Burford MA (2015) Constitutive toxin production under various nitrogen and phosphorus regimes of three ecotypes of Cylindrospermopsis raciborskii ((Wołoszyńska) Seenayya et Subba Raju). Harmful Algae 47:27–34

    CAS  Google Scholar 

  • Willis A, Chuang AW, Woodhouse JN, Neilan BA, Burford MA (2016) Intraspecific variation in growth, morphology and toxin quotas for the cyanobacterium, Cylindrospermopsis raciborskii. Toxicon 119:307–310

    CAS  PubMed  Google Scholar 

  • Yang Y, Jiang Y, Li X, Li H, Chen Y, Xie J, Cai F, Li R (2016) Variations of growth and toxin yield in Cylindrospermopsis raciborskii under different phosphorus concentrations. Toxins (Basel) 9:13

    Google Scholar 

  • Yang Y, Chen Y, Cai F, Liu X, Wang Y, Li R (2018) Toxicity-associated changes in the invasive cyanobacterium Cylindrospermopsis raciborskii in response to nitrogen fluctuations. Environ Pollut 237:1041–1049

    CAS  PubMed  Google Scholar 

  • Žegura B, Straser A, Filipič M (2011) Genotoxicity and potential carcinogenicity of cyanobacterial toxins-a review. Mutat Res 727:16–41

    PubMed  Google Scholar 

  • Zhang W, Lou I, Ung WK, Kong Y, Mok KM (2014) Analysis of cylindrospermopsin-and microcystin-producing genotypes and cyanotoxin concentrations in the Macau storage reservoir. Hydrobiologia 741:51–68

    CAS  Google Scholar 

Download references

Funding

This work was funded by a National Natural Science Foundation of China (NSFC) grant (No. 31770507) and the Water Resource Science and Technology Innovation Program of Guangdong Province (Grant No. 2016-29).

Author information

Authors and Affiliations

  1. Department of Ecology and Institute of Hydrobiology, Jinan University, Guangzhou, 510632, China

    Lamei Lei, Minting Lei, Yan Lu, Liang Peng & Bo-Ping Han

Authors
  1. Lamei Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Minting Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yan Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Liang Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bo-Ping Han
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lamei Lei.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(DOCX 19 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lei, L., Lei, M., Lu, Y. et al. Development of real-time PCR for quantification of Cylindrospermopsis raciborskii cells and potential cylindrospermopsin-producing genotypes in subtropicalreservoirs of southern China. J Appl Phycol 31, 3749–3758 (2019). https://doi.org/10.1007/s10811-019-01898-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10811-019-01898-3

Keywords

  • PCR
  • Real-time PCR
  • rpoC1 gene
  • cyrJ gene
  • Cylindrospermopsin