Elsevier

Harmful Algae

Volume 116, July 2022, 102241
Harmful Algae

7-epi-cylindrospermopsin and microcystin producers among diverse Anabaena/Dolichospermum/Aphanizomenon CyanoHABs in Oregon, USA

https://doi.org/10.1016/j.hal.2022.102241Get rights and content

Highlights

  • Microcystins MC-LR and [Dha7]MC-HtyR and 7-epi-cylindrospermopsin were shown to be produced by Dolichospermum strains in Oregon, USA

  • Congener identity was linked to mcy and cyr cyanotoxin genes

  • Dolichospermum sp. DET69 (cyr+) and Dolichospermum sp. DET73 (mcy+) are the toxin producers in Detroit Reservoir, a major source of drinking water

  • MC-LR is produced by two distinct Dolichospermum strains in three other Oregon lakes

  • Diverse HAB-forming members of the Nostocales ADA clade (Dolichospermum and Aphanizomenon flos-aquae) in the US Pacific Northwest include producers of microcystin, cylindrospermopsin and anatoxin-a, as well as strains lacking cyanotoxin genes.

Abstract

Several genomes of Nostocales ADA clade members from the US Pacific Northwest were recently sequenced. Biosynthetic genes for microcystin, cylindrospermopsin or anatoxin-a were present in 7 of the 15 Dolichospermum/Anabaena strains and none of the 5 Aphanizomenon flos-aquae (AFA) strains. Toxin analyses (ELISA and LC-MS/MS) were conducted to quantitate and identify microcystin (MC) and cylindrospermopsin (CYN) congeners/analogs in samples dominated by Dolichospermum spp. of known genome sequence. MC-LR was the main congener produced by Dolichospermum spp. from Junipers Reservoir, Lake Billy Chinook and Odell Lake, while a congener provisionally identified as [Dha7]MC-HtyR was produced by a Dolichospermum sp. in Detroit Reservoir. A second Dolichospermum sp. from Detroit Reservoir was found to produce 7-epi-CYN, with 7-deoxy-CYN also present, but no CYN. The monitoring history of each of these lakes indicates the capacity for high levels of cyanotoxins during periods when Dolichospermum spp. are the dominant cyanobacteria. The diversity of ADA strains found in the US Pacific NW emphasizes the importance of these cyanobacteria as potentially toxic HAB formers in this temperate climatic region. Our results linking congener and genetic identity add data points that will help guide development of improved tools for predicting congener specificity from cyanotoxin gene sequences.

Introduction

Cyanobacterial harmful algal blooms (CyanoHABs) are a worldwide problem affecting fresh, brackish, and sometimes marine environments. A primary concern connected with such occurrences is the possible presence of cyanotoxins that threaten the health of humans and animals using the affected water bodies as sources of drinking water and foods or for recreation. Several different compound classes are considered cyanotoxins, the most frequently reported being the microcystins (MCs), whose detection often includes the closely related nodularins (NODs), as well as cylindrospermopsins (CYNs), anatoxins (ATXs), and saxitoxins (STXs) (Chorus and Welker, 2021; Graham et al., 2010; Backer et al., 2015). In this study, we have focused on identifying MC and CYN analogs associated with recently characterized Nostocales cyanobacteria occurring in the Pacific Northwest region of the USA (Dreher et al., 2021a,b).

The widespread occurrence of MC/NODs is emphasized by their detection in 32% of randomly sampled lakes (i.e., not necessarily associated with HAB events) analyzed in a recent assessment of lakes across the United States (Loftin et al., 2016). MCs have also been found in 53% of CyanoHAB samples from English water bodies (Turner et al., 2018) and in 85% of lakes sampled in Poland (Kobos et al., 2013). MCs occur as over 250 variants (congeners) of the basic heptapeptide structure, with common variation in the amino acid at the X2 and Z4 positions, as well as numerous variations in other parts of the structure (Fig. 1A) (Dittmann et al., 2013; Bouaïcha et al., 2019; Díez-Quijada et al., 2019). MCs are synthesized by an array of enzymes with nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and tailoring activities that are encoded in large (c. 50 kbp) mcy gene clusters (Dittmann et al., 2013; Cullen et al., 2019).

MCs are potent inhibitors of protein phosphatases and are readily taken up by the liver, where they result in signature hepatocellular damage, although wider toxic symptoms are also observed (e.g., Galey et al. 1987; Briand et al. 2003). Since congener-specific toxicological effects have been reported (van Apeldoorn et al., 2007; Díez-Quijada et al., 2019; Chernoff et al., 2020, 2021), the determination of the MC congener profiles of specific cyanobacterial producers and CyanoHAB events provides information relevant to public health risk assessments. For instance, following oral administration to mice, MC-LA was found to be the most toxic of several common congeners tested, followed by MC-LR and MC-LY, with MC-RR showing much lower toxicity (Chernoff et al., 2020, Chernoff et al., 2021). MC monitoring most commonly employs enzyme linked immunosorbent assays (ELISAs), which do not allow for congener identification, but rather provide group-specific detection. ELISAs with antibodies raised against the Adda amino acid of MC/NODs allow for the detection of approximately 80% of MCs described to date (Bouaïcha et al., 2019), although cross-reactivities vary from >200% for NOD-R (Foss et al., 2017) to < 0.25% for MCs with modifications to Adda (Foss et al., 2020). Identification of MC and NOD congeners requires more specific techniques (esp. LC-MS/MS), sometimes with derivatization for isobaric distinctions, guided by comparison to reference standards (Miles et al., 2013). At the time of this study, only fourteen reference material grade MC congeners and NOD-R were commercially available for instrument calibration.

CYNs are alkaloids that have been reported from fresh waters around the world, associated mainly with cyanobacteria of the order Nostocales (Moreira et al., 2013; Dittmann et al., 2013; Pearson et al., 2010; 2016; Scarlett et al., 2020; Yang et al., 2021). Originally reported in tropical and subtropical habitats of Raphidiopsis (previously Cylindrospermopsis), these toxins are now recognized as important in temperate regions, and are capable of being produced by several genera of the Nostocales (Anabaena, Dolichospermum, Aphanizomenon, Raphidiopsis, Chrysosporum, Umezakia) as well as by a few Oscillatoriales cyanobacteria (Lyngbya/Microseira, Oscillatoria) (Dittmann et al., 2013; Pearson et al., 2016; Adamski et al., 2020; Scarlett et al., 2020). CYNs are made by PKS and other enzymes present in c. 40 kbp cyr gene clusters (Dittmann et al., 2013; Cullen et al., 2019).

There are two commonly reported structural variants of CYN described in the literature: the epimer 7-epi-CYN and their precursor 7-deoxy-CYN (Fig. 1B) (Pearson et al., 2010; Mazmouz et al., 2011; Moreira et al., 2013; Dittmann et al., 2013; Sadler, 2015), though desulphonated variants possibly representing precursor, extraction or metabolism products have also been described (Wimmer et al., 2014; Cullen et al., 2019; Méjean and Ploux, 2021). As with MCs and NODs, CYN analysis using ELISA is broadly specific and variants are not distinguished, with other techniques (esp. LC-MS/MS) required for identification and quantification of specific variants. It is important to distinguish CYN analogs because, while CYN and 7-epi-CYN are considered to possess equivalent toxicity (Runnegar et al., 2002; Norris et al., 1999; Banker et al., 2001), 7-deoxy-CYN is thought to be less toxic (González-Blanco et al., 2020), though oral dosing studies are lacking. CYN specific toxicity in mammals involves varied cytotoxic and genotoxic effects, including inhibition of protein synthesis, increased levels of reactive oxygen species and disruption of cell cycle control. Pathology generally exhibits in the liver and kidney, though multiple organs can be affected (de la Cruz et al., 2013; Moreira et al., 2013; Pearson et al., 2010; Evans et al., 2019; Scarlett et al., 2020; Yang et al., 2021).

In temperate climates, members of the recently described genus-level ADA clade (Anabaena/Dolichospermum/Aphanizomenon) within the Nostocales order are particularly prevalent, and members of these genera are capable of producing each of the four major cyanotoxin classes (Dittmann et al., 2013; Pearson et al., 2016; Driscoll et al., 2018; Dreher et al., 2021b; Österholm et al., 2020). Diverse ADA CyanoHAB strains, both toxigenic and non-toxigenic, have been described in particular from the Pacific Northwest of the United States (Brown et al., 2016; Driscoll et al., 2018; Dreher et al., 2019; 2021a) and from Finland and the neighboring Baltic Sea (Halinen et al., 2007; Wang et al., 2012; Teikari et al., 2019; Österholm et al., 2020). A goal of this work was to document the specific MC and CYN congeners produced by recently described Dolichospermum strains from Oregon. Sequences of the relevant mcy and cyr genes responsible for synthesizing these variants were examined and compared to data acquired through targeted and untargeted LC-MS/MS analyses of environmental samples dominated by Dolichospermum.

Section snippets

Collection and sampling of CyanoHABs

The samples analyzed in this study are listed in Table 1. Samples were 0.5-1 L surface grab samples including surface scum when present. Sampling was opportunistic, with the objective of optimizing the chances for obtaining complete genome sequences, as described (Dreher et al., 2021a). Cyanobacterial mass was usually collected on large-pore filters (1.2 µm glass fiber, or 10 or 35 µm nylon mesh), in some cases after allowing buoyant material to collect in cylinders held at 4°C for several

Diverse ADA cyanobacteria with widely different toxigenicities are present in the same geographic area

We recently characterized several cyanobacteria belonging to the ADA genus-level clade of Nostocales from the US Pacific Northwest by genome sequencing (Driscoll et al., 2018; Dreher et al., 2021a, 2021b). Fig. 2 maps the isolation sites within Oregon and Washington, indicating the toxigenicity of each strain inferred from the genome sequence. These cyanobacteria belong to four different proposed species from the genus-level ADA clade (Table 2), where pairwise genome-wide average nucleotide

Diverse HAB-forming ADA cyanobacteria in a temperate region

Our current and recent studies emphasize that cyanobacteria belonging to four different proposed species from the genus-level ADA clade represent a diverse group that are active in forming contemporary CyanoHABs in Oregon and Washington (Table 2) (Dreher et al., 2021a). Distinct strains were present in the different lakes, except for Dolichospermum sp. OL01 from Odell Lake and Dolichospermum sp. LBC05a from Lake Billy Chinook, which have near-identical genomes. These two lakes are 125 km apart

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank the following for providing cyanotoxin monitoring data: Brandin Hilbrandt and City of Salem, Al Johnson and US Forest Service, and Tina Lundell and US Army Corps of Engineers. This research was supported by the Oregon State University Agricultural Experiment Station.

References (65)

  • A.J. Foss et al.

    Nodularin from benthic freshwater periphyton and implications for trophic transfer

    Toxicon

    (2017)
  • B. Greer et al.

    A validated UPLC–MS/MS method for the surveillance of ten aquatic biotoxins in European brackish and freshwater systems

    Harmful Algae

    (2016)
  • K.A. Loftin et al.

    Cyanotoxins in inland lakes of the United States: Occurrence and potential recreational health risks in the EPA National Lakes Assessment 2007

    Harmful Algae

    (2016)
  • R. Mazmouz et al.

    Characterization of CyrI, the hydroxylase involved in the last step of cylindrospermopsin biosynthesis: binding studies, site-directed mutagenesis and stereoselectivity

    Arch. Biochem. Biophys.

    (2018)
  • T.G. Otten et al.

    Application of molecular tools for microbial source tracking and public health risk assessment of a Microcystis bloom traversing 300 km of the Klamath River

    Harmful Algae

    (2015)
  • K.R. Scarlett et al.

    Global scanning of cylindrospermopsin: Critical review and analysis of aquatic occurrence, bioaccumulation, toxicity and health hazards

    Sci. Total Environ.

    (2020)
  • T. Stachelhaus et al.

    The specificity-conferring code of adenylation domains in non-ribosomal peptide synthetases

    Chem. Biol.

    (1999)
  • D. Tillett et al.

    Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide–polyketide synthetase system

    Chem. Biol.

    (2000)
  • N. Williamson et al.

    Survival of cyanobacteria in rivers following their release in water from large headwater reservoirs

    Harmful Algae

    (2018)
  • K.M. Wimmer et al.

    7-Deoxy-desulfo-cylindrospermopsin and 7-deoxy-desulfo-12-acetylcylindrospermopsin: Two new cylindrospermopsin analogs isolated from a Thai strain of Cylindrospermopsis raciborskii

    Harmful Algae

    (2014)
  • R. Akcaalan et al.

    First report of cylindrospermopsin production by two cyanobacteria (Dolichospermum mendotae and Chrysosporum ovalisporum) in Lake Iznik, Turkey

    Toxins

    (2014)
  • R. Banker et al.

    Uracil moiety is required for toxicity of the cyanobacterial hepatotoxin cylindrospermopsin

    J. Toxicol. Environ. Health Part A

    (2001)
  • L.C. Backer et al.

    Cyanobacteria and algae blooms: review of health and environmental data from the harmful algal bloom-related illness surveillance system (HABISS) 2007–2011

    Toxins

    (2015)
  • K. Blin et al.

    antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline

    Nucleic Acids Res.

    (2019)
  • N. Bouaïcha et al.

    Structural diversity, characterization and toxicology of microcystins

    Toxins (Basel)

    (2019)
  • J.F. Briand et al.

    Health hazards for terrestrial vertebrates from toxic cyanobacteria in surface water ecosystems

    Vet. Res.

    (2003)
  • N.M. Brown et al.

    Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102

    BMC Genom.

    (2016)
  • N. Chernoff et al.

    The comparative toxicity of 10 microcystin congeners administered orally to mice: clinical effects and organ toxicity

    Toxins (Basel)

    (2020)
  • N. Chernoff et al.

    Dose–Response study of microcystin congeners MCLA, MCLR, MCLY, MCRR, and MCYR administered orally to mice

    Toxins

    (2021)
  • I. Chorus et al.

    Toxic cyanobacteria in water: a guide to their public health consequences, monitoring and management

    Toxic Cyanobacteria in Water: a Guide to their Public Health Consequences, Monitoring and Management

    (2021)
  • Dittmann, E., Fewer, D.P. and Neilan, B.A., 2013. Cyanobacterial toxins: biosynthetic routes and evolutionary roots....
  • D.P. Fewer et al.

    Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

    BMC Evol. Biol.

    (2007)
  • Cited by (3)

    • Review: Current understanding on biological filtration for the removal of microcystins

      2023, Chemosphere
      Citation Excerpt :

      When the concentration of MC-LR at the water intake was 118.7 μg/L, the finished water still contained 2.47 μg/L of MC-LR which was above the WHO's guideline limit (1 μg/L) for drinking water. Recently in the United States, two major DWTPs in the states of Ohio and Oregon were forced to issue drinking water bans due to elevated levels of MCs in finished drinking water resulting from chronic HABs in source water from Lake Erie and Detroit Lake, respectively (Dreher et al., 2022). From these events, there is some additional information available for the performance of both SSF and RSF for MC removal.

    View full text