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