Elsevier

Harmful Algae

Volume 93, March 2020, 101767
Harmful Algae

Molecular and morphological characterization of a novel dihydroanatoxin-a producing Microcoleus species (cyanobacteria) from the Russian River, California, USA

https://doi.org/10.1016/j.hal.2020.101767Get rights and content

Highlights

  • Anatoxin-a and dihydroanatoxin-a- producing benthic non-heterocystous filamentous cyanobacterium was isolated from the Russian River, California, USA.

  • Both 16S rRNA and protein sequence phylogenetic analyses showed that the strains represent a distinct new member of the cyanobacterial genus Microcoleus (Oscillatoriales).

  • A novel species, Microcoleus anatoxicus is described and accompanied with light microscope photomicrographs and toxin profiles.

  • The complete anatoxin-a gene cassette with the first description of the anaK gene in Microcoleus is presented.

Abstract

Reports of anatoxins poisoning of wildlife and domestic animals by toxigenic cyanobacteria in streams and rivers are increasing globally. Little is known about the taxonomy, morphology and genomics of anatoxins producing species, limiting our knowledge about their environmental preferences. We isolated three benthic non-heterocystous filamentous cyanobacterial strains from the Russian River in Northern California (USA), which produce anatoxin-a and dihydroanatoxin-a. Both 16S rRNA and protein sequence phylogenetic analyses showed that the strains represent a distinct new member of the cyanobacterial genus Microcoleus (Oscillatoriales). A novel species, Microcoleus anatoxicus is described and accompanied with light microscope photomicrographs, toxin profiles and the complete anatoxin-a gene cassette with the first description of the anaK gene in Microcoleus.

Introduction

Reports of anatoxins producing blooms and animal poisonings from toxic cyanobacteria in rivers, streams, lakes and ponds are a common occurrence in many temperate and tropical systems around the globe (Svirčev et al., 2019). Anatoxins are highly potent neuromuscular-blocking agents (Humbert, 2009) that may be synthesized in at least four distinct structural variants: anatoxin-a (ATX), dihydroanatoxin-a (dhATX), homoanatoxin-a (HTX), and dihydrohomoanatoxin-a (dhHTX), with additional cis and trans configurations possible (Wonnacott and Gallagher, 2006; Mann et al., 2012). The production of benthic anatoxins in streams and rivers is attributed mainly to the mat-forming cyanobacterium Microcoleus autumnalis (Gomont) Strunecký, Komárek and Johansen, 2015 (Syn. Phormidium autumnale Gomont) and related species, observed in North America (Bouma-Gregson et al., 2019), New Zealand (Heath et al., 2010; Wood et al., 2012, 2017, 2018) and Europe (Faassen et al., 2012). On the other hand, studies from relatively low-nutrient lakes and ponds in Italy, Norway, and Germany, identified similar anatoxins with lethal effect on dogs, produced by tychoplanktonic taxa of Tychonema Anagnostidis & Komárek (Shams et al., 2015; Salmaso et al., 2016; Fastner et al., 2018).

Molecular analyses of anatoxin-producing cyanobacterial mats from environmental samples showed that several different genotypes (both toxigenic and non-toxigenic) were contributing to the spatial variation of anatoxins in New Zealand rivers (Heath et al., 2010, 2011). Similarly, Bouma-Gregson et al. (2019) used metagenomics to define four potentially novel Microcoleus species within the Eel River, Northern California, USA, one of which contained genes coding for ATX production. However, these molecular studies of field samples, were not accompanied by unialgal strain isolation in culture, leading to the conclusion that Microcoleus morphospecies are indistinguishable from one another macroscopically, and even microscopically (Heath et al., 2010, 2011; Bouma-Gregson et al., 2019). Bouma-Gregson et al. (2019) further concluded that detecting limiting environmental conditions and thresholds associated with toxigenic Microcoleus proliferations in rivers is challenging because different taxa may have different environmental preferences.

Therefore, we aimed to provide molecular and morphological characterization of three filamentous cyanobacterial strains, producing ATX and dhATX, which were isolated in monocultures from the Russian River in Northern California, collected in response to a dog-poisoning event in the fall of 2015. A separate study on these isolates has already established their broader ecotoxicity for benthic macroinvertebrates (Anderson et al., 2018). Further morphological differentiation of this species in the field, in conjunction with genomic and microscopic identification, should provide support for stream bioassessment and harmful algal bloom mitigation efforts. However, the initial morphospecies identification as Phormidium Kützing ex Gomont (Anderson et al., 2018) is questionable in light of a molecular taxonomic concept of the genus Microcoleus Desmazières ex Gomont (Strunecký et al., 2013), which includes taxa previously placed in Phormidium, Tychonema and Oscillatoria Vaucher Ex Gomont.

The aims of this study were to: 1) determine the generic placement of the three anatoxins- producing strains isolated from the Russian River, based on molecular phylogenetic analysis and comparison with the existing genomic data for other anatoxins-producing cyanobacteria, 2) characterize the genes and pathways for toxin production in the studied strains, and 3) describe the macroscopic and microscopic morphological characteristics of the studied cyanobacterial species, to enable its consistent identification.

Section snippets

Sampling sites and cyanobacteria collection

Two Russian River sites were sampled on October 1st, 2015; near the town of Cloverdale (California State Site Cloverdale #114RR5652, 38.808058, -123.0088 and State Site Camp Rose #114RR3119, 38.613618, -122.831158). Benthic mats from both sites were used for cyanobacteria isolation. Additional samples and underwater pictures for illustration of macroscopic view of the cyanobacterial mats were collected downstream from Camp Rose site on August 15, 2016 (State site #114RR2655, 38.55992,

Results

Three filamentous cyanobacterial strains were isolated in non-axenic monocultures as follows: strains PTRS1 and PTRS2 from California State Site Camp Rose #114RR3119, and strain PTRS3 California State Site Cloverdale #114RR5652. The three strains were established in the fall of 2015 and maintained in culture until recently. Over this period, they were subject to multiple rounds of toxin testing and toxicological experiments with macroinveretbrates (see previous results in Anderson et al., 2018).

Discussion

This study provides molecular, morphological and ecological data for the description of Microcoleus anatoxicus sp. nov., a novel anatoxins-producing member of genus Microcoleus. Following the diagnostic criteria for cyanobacteria taxonomy (González-Resendiz et al., 2019), a total evidence approach, based on 16S rRNA and protein sequencing phylogenetic analyses, secondary structures of different 16 S–23 S ITS regions (D1-D1' helix and Box-B helix), and microscopic observations of field material

Conclusions

Recent work (Borges et al., 2015) emphasized the need to define type strains for species of cyanobacteria based on phylogenetic studies and making sequences available in GenBank in order to advance taxonomic knowledge of these organisms. In this study, we provide both molecular and morphological information used to characterize a novel anatoxin-a and dihydroanatoxin-a producing cyanobacterium Microcoles anatoxicus, which will facilitate its further distinction in the environment, as an

Declaration of Competing Interest

None.

Acknowledgement

Funding for this research was provided from the Surface Water Ambient Monitoring Program of the California State Water Resources Control Board. We are thankful to former CSUSM students Jeanette Nichols, Karri Kirby, Corey Johnson, and Anne Decker for maintaining the cyanobacterial cultures. Bofan Wei (SUNY-ESF) assisted with the LC–MS/MS analysis. The authors are thankful to the editors Michele Burford and Christopher Gobler, as well as two anonymous reviewers for their time and valuable

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