Proliferation and anatoxin production of benthic cyanobacteria associated with canine mortalities along a stream-lake continuum.

Proliferations of benthic cyanobacteria are increasingly in the public eye, with rising animal deaths associated with benthic rather than planktonic blooms. In early June 2021, two dogs died after consuming material on the shore of Shubenacadie Grand Lake, Nova Scotia. Preliminary investigations indicated anatoxins produced by benthic cyanobacterial mats were responsible for the deaths. In this study, we monitored the growth of a toxic benthic cyanobacterial species (Microcoleus sp.) along a stream-lake continuum where the canine poisonings occurred. We found that the species was able to proliferate in both lentic and lotic environments, but temporal growth dynamics and the predominant sub-species were influenced by habitat type, and differed with hydrodynamic setting, nutrient and sunlight availability. Toxin concentration was greatest in cyanobacterial mats growing in the oligotrophic lakeshore environment (maximum measured total anatoxins (ATXs) >20 mg·kg-1 wet weight). This corresponded with a shift in the profile of ATX analogues, which also indicated changing sub-species dominance along the stream-lake transition.

Liquid chromatography-high-resolution tandem mass spectrometry of anatoxins, including new conjugates and reduction products.

Anatoxins (ATXs) are a potent class of cyanobacterial neurotoxins for which only a handful of structural analogues have been well characterized. Here, we report the development of an LC-HRMS/MS method for the comprehensive detection of ATXs. Application of this method to samples of benthic cyanobacterial mats and laboratory cultures showed detection of several new ATXs. Many of these result from nucleophilic addition to the olefinic bond of the α,ß-unsaturated ketone functional group of anatoxin-a (ATX) and homoanatoxin-a (hATX), analogous to the conjugation chemistry of microcystins, which contain similar α,ß-unsaturated amide functionality. Conjugates with glutathione, γ-glutamylcysteine, methanethiol, ammonia, methanol and water were detected, as well as putative C-10 alcohol derivatives. Structural confirmation was obtained by simple and selective analytical-scale semisynthetic reactions starting from available ATX standards. Methanol, water and ammonia conjugates were found to result primarily from sample preparation. Reduction products were found to result from enzymatic reactions occurring primarily after cell lysis in laboratory cultures of Kamptonema formosum and Cuspidothrix issatschenkoi. The relative contributions of the identified analogues to the anatoxin profiles in a set of 22 benthic-cyanobacterial-mat field samples were estimated, showing conjugates to account for up to 15% of total ATX peak area and 10-hydroxyanatoxins up to 38%. The developed methodology, new analogues and insight into the chemical and enzymatic reactivity of ATXs will enable a more comprehensive study of the class than possible previously.