RESUMO
Toxic benthic cyanobacterial mats are increasingly reported worldwide as being responsible for animal mortalities due to their production of the potent neurotoxin anatoxin-a (ATX) and its analogues. Improved analytical methods for anatoxins are needed to address public health and watershed management challenges arising from extremely high spatial and temporal variability within impacted systems. We present the development, validation, and application of a direct analysis in real-time-high-resolution tandem mass spectrometry (DART-HRMS/MS) method for analysis of anatoxins in cyanobacterial field samples, including a simplified sample preparation approach. The method showed excellent sensitivity and selectivity for ATX, homoanatoxin-a, and dihydroanatoxin-a. Isotopically labeled ATX was used as an internal standard for all three analogues and successfully corrected for the matrix effects observed (86 ± 16% suppression). The limit of detection and recovery for ATX was estimated as 5 ng/g and 88%, respectively, using spiked samples. The total analysis time was â¼2 min, and excellent agreement was observed with results from a liquid chromatography-HRMS reference method. Finally, the DART-HRMS/MS method was applied to a set of 45 Microcoleus-dominated benthic cyanobacterial mat samples from the Wolastoq near Fredericton, Canada, demonstrating its power and applicability in enabling broad-scale field studies of ATX distribution.
Assuntos
Cianobactérias , Espectrometria de Massas em Tandem , Animais , Cianobactérias/química , Toxinas de Cianobactérias , Neurotoxinas , Rios/química , TropanosRESUMO
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.
Assuntos
Toxinas Bacterianas , Toxinas de Cianobactérias , Cianobactérias , Tropanos , Cães , Animais , Rios/microbiologia , Toxinas Bacterianas/toxicidade , Lagos/microbiologia , Proliferação de CélulasRESUMO
In July 2018 three dogs died after visiting the Wolastoq (Saint John River) near Fredericton, New Brunswick, in Atlantic Canada. All showed signs of toxicosis, and necropsies revealed non-specific pulmonary edema and multiple microscopic brain hemorrhages. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of vomitus and stomach contents as well as water and biota from the mortality sites confirmed the presence of anatoxins (ATXs), a class of potent neurotoxic alkaloids. The highest levels were measured in a dried benthic cyanobacterial mat that two of the dogs had been eating before falling ill and in a vomitus sample collected from one of the dogs. Concentrations of 357 and 785 mg/kg for anatoxin-a and dihydroanatoxin-a, respectively, were measured in the vomitus. Known anatoxin-producing species of Microcoleus were tentatively identified using microscopy and confirmed by 16S rRNA gene sequencing. The ATX synthetase gene, anaC, was detected in the samples and isolates. The pathology and experimental results confirmed the role of ATXs in these dog mortalities. Further research is required to understand drivers for toxic cyanobacteria in the Wolastoq and to develop methodology for assessing occurrence.
Assuntos
Toxinas Bacterianas , Cianobactérias , Cães , Animais , Toxinas Bacterianas/toxicidade , Toxinas Bacterianas/análise , Novo Brunswick , RNA Ribossômico 16S/genética , Cianobactérias/química , Tropanos/toxicidade , CanadáRESUMO
During ongoing DNA barcode (COI-5P) surveys of the macroalgal flora along the northwest Atlantic coast, we discovered a population of Ceramium secundatum in Narragansett Bay, Rhode Island, USA. This species is regarded as common and widespread in the northeast Atlantic, ranging from Norway to Morocco, but until now has not been reported from the western Atlantic. Several lines of evidence suggest that C. secundatum may be introduced to Narragansett Bay: (1) despite extensive collecting, specimens have only been obtained from a limited geographic range in the northwest Atlantic; (2) three other nonindigenous seaweed species are reportedly introduced in this region, which is thought to be a consequence of shipping; and (3) this species is introduced to South Africa and New Zealand. To investigate this suspected introduction, we applied population genetic analyses (using the cox2-3 spacer) to compare the Narragansett Bay C. secundatum population to native populations in the Republic of Ireland and the United Kingdom. Collectively, analyses of biogeographical and molecular data indicate that C. secundatum is likely introduced to Narragansett Bay. The implications of this discovery are discussed.