Abstract
Microcoleus is a filamentous cyanobacteria genus with a global distribution. Some species form thick, cohesive mats over large areas of the benthos in rivers and lakes. In New Zealand Microcoleus autumnalis is an anatoxin producer and benthic proliferations are occurring in an increasing number of rivers nationwide. Anatoxin content in M. autumnalis-dominated mats varies spatially and temporally, making understanding and managing proliferations difficult. In this study a M. autumnalis-specific TaqMan probe quantitative PCR (qPCR) assay targeting the anaC gene was developed. The assay was assessed against 26 non-M. autumnalis species. The assay had a detection range over seven orders of magnitude, with a limit of detection of 5.14 × 10−8 ng μL−1. The anaC assay and a cyanobacterial specific 16S rRNA qPCR were then used to determine toxic genotype proportions in 122 environmental samples collected from 19 sites on 10 rivers in New Zealand. Anatoxin contents of the samples were determined using LC-MS/MS and anatoxin quota per toxic cell calculated. The percentage of toxic cells ranged from 0 to 30.3%, with significant (p < 0.05) differences among rivers. The anatoxin content in mats had a significant relationship with the percentage of toxic cells (R2 = 0.38, p < 0.001), indicating that changes in anatoxin content in M. autumnalis-dominated mats are primarily related to the dominance of toxic strains. When applied to more extensive samples sets the assay will enable new insights into how biotic and abiotic parameters influence genotype composition, and if applied to RNA assist in understanding anatoxin production.
Highlights
Benthic cyanobacterial proliferations in freshwater ecosystems, in particular Microcoleus spp.and closely related taxa, pose a significant risk to ecosystem, animal, and human health [1,2,3,4].Several Microcoleus and closely related species produce the potent neurotoxins anatoxin-a (ATX) and homoanatoxin-a (HTX), and their dihydro-variants; dihydroanatoxin-a and dihydrohomoanatoxin-a
In vitro tests of specificity did not result in amplification of any non-target species, including a strain of Cuspidothrix issatchenkoi, three Aphanizomenon, two Aphanocapsa, two Raphidiopsis raciborskii, eight Dolichospermum, two Leptolyngbya, two Microcystis, one Nostoc, two Oscillatoria, one Planktothrix, and two Scytonema (Table A1)
The quantitative PCR (qPCR) assay developed in the present study enables the quantification of M. autumnalis anaC
Summary
Benthic cyanobacterial proliferations in freshwater ecosystems, in particular Microcoleus spp. and closely related taxa, pose a significant risk to ecosystem, animal, and human health [1,2,3,4]. Several Microcoleus and closely related species produce the potent neurotoxins anatoxin-a (ATX) and homoanatoxin-a (HTX), and their dihydro-variants; dihydroanatoxin-a (dhATX) and dihydrohomoanatoxin-a (dhHTX; [1,5], hereafter collectively referred to as anatoxins). A significant factor impeding advancements in knowledge on the ecological function of anatoxins and the ability to predict its concentrations in Microcoleus-dominated samples is the coexistence of Toxins 2018, 10, 431; doi:10.3390/toxins10110431 www.mdpi.com/journal/toxins. Improving knowledge of the conditions that favour toxic Microcoleus strains has the potential to help predict when proliferations are more likely to be toxic
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