Detection of Saxitoxin-Producing Cyanobacteria and Anabaena circinalis in Environmental Water Blooms by Quantitative PCR

Abstract

Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by marine "red tide" dinoflagellates and several species of freshwater filamentous cyanobacteria, including Anabaena circinalis, Aphanizomenon spp., Lyngbya wollei, and Cylindrospermopsis raciborskii. A specific quantitative PCR (qPCR) method based on SYBR green chemistry was developed to quantify saxitoxin-producing Anabaena circinalis cyanobacteria, which are major bloom-forming freshwater cyanobacteria. The aim of this study was to infer the potential toxigenicity of samples by determining the copy number of a unique and unusual polyketide synthase (PKS) sequence (sxtA) in the STX biosynthesis gene cluster identified in cyanobacteria. Our qPCR approach was applied to water samples collected from different Australian lakes, dams, and rivers. The STX concentration and cyanobacterial cell density of these blooms were also determined by high-pressure liquid chromatography (HPLC) and microscopic cell counting, respectively. STX concentrations correlated positively with STX gene copy numbers, indicating that the latter can be used as a measure of potential toxigenicity in Anabaena circinalis and possibly other cyanobacterial blooms. The qPCR method targeting STX genes can also be employed for both monitoring and ecophysiological studies of toxic Anabaena circinalis blooms and potentially several other STX-producing cyanobacteria.

Community Composition, Toxigenicity, and Environmental Conditions during a Cyanobacterial Bloom Occurring along 1,100 Kilometers of the Murray River

“…The multiplex qPCR technology described in this study affords the determination of sxtA, cyrA, and mcyE gene copy numbers ml Ϫ1 , which can then be related to the number of toxic cells and the average maximum amount (as quoted in the literature) of saxitoxin, cylindrospermopsin, and microcystin produced. The potential toxicity of a water sample as calculated via toxin gene quantification has been described elsewhere (2). In that study, an average amount of STX produced by a cell of A. circinalis AWQC131C was determined by HPLC (2.1 ϫ 10 Ϫ9 g) and subsequently used to estimate the potential toxicity of environmental samples in STX equivalents, assuming that all STX derivatives are synthesized by the same gene cluster.…”

An overview of diversity, occurrence, genetics and toxin production of bloom-forming Dolichospermum (Anabaena) species

“…4). A specific quantitative PCR (qPCR) method based on SYBR green chemistry was developed to quantify saxitoxin-producing D. circinalis (Al-Tebrineh et al, 2010), and saxitoxin concentrations were positively correlated with sxtA gene copy numbers, indicating that the latter can be used as a measure of potential toxigenicity in D. circinalis and and possibly other cyanobacterial blooms. This assay was expanded and combined into a multiplex qPCR assay to detect sxtA, cyrA, mcyE-ndaF and 16S rRNA from most cyanobacterial species known to produce the saxitoxin, cylindrospermopsin, microcystin and nodularin toxins (Al-Tebrineh et al, 2012).…”

“…The Dolichospermum species are able to produce various kinds of secondary metabolites, notably toxins; about 25% to 75% of cyanobacterial blooms are toxic in natural waters (Chorus and Bartram, 1999). The toxins can be classified into three different categories by chemical structure: cyclic peptides (microcystin), alkaloids (anatoxin-a, anatoxin-a (S), saxitoxin, and cylindrospermopsin) and lipopolysaccharides (LPS); and four groups according to the target organs: neurotoxins (anatoxin-a, anatoxin-a (S) and saxitoxin), hepatotoxins (microcystin), cytotoxins (cylindrospermopsin), and dermatotoxins (LPS) (Krishnamurthy et al, 1986;Chorus and Bartram, 1999;Stü ken et al, 2009;Al-Tebrineh et al, 2010;Ž egura et al, 2011;Singh and Dhar, 2013;Akcaalan et al, 2014;Corbel et al, 2014;Sanchez et al, 2014). Toxins are synthesized at all stages of cyanobacterial growth and remain mostly in the cell until their release into waters after cell lysis (Sivonen and Jones, 1999).…”

Cyanobacterial Toxins of the Laurentian Great Lakes, their Toxicological Effects, and Numerical Limits in Drinking Water

“…Furthermore they purified paralytic toxins from field collections of Gonyaulax catenella. Genes for saxitoxin synthesis have now been discovered in the dinoflagellate Alexandrium and several genera of cyanobacteria [266,267]. This definitively proves that these organisms synthesize saxitoxin de novo.…”