Molecular detection and quantification of the azaspiracid-producing dinoflagellateAmphidoma languida(Amphidomataceae, Dinophyceae)

Abstract

Species of the planktonic dinoflagellates Azadinium and Amphidoma are small, inconspicuous and difficult, if not impossible to be identified and differentiated by light microscopy. Within this group, there are some species that produce the marine biotoxin azaspiracid (AZA) while others are non-toxigenic, therefore a requirement exists for precise species identification. A quantitative polymerase chain reaction (qPCR) assay for molecular detection and quantification of one of the toxigenic species, Amphidoma languida, was designed and extensively tested. The assay was highly specific and sensitive to detect and quantify down to 10 target gene copies (corresponding to ca. 0.05 cells) per reaction. DNA cell quota and copy number cell −1 were constant for four different Am. languida strains, and for one strain they were shown to be stable at various time points throughout the growth cycle. Recovery of known cell numbers of Am. languida spiked into natural samples was 95-103%, and the assay was successfully tested on field samples collected from Irish coastal waters. This new qPCR assay is a valuable tool for routine monitoring for the prevention of AZA-shellfish-poisoning caused by the consumption of contaminated shellfish and is a supportive tool for studies on the biogeography of this AZA-producing species.

Distribution and abundance of azaspiracid-producing dinophyte species and their toxins in North Atlantic and North Sea waters in summer 2018

“…poporum and Am. languida was performed daily on-board as described in Toebe et al [31] and Wietkamp et al [32] using a Roche LightCycler 96 (Roche, Penzberg, Germany). All reactions were carried out in duplicate.…”

“…The final determination of cell densities of the three species for each sample (including samples of the spike experiment) was performed in technical triplicate after the survey at the Alfred-Wegener-Institute (Bremerhaven, Germany) according to procedures described previously [32]. C T values of the three technical replicates per sample were averaged and used for cell abundance calculation.…”

“…qPCR assay specificity. DNA of recently discovered target and non-target species and/or strains from Norway [35] and Denmark [32] was applied to the current qPCR assays on Az. spinosum and Az.…”

“…PCR-based assays have also been designed for Amphidomataceae. Toebe et al [31] and Wietkamp et al [32] developed specific TaqMan assays for three toxigenic species within this family (Az. spinosum, Az.…”

“…languida), and Smith et al [33] designed a SYBR Green assay targeting the DNA of all amphidomatacean species. These qPCR assays have been used to detect Amphidomataceae in field samples, for example on research surveys along the Danish coastline [34], the Norwegian coast [35], the Irish coastline [32], the Argentine shelf [36] and in western Greenland [22], or are in use for the regular monitoring of Az. spinosum and Az.…”