Intercalibration of classical and molecular techniques for identification of Alexandrium fundyense (Dinophyceae) and estimation of cell densities

Abstract

A workshop with the aim to compare classical and molecular techniques for phytoplankton enumeration took place at Kristineberg Marine Research Station, Sweden, in August 2005. Seventeen different techniques -nine classical microscopic-based and eight molecular methods -were compared. Alexandrium fundyense was the target organism in four experiments. Experiment 1 was designed to determine the range of cell densities over which the methods were applicable. Experiment 2 tested the species specificity of the methods by adding Alexandrium ostenfeldii, to samples containing A. fundyense. Experiments 3 and 4 tested the ability of the methods to detect the target organism within a natural phytoplankton community. Most of the methods could detect cells at the lowest concentration tested, 100 cells L À1 , but the variance was high for methods using small volumes, such as counting chambers and slides. In general, the precision and reproducibility of the investigated methods increased with increased target cell concentration. Particularly molecular methods were exceptions in that their relative standard deviation did not vary with target cell concentration. Only two of the microscopic methods and three of the molecular methods had a significant linear relationship between their cell count estimates and the A. fundyense concentration in experiment 2, where the objective was to discriminate that species from a morphologically similar and genetically closely related species. None of the investigated methods were affected by the addition of a natural plankton community background matrix in experiment 3. The results of this study are discussed in the context of previous intercomparisons and the difficulties in defining the absolute, true target cell concentration. #

Application of a sandwich hybridisation assay for rapid detection of the Northern Pacific seastar, Asterias amurensis

“…For New Zealand, A. amurensis is a high-priority, potentially invasive pest. The benefits of the SHA for harmful algae bloom species has been assessed, and the method validated (Godhe et al 2007) and ISO accredited (International Accreditation New Zealand, ISO 17025) for commercial use at the Cawthron Institute (Ayers et al 2005). In this paper, we describe an extension of the technology in designing a SHA probe for A. amurensis using the internal transcribed spacer (ITS) region of the rRNA.…”

Identification and quantification of toxic and nontoxic strains of the harmful dinoflagellate Alexandrium tamarense using fluorescence in situ hybridization and flow cytometry

“…While an individual conducting identification by molecular methods also requires experience, it is faster and easier to become competent in the use of such techniques (Godhe et al 2007). Fortunately, while the regulatory threshold for Alexandrium is "presence," shellfish toxicity is rarely observed at A. tamarense cell densities below ~ 500 cells L -1 in Scottish waters (K. Davidson pers.…”

Molecular genetic tools for environmental monitoring of New Zealand's aquatic habitats, past, present and the future

“…Rhodes et al 2007) due to the high costs of consumables and insufficient detection sensitivities for some species. However, cell counts of toxic species were generally comparable for FISH, SHA and traditional microscope methods (Ayers et al 2005;Godhe et al 2007). …”

“…cell concentrations at which shellfish harvesting would cease until shellfish flesh testing results were available). Phytoplankton monitoring is carried out using microscopy and an Utermo¨hl settling chamber, and considerable taxonomic experience is required to identify species (Utermo¨hl 1958; Godhe et al 2007;Rhodes et al 2012). Additionally, the species of interest may occur as a minor component of the plankton community, and it is often impossible to morphologically differentiate between toxic and non-toxic strains/species (e.g.…”

“…3) has been increasingly utilised for marine phytoplankton monitoring both in New Zealand and worldwide (Mackenzie et al 2005;Dyhrman et al 2006;Godhe et al 2007;Zamor et al 2012). During an Alexandrium catenella bloom in the Marlborough Sounds in 2011, QPCR assays developed by the Australian Water Quality Centre and the University of Tasmania (Mackenzie et al 2005) were used to rapidly determine cell and cyst abundances (MacKenzie et al 2011b).…”