Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy

Abstract

Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella - where two morphologically similar species impossible to separate by light microscopy were distinguished - in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring.

Distribution of Alexandrium fundyense and A. pacificum (Dinophyceae) in the Yellow Sea and Bohai Sea

“…Examples of these methods in studies of Alexandrium include the use of fluorescence in situ hybridization (John et al, 2003;Anderson et al, 2005;Touzet et al, 2010), real-time quantitative PCR (qPCR) (Galac et al, 2003;Galluzzi et al, 2004;Hosoi-Tanabe and Sako, 2005;Dyhrman et al, 2006) and DNA microarrays (Gescher et al, 2008;Galluzzi et al, 2011;Dittami et al, 2013). Each of these methods provide species-specific detection of the targeted toxic Alexandrium species.…”

Development and evaluation of a reverse dot blot assay for the simultaneous detection of common toxic microalgae along the Chinese coast

“…The supposed reason is that excessive labels in the target molecules might impede their accessibility to specific probes, hence producing less intensive hybridization signals. The DNA array technology is also disadvantaged by its high cost (Dittami et al, 2013). A rough estimation showed that BLPM is cheaper than the other two labeling methods.…”

“…One is to produce target molecules by PCR amplification using genomic DNA from target microalgae, with fluorescein added in or after the PCR (Gescher et al, 2008;Galluzzi et al, 2011), and the fluorescein-labeled PCR products are used for DNA array hybridization. The other is to directly label RNAs with fluorescein; the labeled products are then used for hybridization (Dittami et al, 2013;Kegel et al, 2013). Although a DNA array that involves direct RNA labeling could be used to quantify target cells, a relatively high detection limit may be a great disadvantage.…”

“…Hence, the practical applications of DNA array technologies are limited by their relatively high cost and skill requirements. Glass slide-based microarrays that target rRNA have been recently established to detect toxic algae and quantify target cells (Dittami et al, 2013;Kegel et al, 2013); however, high cost and skill requirements again make them not the best choice for analyzing field samples.…”

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species

“…The laser in a microarray scanner scans the slides and the hybridization pattern captured via fluorescent excitation indicates which species are present [60]. DNA microarrays, or phylochips as they have been termed, have been used to identify phytoplankton [63], toxic algae [64,65,66,67,68,69,70,71,72,73,74,75,76,77], bacteria [78,79,80,81,82,83,84], and eggs and larvae from fish species [85]. Phylochip ® , a universal microarray for all prokaryotic organisms is commercially available and circumvents the long analysis time to perform community analysis for the prokaryotes using other molecular tools.…”