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A variety of shellfish toxin-producing Harmful Algal Blooms (HABs) occur every year in coastal temperate waters worldwide. These toxic HABs may cause lengthy (months) harvesting bans of mussels and other suspension feeding bivalves exposed to their blooms. To safeguard public health and the shellfish industry, European Union regulations request periodic monitoring of potentially toxic microalgae in seawater and phycotoxins in live bivalve molluscs from shellfish production areas. Monitoring of other toxic microalgae, e.g., fish killers, is based solely on cell counts. Morphological identification and quantification of microalgal cells with light microscopy is time-consuming, requires a good expertise, and accurate identification to species level (e.g., Pseudo-nitzschia species) may require electron microscopy. Toxicity varies among morphologically similar species; there are toxic and non-toxic strains of the same species. Molecular techniques using ribosomal DNA sequences offer a possibility to identify and detect precisely the potentially toxic genus/species. In an earlier project (MIDTAL), specific probes against rRNA sequences of all HAB taxa, known at the time of the project, affecting shellfish areas worldwide were designed, and those affecting Europe were tested and calibrated against rRNA extracts of clonal cultures and field samples. Microarray technology was adopted to relate to cell numbers the fluorescence signal from the reaction of all target species probes spotted in the microarray slides with those present in a single sample extract. The EMERTOX project aimed to develop a more automatic "Lab on a chip" (LOC) technology, including a non- (cell) disruptive water concentration system and biosensors for HAB cells detection. Here, calibration curves are presented against toxic microalgae (cultures and field samples) causing endemic and emerging toxicity events in Galicia (NW Spain) and Portugal. Results here relating cell numbers to electrochemical signals will be used in an early warning biosensor for toxic algae., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Harmful algal blooms (HABs) are more frequent as climate changes and tropical toxic species move northward, especially along the Iberian Peninsula, a rich aquaculture area. Monitoring programs, detecting the presence of toxic algae before they bloom, are of paramount importance to protect ecosystems, aquaculture, human health and local economies. Rapid, reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention as an alternative to the legally required but impractical microscopic counting-based techniques. Our electrochemical detection system has improved, moving from conventional sandwich hybridization protocols using different redox mediators and signal probes with different labels to a novel strategy involving the recognition of RNA heteroduplexes by antibodies further labelled with bacterial antibody binding proteins conjugated with multiple enzyme molecules. Each change has increased sensitivity. A 150-fold signal increase has been produced with our newest protocol using magnetic microbeads (MBs) and amperometric detection at screen-printed carbon electrodes (SPCEs) to detect the target RNA of toxic species. We can detect as few as 10 cells L -1 for some species by using a fast (~2 h), simple (PCR-free) and cheap methodology (~2 EUR/determination) that will allow this methodology to be integrated into easy-to-use portable systems.

Molecular techniques to detect organisms in aquatic ecosystems are being gradually considered as an attractive alternative to standard laboratory methods. They offer faster and more accurate means of detecting and monitoring species, with respect to their traditional homologues based on culture and microscopic counting. Molecular techniques are particularly attractive when multiple species need to be detected and/or are in very low abundance. This paper reviews molecular techniques based on whole cells, such as microscope-based enumeration and Fluorescence In-Situ Hybridization (FISH) and molecular cell-free formats, such as sandwich hybridization assay (SHA), biosensors, microarrays, quantitative polymerase chain reaction (qPCR) and real time PCR (RT-PCR). Those that combine one or several laboratory functions into a single integrated system (lab-on-a-chip) and techniques that generate a much higher throughput data, such as next-generation systems (NGS), were also reviewed. We also included some other approaches that enhance the performance of molecular techniques. For instance, nano-bioengineered probes and platforms, pre-concentration and magnetic separation systems, and solid-phase hybridization offer highly pre-concentration capabilities. Isothermal amplification and hybridization chain reaction (HCR) improve hybridization and amplification techniques. Finally, we presented a study case of field remote sensing of harmful algal blooms (HABs), the only example of real time monitoring, and close the discussion with future directions and concluding remarks., Competing Interests: The authors declare no conflict of interest.

In Mexican waters, there is no a formal and well-established monitoring program of harmful algal blooms (HAB) events. Until now, most of the work has been focused on the characterization of organisms present in certain communities. Therefore, the development of new techniques for the rapid detection of HAB species is necessary. Capillary electrophoresis finger print technique (CE-SSCP) is a fingerprinting technique based on the identification of different conformers dependent of its base composition. This technique, coupled with capillary electrophoresis, has been used to compare and identify different conformers. The aim of this study was to determine if CE-SSCP analysis of ribosomal RNA (rRNA) gene fragments could be used for a rapid identification of toxic and harmful HAB species to improve monitoring activities along the coasts of Baja California Sur, Mexico.Three different highly variable regions of the 18S and 28S rRNA genes were chosen and their suitability for the discrimination of different dinoflagellate species was assessed by CE-SSCP.The CE-SSCP results obtained for the LSU D7 fragment has demonstrated that this technique with this gene region could be useful for the identification of the ten dinoflagellates species of different genera.We have shown that this method can be used to discriminate species and the next step will be to apply it to natural samples to achieve our goal of molecular monitoring for toxic algae in Mexican waters. This strategy will offer an option to improve an early warning system of HAB events for coastal BCS, allowing the possible implementation of mitigation strategies. A monitoring program of HAB species using molecular methods will permit the analysis of several samples in a short period of time, without the pressure of counting with a taxonomic expert in phytoplankton taxonomy.

A microarray for the detection of toxic algal species was developed in the European Union 7th Framework project MIDTAL. We initially tested all available fluorescence in situ hybridisation probes for toxic algae, which are normally designed to a length of 18 nt, and found that in most cases the signal was rather weak or all probes designed from the second half of the molecule were inaccessible in a microarray format because of secondary structure of the ribosomal RNA molecule We modified the length of the probes, the fragmentation of the rRNA, the stringency of the washing buffers and the length of the spacer molecules linking the probes to the glass surface of the microarray. Because of the secondary structure of the rRNA molecule, regions of the molecule can be difficult to access by the probes. Each of these modifications has improved probe accessibility and probe specificity to reduce false positives.

Dinophysis and Phalacroma species containing diarrheic shellfish toxins and pectenotoxins occur in coastal temperate waters all year round and prevent the harvesting of mussels during several months each year in regions in Europe, Chile, Japan, and New Zealand. Toxicity varies among morphologically similar species, and a precise identification is needed for early warning systems. Molecular techniques using ribosomal DNA sequences offer a means to identify and detect precisely the potentially toxic species. We designed molecular probes targeting the 18S rDNA at the family and genus levels for Dinophysis and Phalacroma and at the species level for Dinophysis acuminata, Dinophysis acuta, and Dinophysis norvegica, the most commonly occurring, potentially toxic species of these genera in Western European waters. Dot blot hybridizations with polymerase chain reaction (PCR)-amplified rDNA from 17 microalgae were used to demonstrate probe specificity. The probes were modified along with other published fluorescence in situ hybridization and PCR probes and tested for a microarray platform within the MIDTAL project ( http://www.midtal.com ). The microarray was applied to field samples from Norway and Spain and compared to microscopic cell counts. These probes may be useful for early warning systems and monitoring and can also be used in population dynamic studies to distinguish species and life cycle stages, such as cysts, and their distribution in time and space.

Harmful algal blooms (HABs), which have expanded worldwide in their occurrence and frequency, are a serious menace to aquatic ecosystems and humans. The development of rapid, accurate and cost-effective detection systems for toxic algal monitoring in aquatic environments is urgently required. Although many efforts have been devoted to develop reliable tools to monitor the entire spectrum of existing toxic algae, a portable semi-automated system that enables HAB monitoring at a low cost is still not available for general purchase. This work reviews the challenges and opportunities in translating the remarkable progress of electrochemical genosensors-based methods towards practical in situ HAB monitoring applications. It is specifically focused on reviewing the optimised methods for a detection system based on a sandwich hybridisation assay (SHA) performed over transducer platforms of different materials, geometries and dimensions and presenting the diverse advantages and disadvantages among them. Probe design and specificity and optimisation of the genosensor in terms of hybridisation conditions and electrochemical signal are discussed as well as their long-term stability and storage and semi-automation attempts. With continuous innovation and attention to key challenges, we expect semi-automatic devices containing DNA-based electrochemical biosensors to have an important impact upon monitoring of serious HAB events.