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© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Manini, E., Baldrighi, E., Ricci, F., Grilli, F., Giovannelli, D., Intoccia, M., Casabianca, S., Capellacci, S., Marinchel, N., Penna, P., Moro, F., Campanelli, A., Cordone, A., Correggia, M., Bastoni, D., Bolognini, L., Marini, M., & Penna, A. Assessment of spatio-temporal variability of faecal pollution along coastal waters during and after rainfall events. Water, 14(3), (2022): 502, https://doi.org/10.3390/w14030502., More than 80% of wastewaters are discharged into rivers or seas, with a negative impact on water quality along the coast due to the presence of potential pathogens of faecal origin. Escherichia coli and enterococci are important indicators to assess, monitor, and predict microbial water quality in natural ecosystems. During rainfall events, the amount of wastewater delivered to rivers and coastal systems is increased dramatically. This study implements measures capable of monitoring the pathways of wastewater discharge to rivers and the transport of faecal bacteria to the coastal area during and following extreme rainfall events. Spatio-temporal variability of faecal microorganisms and their relationship with environmental variables and sewage outflow in an area located in the western Adriatic coast (Fano, Italy) was monitored. The daily monitoring during the rainy events was carried out for two summer seasons, for a total of five sampling periods. These results highlight that faecal microbial contaminations were related to rainy events with a high flow of wastewater, with recovery times for the microbiological indicators varying between 24 and 72 h and influenced by a dynamic dispersion. The positive correlation between ammonium and faecal bacteria at the Arzilla River and the consequences in seawater can provide a theoretical basis for controlling ammonium levels in rivers as a proxy to monitor the potential risk of bathing waters pathogen pollution., This research was funded by WATERCARE project (Water management solutions for reducing microbial environment impact in coastal areas, project ID 10044130, https://www.italy-croatia.eu/web/watercare, accessed on 17 October 2021) funded by the European Union under the Interreg Italy–Croatia CBC Programme.

This study describes the adoption and validation of two innovative methods for the automated count of Ostreopsis spp. concentration in sea water: a molecular assay based on qPCR and an opto-electronic device allowing to automatically acquire images from the whole sample and recognize the target algae on the basis of a machine learning algorithm. The two approaches were tested on samples coming from different locations in 5 countries along the Mediterranean Sea, collected in the framework of the cross-border EU ENPI CBCMED Project M3-HABs, and compared with the standard visual counting method. The results demonstrate the effectiveness of both automatic approaches which provide a valuable tool, mostly cost and time effective, for the establishment of large-scale and effective monitoring of Ostreopsis spp. blooms. Moreover, the two automatic methods demonstrated the ability to discriminate for the presence of a different but similar species, O. fattorussoi, for which new species-specific qPCR primers were developed

Paralytic shellfish poisoning (PSP) is a serious human illness caused by the ingestion of seafood contaminated with saxitoxin and its derivatives (STXs). These toxins are produced by some species of marine dinoflagellates within the genus Alexandrium. In the Mediterranean Sea, toxic Alexandrium spp. blooms, especially of A. minutum, are frequent and intense with negative impact to coastal ecosystem, aquaculture practices and other economic activities. We conducted a large scale study on the sxt gene and toxin distribution and content in toxic dinoflagellate A. minutum of the Mediterranean Sea using both quantitative PCR (qPCR) and HILIC-HRMS techniques. We developed a new qPCR assay for the estimation of the sxtA1 gene copy number in seawater samples during a bloom event in Syracuse Bay (Mediterranean Sea) with an analytical sensitivity of 2.0 × 10° sxtA1 gene copy number per reaction. The linear correlation between sxtA1 gene copy number and microalgal abundance and between the sxtA1 gene and STX content allowed us to rapidly determine the STX-producing cell concentrations of two Alexandrium species in environmental samples. In these samples, the amount of sxtA1 gene was in the range of 1.38 × 105 − 2.55 × 108 copies/L and the STX concentrations ranged from 41−201 nmol/L. This study described a potential PSP scenario in the Mediterranean Sea.

The marine dinoflagellate genus Ostreopsis includes species producing potent toxic compounds, such as paliytoxin and palytoxin analogs, which might cause problematic blooms in the Mediterranean Sea and other tropical or temperate areas. Phylogenetic and phylogeographical studies based on sequences of ribosomal genes, revealed the existence of distinct species and, within them, clades in relation to geographical distribution potentially representing new or cryptic species. The morphological variability of Ostreopsis complicates the identification of species; thus, molecular analyses of isolates or field samples can be helpful. The aim of this study was to improve the characterization of Ostreopsis species and investigate the geographical distribution by using large dataset composed of both new and old sampled isolates. To determine if mitochondrial genes can be used to identify Ostreopsis species, we designed new primers sets then amplified and sequenced representative regions of the COI (cytochrome c oxydase 1) and cob (cytochrome b) genes. Phylogenetic analyses of the resulting mitochondrial DNA (mtDNA) and existing or new ribosomal DNA (rDNA) sequence data showed little divergence in the mtDNA sequences among Ostreopsis species indicating that neither the COI or cob genes are phylogenetically informative. In contrast, the ribosomal gene phylogeny indicated the existence of distinct Ostreopsis species. A network of haplotypes (based on ITS-5.8S rDNA) inferred from O. cf. ovata isolates collected worldwide revealed that Atlantic/Mediterranean and Indo/Pacific areas might host two separated large populations. In conclusion, it appears that rDNA gene sequences provide an effective molecular means of distinguishing the phylogenetic and phylogeographical relationships among Ostreopsis species.