Okadaic acid as a major problem for the seafood safety (Mytilus galloprovincialis) and the dynamics of toxic phytoplankton in the Slovenian coastal sea (Gulf of Trieste, Adriatic Sea).

This article presents the first results on shellfish toxicity in the Slovenian sea (Gulf of Trieste, Adriatic Sea) since the analytical methods for the detection of biotoxins (PSP, ASP, DSP and other lipophilic toxins) in bivalve molluscs were included in the national monitoring program in 2013. In addition to toxins, the composition and abundance of toxic phytoplankton and general environmental characteristics of the seawater (surface temperature and salinity) were also monitored. During the 2014-2019 study period, only lipophilic toxins were detected (78 positive tests out of 446 runs), of which okadaic acid (OA) predominated in 97 % of cases, while dinophysistoxin-2 and yessotoxins only gave a positive result in one sampling event each. The number of samples that did not comply with the EC Regulation for the OA group was 17 or 3.8 % of all tests performed, all of which took place from September to November, while a few positive OA tests were also recorded in December, April, and May. This toxicity pattern was consistent with the occurrence pattern of the five most common DSP-producing dinoflagellates, which was supported by the development of warm and thermohaline stratified waters: Dinophysis caudata, D. fortii, D. sacculus, D. tripos and Phalacroma rotundatum. The strong correlation (r = 0.611, p < 0.001) between D. fortii, reaching abundances of up to 950 cells L-1, and OA suggests that D. fortii is the main cause of OA production in Slovenian waters. Strong interannual variations in OA and phytoplankton dynamics, exacerbated by the effects of anthropogenic impacts in this coastal ecosystem, reduce the predictability of toxicity events and require continuous and efficient monitoring. Our results also show that the introduction of the LC-MS/MS method for lipophilic toxins has improved the management of aquaculture activities, which was not as accurate based on mouse bioassays.

Exploring the mesoscale connectivity of phytoplankton periodic assemblages' succession in northern Adriatic pelagic habitats.

An appropriate model for phytoplankton distribution patterns is critical for understanding biogeochemical cycles and trophic interactions in the oceans and seas. Because phytoplankton dynamics in coastal waters are more complex due to shallow depth and proximity to land, more accurate models applied to the correct spatial and temporal scales are needed. Our study investigates the role of the atmosphere and hydrosphere in pelagic habitat by modelling phytoplankton assemblages at two Long Term Ecological Research sites in the northern Adriatic Sea using niche-forming environmental variables (wind, temperature, salinity, river discharge, rain, and water column stratification). To study the synchronization between the phytoplankton community and these environmental variables at the two LTER sites, we applied current linear and nonlinear numerical methods for ecological modelling. The aim was to use periodic and/or non-periodic properties of the environmental variables to classify the phytoplankton assemblages at one LTER site (Gulf of Trieste - Slovenia) and then predict them at another LTER site 100 km away (Gulf of Venice - Italy). We found that periodicity played a role in the explanatory and predictive power of the environmental variables and that it was more important than non-periodic events in defining the common structure of the two pelagic habitats. The non-linear classification functions of the neural networks further increased the predictive power of these variables. We observed partial synchronization of communities at the mesoscale and differences between the original and predicted assemblages under similar environmental conditions. We conclude that mesoscale connectivity plays an important role in phytoplankton communities in the northern Adriatic. However, the loss of periodicity of niche-forming variables due to more frequent extreme meteorological and hydrological events could loosen these connections and affect the temporal succession of phytoplankton assemblages.

Corrigendum: Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected genera.

[This corrects the article DOI: 10.3389/fmicb.2023.1071379.].

Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected genera.

Diatoms are one of the most important phytoplankton groups in the world's oceans. There are responsible for up to 40% of the photosynthetic activity in the Ocean, and they play an important role in the silicon and carbon cycles by decoupling carbon from atmospheric interactions through sinking and export. These processes are strongly influenced by the taxonomic composition of diatom assemblages. Traditionally, these have been assessed using microscopy, which in some cases is not reliable or reproducible. Next-generation sequencing enabled us to study diversity in a high-throughput manner and uncover new distribution patterns and diversity. However, phylogenetic markers used for this purpose, such as various 18S rDNA regions, are often insufficient because they cannot distinguish between some taxa. In this work, we demonstrate the performance of the chloroplast-encoded rbcL marker for metabarcoding marine diatoms compared to microscopy and 18S-V9 metabarcoding using a series of monthly samples from the Gulf of Trieste (GoT), northern Adriatic Sea. We demonstrate that rbcL is able to detect more taxa compared to 18S-V9 metabarcoding or microscopy, while the overall structure of the diatom assemblage was comparable to the other two methods with some variations, that were taxon dependent. In total, 6 new genera and 22 new diatom species for the study region were identified. We were able to spot misidentification of genera obtained with microscopy such as Pseudo-nitzschia galaxiae, which was mistaken for Cylindrotheca closterium, as well as genera that were completely overlooked, such as Minidiscus and several genera from the Cymatosiraceae family. Furthermore, on the example of two well-studied genera in the region, namely Chaetoceros and particularly Pseudo-nitzschia, we show how the rbcL method can be used to infer even deeper phylogenetic and ecologically significant differences at the species population level. Despite a very thorough community analysis obtained by rbcL the incompleteness of reference databases was still evident, and we shed light on possible improvements. Our work has further implications for studies dealing with taxa distribution and population structure, as well as carbon and silica flux models and networks.

Toxicity of the Diatom Genus Pseudo-nitzschia (Bacillariophyceae): Insights from Toxicity Tests and Genetic Screening in the Northern Adriatic Sea.

Diatoms of the genus Pseudo-nitzschia H.Peragallo are known to produce domoic acid (DA), a toxin involved in amnesic shellfish poisoning (ASP). Strains of the same species are often classified as both toxic and nontoxic, and it is largely unknown whether this difference is also genetic. In the Northern Adriatic Sea, there are virtually no cases of ASP, but DA occasionally occurs in shellfish samples. So far, three species-P. delicatissima (Cleve) Heiden, P. multistriata (H. Takano) H. Takano, and P. calliantha Lundholm, Moestrup, & Hasle-have been identified as producers of DA in the Adriatic Sea. By means of enzme-linked immunosorbent assay (ELISA), high-performance liquid chromatography with UV and visible spectrum detection (HPLC-UV/VIS), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), we reconfirmed the presence of DA in P. multistriata and P. delicatissima and detect for the first time in the Adriatic Sea DA in P. galaxiae Lundholm, & Moestrup. Furthermore, we attempted to answer the question of the distribution of DA production among Pseudo-nitzschia species and strains by sequencing the internal transcribed spacer (ITS) phylogenetic marker and the dabA DA biosynthesis gene and coupling this with toxicity data. Results show that all subclades of the Pseudo-nitzschia genus contain toxic species and that toxicity appears to be strain dependent, often with geographic partitioning. Amplification of dabA was successful only in toxic strains of P. multistriata and the presence of the genetic architecture for DA production in non-toxic strains was thus not confirmed.

Toxic marine microalgae and noxious blooms in the Mediterranean Sea: A contribution to the Global HAB Status Report.

We review the spatial distribution of toxic marine microalgal species and the impacts of all types of harmful algal events (Harmful Algal Blooms, HABs) in the Mediterranean Sea (MS), including the Black Sea, the Sea of Marmara, coastal lagoons and transitional waters, based on two databases compiled in the Ocean Biogeographic Information System (OBIS). Eighty-four potentially toxic species have been detected in the MS (2,350 records), of which 16 described from these waters between 1860 and 2014 and a few suspected to have been introduced. More than half of these species (46) produce toxins that may affect human health, the remainders ichthyotoxic substances (29) or other types of toxins (9). Nevertheless, toxicity-related events are not frequent in the MS (308 records in 31 years), and mainly consist of impacts on aquaculture, caused by the dinoflagellates Dinophysis and Alexandrium, along with a few actual shellfish poisoning cases. Pseudo-nitzschia blooms are widespread, but domoic acid in shellfish rarely exceeds regulatory levels. Fish kills are probably less sporadic than reported, representing a problem at a few places along the southern MS coasts and in the Ebro River Delta. Since the last decade of the 20th century, blooms of the benthic dinoflagellates Ostreopsis cf. ovata have regularly occurred all along rocky shores of the MS, at times with human health problems caused by toxic aerosol. New records of Gambierdiscus and Fukuyoa, until now reported for the westernmost and easternmost MS coasts, raise concerns about the risk of ciguatera, a syndrome so far known only for subtropical and tropical areas. Recent discoveries are the dinoflagellates Vulcanodinium rugosum, responsible for the presence of pinnatoxins in French lagoons' shellfish, and the azaspiracid-producers Azadinium spp. Mucilages and discolorations have a major impact on tourism in summer. Reports of toxic species and HABs have apparently increased in the MS over the last half century, which is likely related to the increased awareness and monitoring operations rather than to an actual increase of these phenomena. Indeed, while the case of Ostreopsis appears as a sudden upsurge rather than a trend, no actual increase of toxic or noxious events has so far emerged in intensively studied areas, such as the French and Spanish coasts or the Adriatic Sea. Moreover, some cases of decrease are reported, e.g., for Alexandrium minutum blooms disappearing from the Harbour of Alexandria. Overall, main HAB risks derive from cases of massive development of microalgal biomass and consequent impacts of reduced coastal water quality on tourism, which represents the largest part of the marine economy along the MS coasts.

Ecological time series and integrative taxonomy unveil seasonality and diversity of the toxic diatom Pseudo-nitzschia H. Peragallo in the northern Adriatic Sea.

Pseudo-nitzschia H. Peragallo (1900) is a globally distributed genus of pennate diatoms that are important components of phytoplankton communities worldwide. Some members of the genus produce the neurotoxin domoic acid, so regular monitoring is in place. However, the identification of toxic members in routine samplings remains problematic. In this study, the diversity and seasonal occurrence of Pseudo-nitzschia species were investigated in the Gulf of Trieste, a shallow gulf in the northern Adriatic Sea. We used time series data from 2005 to 2018 to describe the seasonal and inter-annual occurrence of the genus in the area and its contribution to the phytoplankton community. On average, the genus accounted for about 15 % of total diatom abundance and peaked in spring and autumn, with occasional outbreaks during summer and large inter-annual fluctuations. Increased water temperature and decreased salinity positively affected the presence of some members of the genus, while strong effects could be masked by an unsuitable definition of the species complexes used for monitoring purposes. Therefore, combining morphological (TEM) and molecular analyses by sequencing the ITS, 28S and rbcL markers, eight species were identified from 83 isolated monoclonal strains: P. calliantha, P. fraudulenta, P. delicatissima, P. galaxiae, P. mannii, P. multistriata, P. pungens and P. subfraudulenta. A genetic comparison between the isolated strains and other strains in the Mediterranean was carried out and rbcL was inspected as a potential barcode marker in respect to our results. This is the first study in the Gulf of Trieste on Pseudo-nitzschia time series from a long-term ecological research (LTER) site coupled with molecular data. We show that meaningful ecological conclusions can be drawn by applying integrative methodology, as opposed to the approach that only considers species complexes. The results of this work will provide guidance for further monitoring efforts as well as research activities, including population genetics and genomics, associated with seasonal distribution and toxicity profiles.

Phytoplankton diversity in Adriatic ports: Lessons from the port baseline survey for the management of harmful algal species.

An inventory of phytoplankton diversity in 12 Adriatic ports was performed with the port baseline survey. Particular emphasis was put on the detection of harmful aquatic organisms and pathogens (HAOP) because of their negative impact on ecosystem, human health, and the economy. Phytoplanktonic HAOP are identified as species, either native or non-indigenous (NIS), which can trigger harmful algal blooms (HAB). A list of 691 taxa was prepared, and among them 52 were classified as HAB and five as NIS. Records of toxigenic NIS (Pseudo-nitzschia multistriata, Ostreopsis species including O. cf. ovata) indicate that the intrusion of non-native invasive phytoplankton species has already occurred in some Adriatic ports. The seasonal occurrence and abundance of HAOP offers a solid baseline for a monitoring design in ports in order to prevent ballast water uptake and possible expansion of HAOP outside their native region.

Strategy of port baseline surveys (PBS) in the Adriatic Sea.

Port baseline surveys (PBS) provide species inventories in and around ports, with a focus on non-indigenous species that may have been introduced by vessels, primarily via ballast water. PBS are an essential tool to support effective management strategies for non-indigenous as well as native harmful aquatic organisms and pathogens (HAOP). This paper describes the methodology of PBS that were conducted in 12 Adriatic ports. The PBS employed existing protocols that were adapted to meet the characteristics of the Adriatic sites. Their results are reported in several papers included in this special issue, each of which is devoted to a specific community. An overview of existing surveys protocols - which provide valuable support to decision-making and to design effective monitoring of non-indigenous species - is also supplied.

NanoSIMS and tissue autoradiography reveal symbiont carbon fixation and organic carbon transfer to giant ciliate host.

The giant colonial ciliate Zoothamnium niveum harbors a monolayer of the gammaproteobacteria Cand. Thiobios zoothamnicoli on its outer surface. Cultivation experiments revealed maximal growth and survival under steady flow of high oxygen and low sulfide concentrations. We aimed at directly demonstrating the sulfur-oxidizing, chemoautotrophic nature of the symbionts and at investigating putative carbon transfer from the symbiont to the ciliate host. We performed pulse-chase incubations with 14C- and 13C-labeled bicarbonate under varying environmental conditions. A combination of tissue autoradiography and nanoscale secondary ion mass spectrometry coupled with transmission electron microscopy was used to follow the fate of the radioactive and stable isotopes of carbon, respectively. We show that symbiont cells fix substantial amounts of inorganic carbon in the presence of sulfide, but also (to a lesser degree) in the absence of sulfide by utilizing internally stored sulfur. Isotope labeling patterns point to translocation of organic carbon to the host through both release of these compounds and digestion of symbiont cells. The latter mechanism is also supported by ultracytochemical detection of acid phosphatase in lysosomes and in food vacuoles of ciliate cells. Fluorescence in situ hybridization of freshly collected ciliates revealed that the vast majority of ingested microbial cells were ectosymbionts.

Dispersal similarly shapes both population genetics and community patterns in the marine realm.

Dispersal plays a key role to connect populations and, if limited, is one of the main processes to maintain and generate regional biodiversity. According to neutral theories of molecular evolution and biodiversity, dispersal limitation of propagules and population stochasticity are integral to shaping both genetic and community structure. We conducted a parallel analysis of biological connectivity at genetic and community levels in marine groups with different dispersal traits. We compiled large data sets of population genetic structure (98 benthic macroinvertebrate and 35 planktonic species) and biogeographic data (2193 benthic macroinvertebrate and 734 planktonic species). We estimated dispersal distances from population genetic data (i.e., FST vs. geographic distance) and from ß-diversity at the community level. Dispersal distances ranked the biological groups in the same order at both genetic and community levels, as predicted by organism dispersal ability and seascape connectivity: macrozoobenthic species without dispersing larvae, followed by macrozoobenthic species with dispersing larvae and plankton (phyto- and zooplankton). This ranking order is associated with constraints to the movement of macrozoobenthos within the seabed compared with the pelagic habitat. We showed that dispersal limitation similarly determines the connectivity degree of communities and populations, supporting the predictions of neutral theories in marine biodiversity patterns.

Phytoplankton across Tropical and Subtropical Regions of the Atlantic, Indian and Pacific Oceans.

We examine the large-scale distribution patterns of the nano- and microphytoplankton collected from 145 oceanic stations, at 3 m depth, the 20% light level and the depth of the subsurface chlorophyll maximum, during the Malaspina-2010 Expedition (December 2010-July 2011), which covered 15 biogeographical provinces across the Atlantic, Indian and Pacific oceans, between 35°N and 40°S. In general, the water column was stratified, the surface layers were nutrient-poor and the nano- and microplankton (hereafter phytoplankton, for simplicity, although it included also heterotrophic protists) community was dominated by dinoflagellates, other flagellates and coccolithophores, while the contribution of diatoms was only important in zones with shallow nutriclines such as the equatorial upwelling regions. We applied a principal component analysis to the correlation matrix among the abundances (after logarithmic transform) of the 76 most frequent taxa to synthesize the information contained in the phytoplankton data set. The main trends of variability identified consisted of: 1) A contrast between the community composition of the upper and the lower parts of the euphotic zone, expressed respectively by positive or negative scores of the first principal component, which was positively correlated with taxa such as the dinoflagellates Oxytoxum minutum and Scrippsiella spp., and the coccolithophores Discosphaera tubifera and Syracosphaera pulchra (HOL and HET), and negatively correlated with taxa like Ophiaster hydroideus (coccolithophore) and several diatoms, 2) a general abundance gradient between phytoplankton-rich regions with high abundances of dinoflagellate, coccolithophore and ciliate taxa, and phytoplankton-poor regions (second principal component), 3) differences in dominant phytoplankton and ciliate taxa among the Atlantic, the Indian and the Pacific oceans (third principal component) and 4) the occurrence of a diatom-dominated assemblage (the fourth principal component assemblage), including several pennate taxa, Planktoniella sol, Hemiaulus hauckii and Pseudo-nitzschia spp., in the divergence regions. Our findings indicate that consistent assemblages of co-occurring phytoplankton taxa can be identified and that their distribution is best explained by a combination in different degrees of both environmental and historical influences.

Quantifying indicatively living phytoplankton cells in ballast water samples--recommendations for Port State Control.

Different phytoplankton analysis methods (pulse-amplitude modulated fluorometry (PAM) and microscopy) were compared in preparation for compliance monitoring and enforcement with ballast water discharge standards. The key objective was to practically evaluate the performance of different new methods and tools to identify indicatively living phytoplankton cells of the size <50 µm in minimum dimension and ≥ 10 µm in minimum dimension as addressed by the Ballast Water Performance Standard (Regulation D-2, International Convention for the Control and Management of Ships' Ballast Water and Sediments, 2004). Four different PAM instruments were selected for the tests based upon knowledge and experience gained in different ballast water sample processing studies. The measurements of the PAM instruments were compared with epifluorescence microscope algae cell counts using fluorescein diacetate as viability stain. It was concluded that PAM fluorometry is a suitable method for indicative phytoplankton analysis of ballast water and the most accurate PAM instruments were identified.

Ecological characterization of toxic phytoplankton species (Dinophysis spp., Dinophyceae) in Slovenian mariculture areas (Gulf of Trieste, Adriatic Sea) and the implications for monitoring.

Diarrhetic shellfish poisoning (DSP) events are often registered in Slovenian mariculture areas (Gulf of Trieste, Adriatic Sea) and are related to the occurrence of Dinophysis spp. The annual dynamic of this genus and succession of the most important species were studied at two shellfish farms during monitoring fieldwork in the period 1995-2003. Results indicate that the Dinophysis genus maintains a relatively stable inter-annual dynamic at both sites. The Dinophysis community is characterized by two surface maxima in June and September, while in the middle layer only the autumn peak is pronounced (peak median 92 cells l(-1)). Occasional abundance maxima of around 2000 cells l(-1) in the surface layer indicate that potential outbursts of toxic species are less predictable than their seasonal dynamic. On the basis of multivariate analysis, Dinophysis sacculus was characterized as a typical late spring-early summer species, and Dinophysis caudata and Dinophysis fortii as autumn species. Correlation analysis revealed the influence of stratified conditions only on the most abundant species, D. sacculus. Ecological characteristics of the species were combined with shellfish safety requirements towards a more effective monitoring.