The northeastern Adriatic is a shallow (depth up to 40 m), northernmost coastal area of the Mediterranean. The region is significantly influenced by riverine freshwater inputs and the established circulation patterns of the Adriatic Sea, characterised primarily by inflows of high-salinity water from the south, transported by the Eastern Adriatic Current (EAC). As a result, steep ecological gradients, driven mainly by rapid, pronounced spatial and temporal variations in environmental parameters, are present in the northeastern Adriatic. Therefore, complex monitoring approaches and advanced data acquisition are necessary for reliable assessments of this dynamic marine ecosystem. During a perennial (5-year) case study, designed to test and improve the existing monitoring approach for the area, the detailed dataset obtained for salinity, temperature, and oxygen concentration enabled the characterisation of measurement trends and the detection of environmental changes in the region. The expansion of the monitoring area, the increase in measurement frequency, and the implementation of new, modern measuring techniques and instruments (oceanographic buoy) have contributed to creating a robust, reliable dataset for present state analyses and future environmental predictions in the northeastern Adriatic.

The northern Adriatic is a highly dynamic marine ecosystem where multiple environmental stressors, particularly phosphorus limitation, shape phytoplankton communities. Previous studies have established annual phytoplankton succession patterns primarily using light microscopy, while metatranscriptomic analyses have been lacking. This study used a metatranscriptomic approach to investigate the taxonomic and functional dynamics of the northern Adriatic phytoplankton community, focusing on the predominant group of dinoflagellates. Monthly sampling from April 2021 to March 2022 at two coastal stations revealed dinoflagellates as the most metabolically active phylum throughout the year in the size fraction > 50 μm. Peaks in metabolic activity of other studied phyla aligned with the characteristic seasonal species succession observed in previous studies. Community ordination indicated distinct seasonal shifts driven by environmental factors, notably phosphorus and silicon availability, as well as species interactions. Both photosynthesis and phagotrophy emerged as important trophic strategies for dinoflagellates. Nitrogen and phosphorus metabolism showed clear seasonal trends, with dinoflagellates employing various strategies for nutrient acquisition and recycling depending on resource availability. Changes in the activation of different cellular processes highlighted a seasonal shift in metabolic investment, with spring favouring rapid population expansion, while the rest of the year was characterised by prevalent transcription of genes indicative of cellular maintenance and adaptation. This study provides critical new insights into dinoflagellate phytoplankton ecology and emphasises the need for further multi-method research to fully understand their role in the northern Adriatic ecosystem.

Over the past two decades, omics approaches have been widely applied to investigate the molecular diversity and physiological responses of marine eukaryotic phytoplankton to changing environmental conditions. The northern Adriatic stands as a highly dynamic marine ecosystem in which multiple environmental stressors and steep ecological gradients shape phytoplankton communities, particularly through phosphorus limitation. In this study, metatranscriptomic and metabarcoding approaches, complemented by light microscopy, were used to investigate the taxonomic composition and functional dynamics of the phytoplankton community in the northern Adriatic Sea, with a focus on toxic and potentially toxic genera. Monthly sampling, from April 2021 to January 2023, was conducted at two coastal stations. The three-method approach enabled differentiation between the two stations according to presence and metabolic contribution of toxic and potentially toxic genera. We demonstrate that molecular approaches (metatranscriptomics and metabarcoding) nearly doubled the detected species richness compared to traditional microscopy, highlighting a significant underestimation of cryptic, nanoplanktonic, and toxigenic species in current monitoring frameworks. Metatranscriptomic data reveal that many HAB taxa maintain active metabolic states throughout the year, even when their biomass is undetectable by light microscopy, suggesting the persistence of low-abundance active populations rather than exclusive reliance on quiescent resting stages.For the studied area, Pseudo-nitzschia species metabolically dominated the autumn diatom bloom. In spring 2021, a Noctiluca outbreak was detected, along with most other toxic and potentially toxic dinoflagellate genera recorded during the study period, notably Alexandrium, Amphidinium, Azadinium, Dinophysis, Gambierdiscus and Karenia. The results indicate that intense N. scintillans blooms markedly reshape phytoplankton metabolic activity, with implications for nutrient and carbon cycling.Based on patterns of metabolic pathway activity, including photosynthesis, carbon fixation, nitrogen metabolism, glycolysis, and oxidative phosphorylation, together with associated physiological responses, we identified contrasting life strategies and adaptive responses among phytoplankton taxa. This study provides critical new insights into the inventory of toxic species and their metabolic activities in the Northern Adriatic, including the detection of several toxigenic and potentially invasive species previously unrecorded or poorly resolved in the region, such as Azadinium poporum, Karlodinium veneficum, Gambierdiscus australes, and Prymnesium parvum. However, the results also underscore the need for further long term, multi-method research to fully understand the ongoing dramatic ecological changes in the northern Adriatic ecosystem in particular with respect to HAB species.

Mucilage aggregation is a striking phenomenon in the northern Adriatic Sea, reappearing massively in surface waters on the Istrian coast in 2024 after 20 years. Formed through polymerization of exuded sugars during microphytoplankton blooms, mucilage events are hard to capture with traditional monitoring. Here, we used real-time in situ sensors, satellite data and daily pulse-shape flow cytometry to analyze phytoplankton dynamics during this event. Mucilage formation was linked to rising temperatures and Po River-induced salinity drops. Microphytoplankton species like Cerataulina pelagica, Cylindrotheca closterium, Thalassionema spp., and Gonyaulax fragilis showed as important taxa in each different phase of the phenomenon. Aggregates consisted mainly of single cells. Mucilage periods featured low diversity and thicker, more complex cells, unlike autumn blooms which showed higher diversity, chain-forming colonies, and more pigments. Our findings highlight the key role of microphytoplankton and single cells in mucilage dynamics and the influence of environmental factors like temperature, wind and freshwater inputs on phytoplankton structure and biomass in coastal ecosystems.

We present a new version of a barcoding reference library dedicated to diatoms, Diat.barcode v12, with newly published sequences, annotated with ecological, and biological traits (size class, life forms, ecological guilds, etc) and curated by a college of experts. Diat.barcode incorporates rbcL data on all diatoms, though freshwater taxa are better represented. We used this library in two different areas, one where the taxonomic coverage of the library was good (mainland France) and another where it was poor (French Guyana) with about 320 diatom samples collected for river monitoring across both regions. We show that a direct bioinformatic assignment of environmental sequences to traits (ecological guilds, habitats, morphology) has great potential in French Guyana where species knowledge is poor and therefore the proportion of assigned environmental sequences is much lower (12.8%) than trait assignation (30%). We used co-correspondence analyses to show that, unlike mainland France where all species and trait assignation datasets were significantly correlated, in French Guyana only 7 out of 13 trait categories showed a significant correlation. This indicates a significant loss of ecological information through species assignment in French Guyana. Consequently, directly assigning environmental sequences to traits can be useful and provide more ecological information in regions with poor taxonomic knowledge because of the many rbcL sequences without taxonomic assignations.

DNA metabarcoding of benthic diatoms has been successfully applied for biomonitoring at the national scale and can now be considered technically ready for routine application. However, protocols and methods still vary between and within countries, limiting their transferability and the comparability of results. In order to overcome this, routine use of DNA metabarcoding for diatom biomonitoring requires knowledge of the sources of variability introduced by the different steps of the procedure. Here, we examine how elements of routine procedures contribute to variability between European laboratories. A set of four experiments were performed focusing on DNA extraction and PCR amplification steps to evaluate their reproducibility between different laboratories and the variability introduced by different protocols currently applied by the scientific community. Under the guidance of a reference laboratory, 17 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using the same fixed protocol and their own choice of protocol. Experiments were performed by each participant on a set of standardised DNA and biofilm samples (river, lake and mock community) to investigate potential systematic and random errors. Our results revealed the successful transferability of a protocol amongst labs and a highly similar and consistent ecological assessment outcome obtained regardless of the protocols used by each participant. We propose an “all for one but prove them all” strategy, suggesting that distinct protocols can be used within the scientific community, as long as their consistency is be proven by following minimum standard requirements.

The Northern Adriatic is a dynamic and the most productive part of the Adriatic Sea. The phytoplankton of the Northern Adriatic is, as many studies in this area showed, under heavy influence of freshwater inputs and anthropogenic pressures. Cluster analysis of a more than 20 year phytoplankton data set across steep spatio-temporal, ecological gradients on a longitudinal transect across the northern Adriatic, allowed us to identify significant and reoccurring phytoplankton species assemblages. Those phytoplankton clusters show stable seasonality and relation to environmental conditions and are presumed to be cornerstones of important ecosystem services provided by phytoplankton. Our results demonstrate that the stable seasonality of important phytoplankton clusters is disrupted, leading to a destabilisation of seasonal patterns in the biodiversity of primary producers. During the last 20 years, clusters characterised by dominating species were replaced by clusters characterised by the coexistence of several species. Overall the frequency of dense blooms of single species appears to decline. Multivariate analysis showed that possible reasons might lie in oligotrophication and increased salinity dynamics. Our results suggest the importance and applicability of cluster analysis in taxon based biodiversity long term data sets to elucidate functional biodiversity changes.

Plankton studies serve as a basis for marine ecosystem research, but knowledge of marine plankton is still incomplete due to its extreme taxonomic and functional complexity. The application of metabarcoding is very valuable for the characterisation of the plankton community. The plankton community of the Southern Adriatic is subject to strong environmental fluctuations and changes, which underlines the need for frequent, reliable and comprehensive characterisation of the plankton. The aim of this study was to determine the taxonomic composition and seasonal distribution of eukaryotic plankton in the Southern Adriatic. Plankton samples were collected monthly for one year at the coastal station of the Southern Adriatic and metabarcoding was used for taxonomic identification. The results showed a high taxonomic diversity and dynamic seasonal distribution patterns for both the protist and metazoan plankton communities. Metabarcoding revealed both the core, year-round plankton community and previously unrecorded plankton organisms in the Southern Adriatic. The results provide for the first time a comprehensive overview of the plankton community in this area by metabarcoding. The identified seasonal patterns of plankton genera and species in the Southern Adriatic will contribute to the understanding of plankton interactions and future changes in community diversity characterisation.

Marine microbial communities form the basis for the functioning of marine ecosystems and the conservation of biodiversity. With the application of metagenomics and metatranscriptomics in marine environmental studies, significant progress has been made in analysing the functioning of microbial communities as a whole. These molecular techniques are highly dependent on reliable, well-characterised, comprehensive and taxonomically diverse sequenced reference transcriptomes of microbial organisms. Here we present a set of 12 individual transcriptome assemblies derived from 6 representative diatom species from the northern Adriatic Sea grown under 2 environmentally relevant growth conditions (phosphate replete vs. phosphate deprived). After filtering the reads and assembly, an average number of 64,932 transcripts per assembly was obtained, of which an average of 8856 were assigned to functionally known proteins. Of all assigned transcripts, an average of 6483 proteins were taxonomically assigned to diatoms (Bacillariophyta). On average, a higher number of assigned proteins was detected in the transcriptome assemblies of diatoms grown under replete media condition. On average, 50% of the mapped proteins were shared between the two growth conditions. All recorded proteins in the dataset were classified into 24 COG categories, with approximately 25% belonging to the unknown function and the remaining 75% belonging to all other categories. The resulting diatom reference database for the northern Adriatic, focussing on the response to nutrient limitation as characteristic for the region and predicted for the future world oceans, provides a valuable resource for analysing environmental metatranscriptome and metagenome data. Each northern Adriatic transcriptome can also be used by itself as a reference database for the (meta)transcriptomes and gene expression studies of the associated species that will be generated in the future.

Two main parameters that structure the marine ecosystem by affecting species distribution, abundance, community structure, timing of major life cycle events, and trophic state of the ecosystem are temperature and nutrient availability. Faced with climate change, eukaryotic plankton cope at multiple levels through physiological accommodation, adaptive evolution, shift in time and/or space of habitat, and/or community responses. Thirty‐two years of our phytoplankton research indicate that Chaetoceros curvisetus / pseudocurvisetus adjust to climate change more successfully than the majority of the accompanying phytoplankton taxa in the mesotrophic part of the NW Adriatic Sea, the Mediterranean. While the abundance of the entire accompanying phytoplankton community has decreased significantly since 2003 (the period of the northern Adriatic warming and oligotrophication) compared to the previous period (1986–2003), the abundance of C. curvisetus / pseudocurvisetus remained unchanged, while its contribution to the community increased significantly. Accommodation strategies include a change in the timing of high abundance and blooms in the surface layer and successful blooming in the deeper layers during warm months. Apart from the observed in situ accommodation, physiological acclimation to warming may involve changes in photosynthesis, respiration, growth, and cell biochemistry. Here, we conducted laboratory experiments with C. pseudocurvisetus to investigate how warming affects its biochemical response through the fatty acid remodeling of phospholipid classes. Long‐term field observations and short‐term laboratory experiments suggest that marine diatoms C. curvisetus / pseudocurvisetus are potential global winners with the ability to acclimate/adapt to climate change.

The northern Adriatic is characterised as the coldest and most productive marine area of the Mediterranean, which is due to high nutrient levels introduced by river discharges, the largest of which is the Italian Po River (at the same time also the largest freshwater input into the Mediterranean). The northern Adriatic is a very shallow marine ecosystem with ocean current patterns that result in long retention times of plankton in the area. The northern Adriatic phytoplankton biodiversity and abundance are well-studied, through many scientific and long-term monitoring reports. These datasets were based on phytoplankton morphological traits traditionally obtained with light microscopy. The most recent comprehensive eastern Adriatic phytoplankton checklist was published more than 20 years ago and is still valuable today. Since phytoplankton taxonomy and systematics are constantly being reviewed (partly also due to new molecular methods of species identification that complement classical methodologies), checklists need to be updated and complemented. Today, metabarcoding of molecular markers gains more and more importance in biodiversity research and monitoring. Here, we report the use of high throughput sequencing methods to re-examine taxonomic richness and provide updated knowledge of phytoplankton diversity in the eastern northern Adriatic to complement the standardised light microscopy method.This study aimed to report an up-to-date list of the phytoplankton taxonomic richness and phylogenetic relationships in the eastern northern Adriatic, based on sequence variability of barcoding genes resolved with advanced molecular tools, namely metabarcoding. Here, metabarcoding is used to complement standardised light microscopy to advance conventional monitoring and research of phytoplankton communities for the purpose of assessing biodiversity and the status of the marine environments. Monthly two-year net sampling targeted six phytoplankton groups including Bacillariophyceae (diatoms) and Chrysophyceae (golden algae) belonging to Ochrophyta, Dinophyceae (dinoflagellates), Cryptophyceae (cryptophytes), Haptophyta (mostly coccolithophorids) and Chlorophyta with Prasinophyceae (prasinophytes) and Chlorophyceae (protist green algae). Generated sequence data were taxonomically assigned and redistributed in two kingdoms, five classes, 32 orders, 49 families and 67 genera. The most diverse group were dinoflagellates, comprising of 34 found genera (48.3%), following by diatoms with 23 (35.4%) and coccolithophorids with three genera (4.0%). In terms of genetic diversity, results were a bit different: a great majority of sequences with one nucleotide tolerance (ASVs, Amplicon sequence variants) assigned to species or genus level were dinoflagellates (83.8%), 13.7% diatoms and 1.6% Chlorophyta, respectively. Although many taxa have not been detected that have been considered as common in this area, metabarcoding revealed five diatoms and 20 dinoflagellate genera that were not reported in previous checklists, along with a few species from other targeted groups that have been reported previously. We here describe the first comprehensive 18S metabarcode inventory for the northern Adriatic Sea.

Heterotrophic flagellates (HF) represent an important protist group in marine ecosystem functioning. Characterised by high taxonomic diversity, identification and classification of HF is often difficult using classical methods of light microscopy (LM). Complementing LM with molecular methods, such as barcoding, enables reliable taxonomic identification of even small size nanoflagellates that share similar or unnoticeable morphological features. The order Bicosoecida is a group of heterotrophic nanoflagellates that are important part of protist plankton and benthic communities of the world oceans. Recently, on the basis of high-resolution light microscopy and barcoding, a new bicosoecid genus, Bilabrum, was described with B. latius sp. as a type species. Our study reports on identification of B. latius from co-culture with the diatom species Chaetoceros affinis isolated from fresh plankton samples collected in the southern Adriatic. This detection of the Adriatic B.latius represents first record of this species outside itś up to now known and described habitat (deep-sea sediment of the South - East Atlantic Ocean) and in diatom co-culture.

We studied the decapod communities at pillars of gas platforms in the Northern Adriatic sorted from samples of 20 x 20 centimetre biofouling scratched at 3, 10, and 20 metres depth at seven platforms named: Ana, Ivana A, Ivana B, Ivana C, Ivana D, and Vesna. In the samples, we found nine species: Alpheus dentipes Guerin, 1832, Alpheus macrocheles (Hailstone, 1835), Athanas nitescens (Leach, 1814), Galathea intermedia Lilljeborg, 1851, Pachygrapsus marmoratus (Fabricius, 1787), Pilumnus hirtellus (Linnaeus, 1761), Pilumnus spinifer H. Milne Edwards, 1834, Pisidia bluteli (Risso, 1816), and Pisidia longicornis (Linnaeus, 1767). Alpheus dentipes was the most abundant species in our samples with 62 individuals recorded. Most of the species might have colonised the pillars from the surrounding benthic communities. The presence of the intertidal crab Pachygrapsus marmoratus, of one megalopa of Pilumnus spinifer, and numerous juvenile crabs of Pilumnus hirtellus and P. spinifer (CW < 1 cm) indicates that colonisation via planktonic drift might also occur. We discuss possible colonisation pathways and relate them to the general and wind-driven currents in the Northern Adriatic.

The development of DNA metabarcoding and High-Throughput Sequencing for diatoms is nowadays offering an interesting approach to assess their communities in freshwater and marine ecosystems. In the context of the implementation of these genomic methods to environmental monitoring, protocol constraints are moving from scientific to operational applications, requiring operational guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design included four freshwater and two marine samples from sites of diverse ecological characteristics. The impact of the preservation and storage was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, community composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflects the site origin. Only rare taxa (below 100 reads) differed among methods and durations. Thus, importance of preservation method choice is important for low-density species (rare, invasive, threatened or toxic species). However, for biomonitoring purposes, freshwater ecological index values were not affected whatever the preservation method and duration considered (including ethanol preservation), reflecting the site ecological status. This study proved that diatom metabarcoding is robust enough to replace or complement the current approach based on morphotaxonomy, paving the way to new possibilities for biomonitoring. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring.

The northern Adriatic is highly productive and shallow area characterized by numerous spatio-temporal gradients (e.g. nutrients, salinity, temperature). It is strongly influenced by numerous freshwater inputs, mainly from Po river. Its current systems as well as Po river, generates gradients of phosphate availability with an expressed N/P imbalance and phosphate limitation. A number of recent studies characterized these gradients as major factors affecting abundance and composition of microphytoplankton communities. Focus of this study is on two Leptocylindrus species, Leptocylindrus aporus (F.W. French & Hargraves) D. Nanjappa & A. Zingone 2013 and Leptocylindrus hargravesii D. Nanjappa & A. Zingone 2013. Species belonging to Leptocylindrus genus are frequently observed and have high abundances and also high contributions to the microphytoplankton community in this area. We focused on their morphological and physiological responses to phosphate limitation in situ and also performed in vitro experiments. In this study we report data on species specific growth rates under phosphorus (P) deplete and P rich conditions, localization and characteristics of alkaline phosphate activity, phosphate uptake rates as well as their morphological differences in P deplete versus P rich conditions. Our in vitro experiments showed that both Leptocylindrus species morphologically reacted similarly to phosphorus depletion and showed significantly elongated pervalvar axis in P depleted conditions if compared to P rich conditions. Also average chain lengths increased when in P depleted conditions. Two previously mentioned adaptations indicate their tendency to increase cellular surface areas available for alkaline phosphatase. Chlorophyll fluorescence of both species significantly decreased in P depleted medium. Although both species morphologically reacted similarly, our experiment demonstrated significant differences in physiological reactions to P depleted conditions.

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa (< 100 reads) differed among preservation methods and storage durations. For biomonitoring purposes, freshwater ecological index values were not affected by the preservation method and storage duration tested (including ethanol preservation), all treatments returning the same ecological status for a site. This study contributes to consolidating diatom metabarcoding. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring.

Diatoms of the genus Pseudo-nitzschia are cosmopolitans spread in seas and oceans worldwide, with more than 50 described species, dozens of which have been confirmed to produce domoic acid (DA). Here, we characterized and investigated the toxicological activity of secondary metabolites excreted into the growth media of different Pseudo-nitzschia species sampled at various locations in the northern Adriatic Sea (Croatia) using human blood cells under in vitro conditions. The results revealed that three investigated species of the genus Pseudo-nitzschia were capable of producing DA indicating their toxic potential. Moreover, toxicological data suggested all three Pseudo-nitzschia species can excrete toxic secondary metabolites into the surrounding media in addition to the intracellular pools of DA, raising concerns regarding their toxicity and environmental impact. In addition, all three Pseudo-nitzchia species triggered oxidative stress, one of the mechanisms of action likely responsible for the DNA damage observed in human blood cells. In line with the above stated, our results are of great interest to environmental toxicologists, the public and policy makers, especially in light of today's climate change, which favours harmful algal blooms and the growth of DA producers with a presumed negative impact on the public health of coastal residents.