The introduction of non-indigenous species presents an increasing threat to marine ecosystems and aquaculture activities worldwide. We report the first confirmed record of the invasive polyclad flatworm Postenterogonia orbicularis in the eastern Adriatic Sea, based on samples collected from mussel farms in Slovenia, Croatia and Montenegro. Specimens were identified through morphological examination and phylogenetic analyses using multiple markers (COI, 16S, 28S) with maximum likelihood and Bayesian inference. Results for all genetic markers were consistent, confirming their taxonomic assignment within a well-supported clade, comprising the Adriatic specimens and previously reported specimens of P. orbicularis from the sea around Shikoku Island (Japan) and the Bay of Biscay (coast of Spain and France). Phylogenetic results support the proposed synonymy between Idiostylochus tortuosus and P. orbicularis, further highlighting the taxonomic complexity within this group. Postenterogonia orbicularis was misidentified during the first observations in 2020, and the economic loss was generally attributed anecdotally to flatworms. There is an urgent need for monitoring at mussel farms and focused research on this species to develop effective management strategies.

The aim of the present study was to investigate the diversity, distribution of virulence factor genes and antimicrobial resistance phenotypes of Vibrio Harveyi clade species from two distinct and spatially distant near-shore bivalve farming environments located in protected marine reserves of the eastern Adriatic. Tissues of farmed Mediterranean mussel Mytilus galloprovincialis and European flat oyster Ostrea edulis, seawater and sediment samples were collected during six bi-monthly sampling events conducted over one year from farming locations in Lim Bay and Mali Ston Bay, Croatia. Eight distinctive Harveyi subclades, namely, V. harveyi, V. alginolyticus, V. diabolicus, V. jasicida, V. parahaemolyticus, V. rotiferianus, V. campbellii and V. communis, were detected by Phylogenetic Multilocus Sequence Analysis (MLSA) based on rpoD, toxR and rctB concatenated genes sequences. Genomic fingerprinting of MLSA-classified samples revealed substantial diversity of environmental isolates. The virulence-related genes considered typical for the Harveyi clade and their homologues were represented in all subclades with varying frequencies. Of twelve antibiotics commonly used in human and veterinary medicine, the most frequent resistance phenotypes were resistance to ampicillin (90%), erythromycin (39%), imipenem (29%) and streptomycin (22%). >60% of isolates were resistant to at least two classes of antibiotics. Seasonal or site-specific differences were found in the proportion of isolates carrying virulence genes and displaying specific antibiotic resistance phenotype.This study is the first to provide a broader perspective on the properties of Vibrio Harveyi clade species associated with ecosystems under presumably low anthropogenic pressures, such as marine protected areas. New data on virulence and antibiotic resistance are presented of Harveyi clade species coexisting in different environmental compartments, including those that have been largely overlooked or understudied in the context of bivalve aquaculture. Taken together, these results highlight the importance of monitoring the presence of potentially pathogenic strains in the natural Vibrio population of bivalve farming areas to reduce the risk to seafood consumers.

Vibrio species are ubiquitous in marine environments, with several taxa acting as opportunistic pathogens affecting both human and aquatic health. Antimicrobial resistance (AMR) in marine Vibrio spp. poses an emerging risk to environmental and public health, particularly within aquaculture-intensive coastal regions. This preliminary study investigates antimicrobial resistance (AMR) patterns in Vibrio spp. and presents the first integrated One Health-based assessment of spatial variability within aquaculture systems of the eastern Adriatic Sea. A total of 258 isolates were recovered from farmed fish, seawater, sediments, and biofouling mussels. These isolates were classified into ecologically relevant clades, with Harveyi (47.29 %) and Splendidus (43.02 %) being the most prevalent. Antibiotic susceptibility testing revealed high resistance to ampicillin (62.8 %), followed by erythromycin (33.3 %), streptomycin (29.1 %), imipenem (23.3 %), and oxytetracycline (19.4 %). Notably, 58.5 % of isolates were resistant to >= 2 antibiotics, and 37.6 % exhibited a multiple antibiotic resistance (MAR) index of >= 0.25. Distinct spatial resistance patterns were observed: Harveyi clade isolates from northern and central Adriatic sites exhibited elevated resistance to ampicillin, while Splendidus clade isolates from the southern Adriatic showed greater resistance to florfenicol and tetracycline. These findings suggest that local environmental conditions and aquaculture practices shape resistance dynamics, even in the absence of direct antibiotic concentration data. This study highlights the dominant role of Harveyi and Splendidus clades in AMR development and underscores the urgent need for spatially informed, region-specific antibiotic stewardship. The results provide essential baseline data to inform future AMR monitoring frameworks and public health policies across the Mediterranean aquaculture sector, using a One Health framework that promotes integrated collaboration across human, animal, and environmental health sectors, particularly in the context of climate change.

Changes in natural conditions and anthropogenic pollutants, alone or in combination, pose a significant challenge to coastal bivalve populations. The susceptibility of economically important bivalves to potential stressors in their farming environment has not been sufficiently investigated, despite the increase in anthropogenic pressure along the coast and the remarkable warming of seawater in recent years. Thus, the aim of this study was to evaluate the performance of mussel (Mytilus galloprovincialis) from two important farming sites in the eastern Adriatic, namely Mali Ston Bay (MSB) and Lim Bay (LB), in relation to variations of seawater parameters, reproductive cycle dynamics and tissue content of potentially harmful pollutants. The complex seasonal and site-specific patterns of chemical pollutants were determined, with tissue levels of metals, As, PAHs and PCBs largely comparable to those previously reported for the Mediterranean region. Concentrations of organochlorinated pesticides were below the level of detection. Significantly higher Cd, As and Hg concentrations were detected in the tissues of the MSB mussels. The reproductive cycle was clearly associated with the bioaccumulation of pollutants. All biochemical response parameters varied to some extent across seasons and/or between farming sites. A very pronounced seasonality was recorded for acetylcholinesterase and glutathione S-transferase activity at both sites. Metallothionein concentration and superoxide dismutase activity were generally steady throughout the study period. The most striking difference between the two sites was recorded for lipid peroxides concentrations which were predominantly significantly higher in the MSB mussels, indicating expressed pro-oxidant conditions at this site. In particular, significant correlations were found between lipid peroxides and the potentially toxic metals (Cd, As, Hg) accumulated in the mussel tissue. Data reported here are valuable as baseline information for further studies related to stress in farmed bivalves caused by oscillations of environmental factors and increasing anthropogenic pressure along the coastline.

The aim of the present study was to determine the levels of acetylcholinesterase (AChE) activity in the tail muscle tissue of wild populations of Nephrops norvegicus from the Northern Adriatic, and correlate it to body size, seasons, sex and the content of mercury, arsenic, cadmium, lead and copper. The animals of both sexes were collected in spring and autumn from two relatively distant fishing grounds. A marked variability of muscle AChE activity was found (0.49 to 11.22 nmol/min/mg prot.), displaying the opposite seasonal trend between two sampling sites. Small, but significant negative correlation has been found between AChE activity and carapace length (rs = -0.35, p<0.05). Data reported here provide an essential baseline for future studies of neurotoxicity in crustaceans. The study highlights the necessity for continuous monitoring of potentially toxic metals in edible marine species to avoid possible repercussions of seafood consumption on human health.

Mediterranean Sea is the sixth largest area of marine litter accumulation in the world, and plastic pollution is a growing problem in its Adriatic sub-basin. The aim of the present study was to evaluate the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers in the south-eastern Adriatic Sea in comparison with microbiota in seawater and sediment. Plastic litter in the sea contains an autochthonous microbiota that is different from that of the surrounding seawater and sediment. Vibrio abundance was higher on plastic litter than in surrounding seawater and sediment. All isolated Vibrio showing resistance to ampicillin and vancomycin, while resistance to other antibiotics depended on the isolated species. Overall, this study provides for the first time information on the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers and provides a data base for further studies.

Vibrio species are widely distributed and can be potentially pathogenic to aquatic organisms. In this study, we isolated Vibrio spp. from environmental samples (seawater, sediment, and fish swabs) collected over a three-year period from a fish farm in Mali Ston Bay in the Adriatic Sea, Croatia, and assess their distribution. A total of 48 seawater samples and 12 sediment samples, as well as gill and skin swabs from 110 farmed European seabass, were analysed for the presence of Vibrio. Vibrio strains were identified to the species level by MALDI TOF MS. The analysis revealed that V. alginolyticus was the predominant species in European seabass, followed by V. anguillarum. V. alginolyticus was isolated from the sediments, along with V. gigantis and V. pomeroyi, while V. chagasii, V. cyclitrophicus, V. fortis, V. gigantis, V. harveyi, V. pelagius, and V. pomeroyi were isolated from seawater. V. anguillarum was isolated only twice during two different spring seasons, once from a diseased sea bass and the second time from a healthy sea bass. We analysed these two isolates and found that they differ both genetically and in terms of resistance to antibiotics. Our results confirm the seasonality of vibriosis incidence and the presence of the pathogenic V. anguillarum, which increases the risk of vibriosis.

This study examined culturable skin microbiota that was associated with farmed European seabass (Dicentrarchus labrax). Healthy European seabass were sampled during summer commercial harvest from one conventional fish farm where antibiotics are used, and from another practicing a certified antibiotic-free fish aquaculture. Physicochemical and microbiological analysis of seawater and sediment were performed, as well as determination of culturable bacteria, including Vibrio, from skin swabs of European seabass and seawater and sediment at both farms. Samples were processed for isolation of bacteria and their characterization by molecular and antibiotic susceptibility tests. In both fish farms, most of the bacteria that were identified in the skin belonged to the genera Pseudomonas and Vibrio. Some of the microbiota that were identified are known to be pathogenic to fish: V.alginolyticus, V. anguillarum, and V. harveyi. Vibrio strains showed higher resistance to certain antibiotics compared to previous studies. This study provides, for the first time, information on the culturable skin bacteria that is associated with healthy European seabass under culture conditions with and without the use of antibiotics. This information will be useful in assessing how changes in culturable microbiota may affect the health of farmed European seabass, indicating a potential problem for fish health management during disease outbreaks.

Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

This research presents a comprehensive study of bacterial assemblages within the water column and in the surface sediments in the zone of two European sea bass cage farms. By the application of the high-throughput amplicon sequencing of 16S rRNA gene, and further implementing microbial ecology tools, a bacterial segment from cage culturing systems and their respective controls were analyzed, with special reference to potential impact on animal, human and environmental health. Samples of seawater and sediments were collected seasonally, at locations situated in the central and southern Adriatic Sea. Bacterial composition was significantly different in the seawater vs. sediment. No significant differences in alpha diversity in sediments were indicated between aquaculture and control sites, and it appears that it is not affected by farming practices. Control sediments have higher relative abundance of aerobic and facultative anaerobic bacteria, while aquaculture sediments are markedly anaerobic. Sediments largely contain functional groups for respiration of sulfate and sulfur compounds, though doubly more in aquaculture sites. Seasonal groupings of bacterial assemblages were confirmed in the seawater, with higher relative abundance of known aquaculture pathogens (except Photobacterium in the winter samples) detected in the winter and summer, opposed to other two seasons. Rare taxa were analyzed in the sediment and in the water column in the search for known fish pathogens, with five genera detected: Vibrio, Pseudomonas, Photobacterium, Tenacibaculum and Mycobacterium. Biomarkers important for the impact of aquaculture on the environment were identified, e.g. Blastopirullela, Sva0081, Suflurovum, Spirochaeta 2, etc., as well as human and fish potential pathogens: Vibrio ichtyoentery, V. harvey, Acinetobacter lwoffi, A. johnsonii, Clostridium perfringens, etc. Chemoheterotrophy has emerged as the dominant functional group in both environments. Regarding priorities for aquaculture microbial management, seawater seems to contain a higher percentage of taxa connected to health-related functional groups.

Karst rivers are of great interest for commercial fishing and aquaculture, yet they are quite vulnerable aquatic environments because the permeable karst rocks do not effectively filter out contaminants. To understand the current state of karst rivers water quality, we analysed the physico-chemical and microbiological parameters, focusing on antibiotic pollution and the emergence of antibiotic-resistant microbes of three such rivers in Croatia. Water quality varied between classes I and II across sampling sites, and the numbers of total coliforms, enterococci and heterotrophic bacteria varied substantially among sites. Swabs from fish gills, spleen, liver and kidneys were cultured and 94 isolates identified by MALDI-TOF mass spectrometry. The predominant genus was Aeromonas (42.5% of all identified isolates), known for its adaptability to polluted environments and its frequent association with antibiotic resistance. Of the selected Aeromonas isolates known as most pathogenic, half were resistant to at least three antibiotic categories. The Enterobacteriaceae family was represented by the greatest number of genera, most of which are pathogenic for humans and animals and are spoilage bacteria for fish. The results of this study highlight the extent of antibiotic contamination in aquatic environments and the increasing threat of pathogenic and spoilage bacteria in traditionally high-quality karst rivers.

Metals and organophosphorous pesticides commonly co-occur in marine environment, but the effect of their mixtures on non-target organisms is still poorly understood. This study investigated the combined effect of the essential metal copper (Cu) and organophosphorous pesticide chlorpyrifos (Chp) in mussels (Mytilus galloprovincialis) after short-term exposure to their sublethal concentrations. Mussels were exposed for four days to 5 and 15 μg L-1 Cu and 0.05 and 5 μg L-1 Chp, and to their binary mixtures. The investigated biomarkers, namely acetylcholinesterase activity (AChE), glutathione S-transferase activity (GST), metallothioneins content (MTs) and lipid peroxide levels (LPO) displayed unspecific and inconsistent response patterns that varied depending on the concentration of chemicals and composition of mixtures. The exposure to Cu or Chp alone did not induce AChE activity changes, whereas only Cu provoked a significant GST activity increase. Exposure to lower and higher concentration of Chp resulted in MTs content and LPO level increase, respectively. Response of biomarkers to mixtures was generally inconsistent. Data integration by IBR index and PCA revealed different stress levels for given exposure conditions, but no explicit differentiation between single and joint exposures was found. The present results showed that low and environmentally relevant concentrations of Cu and Chp in mixtures may result in a detectable biological response, stressing the need for further investigation of joint effects of widespread marine contaminants in sentinel organisms.

Semi-enclosed bays are generally considered important environmental resources for mariculture. This study was conducted within cage fish farms in two semi-enclosed bays of the Eastern Adriatic Sea, at Boka Kotorska Bay (Montenegro), and at Mali Ston Bay (Croatia). A total of 16 sea water samples, and 46 swabs from the gills and skin of European seabass were collected from two sea bass farms during two samplings in autumn and spring. The aim of this study was to determine the health status of European seabass in Montenegro and Croatia, to evaluate the environmental conditions in both farming areas, and to assess the presence of Vibrio and its impact on fish health. Most of the isolated bacteria from the samples were Gram-negative and comprised of Vibrio, Photobacterium and Pseudomonas genera. Assessment of fish health status showed that fish were clinically healthy and results present a bacterial community associated with healthy farmed European seabass. At the same time, some of the isolated bacterial strains are known to be pathogenic (V. alginolyticus, V. harveyi, V. anguillarum, P. damselae), and present a potential reservoir of infection. Simultaneous survey confirmed microbial impact in both bays due to an anthropogenic influence. The approach presented in this study is valuable in the assessment of farming conditions in semi-enclosed aquatic environments and is easily applicable to other similar locations worldwide.

Marine pollution favours the invasion and spreading of non-indigenous species in their new habitats. Comparative advantages of introduced and potentially invasive bivalves over their native counterparts might be reflected in better ability to cope with chemical stressors. The objective of the present study was to investigate differences in the biochemical stress response to Cd and Cu between the flat oyster Ostrea edulis (Linnaeus, 1758), native to European coasts, and its non-indigenous congener, the Pacific oyster Crassostrea gigas (Thunberg, 1793), recently detected in the eastern Adriatic. Oysters were co-exposed in vivo to sub lethal concentrations of metals. The stress response of two oyster species was evaluated using biomarkers of neurotoxicity (acetylcholinesterase, AChE), detoxification (metallothioneins, MTs) and oxidative stress (glutathione S-transferase, GST; lipid peroxidation, LPO). Biomarkers related to oxidative stress response were the most informative and suggested comparably lower capability of the non-indigenous C. gigas for handling pro-oxidant conditions after Cu exposure. Overall, the species-specific biomarker alterations displayed by oysters exposed simultaneously to the same experimental conditions represent the first evidence of differences between these two bivalves in the ability to overcome the chemically induced stress. The present research highlights the need for monitoring of biochemical features that might determine the behaviour of oysters in newly colonised habitats under environmental challenges foreseen in the upcoming years.