Performance of mussel Mytilus galloprovincialis under variable environmental conditions and anthropogenic pressure: A survey of two distinct farming sites in the Adriatic Sea.

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.

Acetylcholinesterase activity in muscle tissue of Norway lobster Nephrops norvegicus: Importance of body size, season, sex and naturally occurring metals.

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.

A preliminary study of the cultivable microbiota on the plastic litter collected by commercial fishing trawlers in the south-eastern Adriatic Sea, with emphasis on Vibrio isolates and their antibiotic resistance.

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.

Characterization of Vibrio Populations from Cultured European Seabass and the Surrounding Marine Environment with Emphasis on V. anguillarum.

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.

Stem cells of aquatic invertebrates as an advanced tool for assessing ecotoxicological impacts.

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.

Microbiological Quality Assessment of Water and Fish from Karst Rivers of the Southeast Black Sea Basin (Croatia), and Antimicrobial Susceptibility of Aeromonas Isolates.

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.

The effect of copper and chlorpyrifos co-exposure on biomarkers in the marine mussel Mytilus galloprovincialis.

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.

A baseline study of the metallothioneins content in digestive gland of the Norway lobster Nephrops norvegicus from Northern Adriatic Sea: Body size, season, gender and metal specific variability.

Metallothioneins content was investigated in digestive gland of two wild-caught Norway lobster Nephrops norvegicus populations from the Northern Adriatic Sea, in relation to body size, season and gender. Concomitant accumulation of cadmium, mercury, arsenic, lead, chromium and manganese, reactive oxygen species concentration and energy reserves in digestive gland were also assessed. While differences between genders were not recorded, metallothioneins content seasonal trends were affected by body size. Most of parameters displayed inconsistent trends across sampling sites. Significant correlation between metallothioneins content and cadmium, arsenic and mercury concentrations was recorded only for larger lobsters. A negative correlation of reactive oxygen species concentration and metallothioneins content was observed for small, but not large lobsters. Energy reserves, in particular lipids, could considerably influence biochemical and chemical parameters variations. The present results constitute the essential baseline for future studies aimed at evaluating the N. norvegicus health in relation to metal contamination of coastal sediments.

Effect of Environmental Pollutant Mixtures on Acid DNase Activity in Mussel Mytilus galloprovincialis: Ex Situ and In Situ Study.

Acid DNase activity in marine organisms displays a sensitive response to pollutants. In the present study, ex situ effect of toxic seawater organic extracts (Rijeka, Vranjic, Ploce) and in situ impact of environmental factors at referent (St. Andrew) and a polluted site (ACI Rovinj) on acid DNase in the digestive gland and gills of mussels was investigated. In the digestive gland, enzyme activity was related to potential toxicity of seawater organic extracts. Seawater organic extracts showed an increase of the enzyme activity in digestive gland following exposure. In in situ study, polluted environment affected mussel's acid DNase activity in digestive gland, when compared to gills. Acid DNase activity in the digestive gland expressed significant difference between the referent and polluted site, particularly from early spring through summer. Both ex situ and in situ studies displayed more sensitive acid DNase response to the presence of toxic mixtures in the digestive gland.

Variations of biomarkers response in mussels Mytilus galloprovincialis to low, moderate and high concentrations of organic chemicals and metals.

The changes of acetylcholinesterase activity (AChE), metallothioneins content (MTs), catalase activity (CAT) and lipid peroxidation (LPO) were assessed after 4 days exposure of mussels Mytilus galloprovincialis to a wide range of sublethal concentrations of chlorpyrifos (CHP, 0.03-100 µg/L), benzo(a)pyrene (B(a)P, 0.01-100 µg/L), cadmium (Cd, 0.2-200 µg/L) and copper (Cu, 0.2-100 µg/L). The activity of AChE in the gills decreased after exposure to CHP and Cu, whereas no change of activity was detected after exposure to B(a)P and Cd. Both induction and decrease of MTs content in digestive gland occurred after exposure to CHP and B(a)P, while a marked increase was evident at highest exposure concentrations of Cd. The content of MTs progressively decreased of MTs with increasing concentration of Cu. CAT activity and LPO in the gills did not change after exposure to any of the chemicals. The results demonstrate different response profile in relation to the type of chemical compound, and highlight the potential implications for evaluation of biological effect of contaminants in marine environment. Furthermore, the AChE activity in the gills and MTs content in the digestive gland could be modulated by CHP and Cu at environmentally relevant concentrations indicating the potential risks of short-term transient mussels exposure that may occur due to run-off from land or accidental releases.

Biochemical and proteomic characterisation of haemolymph serum reveals the origin of the alkali-labile phosphate (ALP) in mussel (Mytilus galloprovincialis).

Mollusc haemolymph proteins are known to play several important physiological roles in the immune system, heavy metal transport and the tissue distribution of lipophilic compounds. In this study, we analysed acetone-extracted proteins from mussel haemolymph by one- and two-dimensional gel electrophoresis. The proteins were identified by comparing mass spectrometry data with the invertebrate EST database, allowing us to establish the mussel haemolymph serum proteome. Extrapallial protein (EP) precursor represents the most abundant serum protein; astacin and CuZn superoxide dismutase were also detected. Slight contamination from muscle proteins, due to the sampling method, was also found. No differences were observed in the profiles obtained for male and female serum proteins. One aspect of interest was the previously reported finding that alkali-labile phosphate (ALP) from haemolymph serum may be representative of vitellogenin (vtg)-like protein content in the circulatory fluid of molluscs. In our analysis of mussel haemolymph serum, vitellogenin-like proteins were never found. To confirm these data, a typical methyl-tert-butyl-ether (MTBE) extraction, which is specific for vtg-like proteins, was performed, and the results of the electrophoretic analyses were compared with those obtained by acetonic precipitation. The results showed that the electrophoretic profiles are similar and that vtg-like proteins cannot be identified. Moreover, the main phosphoprotein present in female and male extracts is EP protein precursor. In addition, agarose gel electrophoresis demonstrates that high-molecular-weight forms of vtg-like proteins are not detectable.

Cholinesterase activity in the tissues of bivalves Noah's ark shell (Arca noae) and warty venus (Venus verrucosa): characterisation and in vitro sensitivity to organophosphorous pesticide trichlorfon.

Cholinesterase (ChE, EC 3.1.1.7) activity was investigated in gills and adductor muscle of two bivalve species: Arca noae and Venus verrucosa. The properties of ChEs were investigated using acetylcholine iodide (ASCh), butyrylcholine iodide (BSCh) and propionylcholine iodide (PrSCh) as substrates and eserine, BW254c51 and iso-OMPA as specific inhibitors. The highest level of ChE activity in crude tissue extracts was detected with PrSCh followed by ASCh, while values obtained with BSCh were apparently low, except in A. noae adductor muscle. The enzyme activity in A. noae gills and V. verrucosa gills and adductor muscle was significantly inhibited by BW254c51, but not with iso-OMPA. ChE activity in adductor muscle of A. noae was significantly reduced by both diagnostic inhibitors. The effect of organophosphorous pesticide trichlorfon on ChE activity was investigated in vitro in both species as well as in the gills of mussels Mytilus galloprovincialis. The highest sensitivity of ChE to trichlorfon was observed in A. noae gills and adductor muscle (IC50 1.6×10(-7)M and 1.1×10(-7)M, respectively), followed by M. galloprovincialis gills (IC50 1.0×10(-6)M) and V. verrucosa gills and adductor muscle (IC50 1.7×10(-5)M and 0.9×10(-5)M, respectively). The results of this study suggest the potential of ChE activity measurement in the tissues of A. noae as effective biomarker of OP exposure in marine environment.

Effect of hypoosmotic stress by low salinity acclimation of Mediterranean mussels Mytilus galloprovincialis on biological parameters used for pollution assessment.

In the present study, we investigated the progressive acclimation of the mussel Mytilus galloprovincialis to different reduced seawater (SW) salinities and its effect on several biochemical markers and biotests. Mussels were purchased from a local mariculture facility during summer (SW temperature 27 degrees C, salinity 37.5 psu) and winter (13 degrees C, 37 psu) seasons, and transferred to the laboratory for acclimation to reduced SW salinities (37, 28, 18.5 and 11 psu). At the beginning and at the end of acclimation processes tests of mussel survival in air were provided. After 14 days of acclimation the DNA integrity, p38-MAPK activation, metallothionein induction, oxygen consumption rate, and condition index were measured. Survival in air (SOS test), as a physiological index of mussel's health and vitality, had significantly lower LT50 values (11 psu) in the summer than in the winter, and it seems to be negatively affected by acclimation in comparison to controls (37 psu and mariculture). Condition indexes (CIs) were not significantly different, but mussel's acclimation resulted in decline (i.e., a negative trend), especially of CI-2 and CI-3 calculated on the basis of mussel tissue weight and shell sizes. Oxygen consumption rate (VO2) of M. galloprovincialis acclimated to reduced salinities was a concentration-dependent process and increased considerably to about 51 and 65% in lower SW concentrations (28 and 18 psu) compared to control mussels (37 psu). DNA integrity, determined by Fast Micromethod, was negatively impacted by salinity acclimation and corresponding physiological stress as well. Some differences in 1D protein expression patterns between control groups and mussels acclimated to 28, 18.5 and 11 psu (SW) were established. Reduced SW salinities (18.5 and 11 psu) resulted in significantly higher p38-MAPK phosphorylation, whereas the SW salinity of 28 psu decreased p-p38 significantly compared to control (37 psu). The concentration of metallothioneins in mussels' gills was reduced at 28 and 18.5 psu, while it was significantly higher at 11 psu. Results indicated that SW salinity variation (i.e., hypoosmotic stress) in the marine environment can affect all investigated parameters. This investigation expands our understanding of multifactorial effects of the physical marine environment on the specificity of investigated biomarkers and biotests, providing insight into the acclimation, adaptive and stress response processes of mussels. Effects of environmental factors have to be considered in sampling strategies for monitoring programmes to prevent false interpretation of results.

Effect of marine pollutants on the acid DNase activity in the hemocytes and digestive gland of the mussel Mytilus galloprovincialis.

The level of the acid DNase activity in the hemocytes and digestive gland of the mussel Mytilus galloprovincialis after exposure to model marine pollutants, a detergent, gasoline and a copper salt, as well as to unknown environmental mixture at selected sampling sites, was investigated. The specific enzyme activity in unexposed mussels from mariculture area was higher in hemocytes than in digestive gland. Concentration and time effect patterns of DNase activity revealed tissue- and pollutant-specific responses to model marine pollutants. Since in some cases the pollutant effect could not be detected by measurement of acid DNase in single tissue only, digestive gland/hemocyte (Hep/Hem) ratio was introduced. The Hep/Hem ratio enabled the detection of pollutant effect at the significance level. Field investigations indicated that the digestive gland is a suitable tissue for discrimination of polluted areas from maricultured area. Additionally, the Hep/Hem ratio enabled differentiation within a group of polluted sampling sites that differ in the type of pollutants and/or environmental conditions.

Tissue distribution of neutral deoxyribonuclease (DNase) activity in the mussel Mytilus galloprovincialis.

The presence of neutral DNase activity in bivalves is reported for the first time. The enzyme activity in four tissues of the mussel Mytilus galloprovincialis was analyzed by three different methods (i) specific denaturating SDS-PAGE zymogram, (ii) sensitive single radial enzyme diffusion (SRED) assay and (iii) rapid and sensitive fluorimetric determination of DNase activity with PicoGreen. The fluorimetric assay was rapid and sensitive enough for determination of hydrolytic activity of dsDNA in mussel hepatopancreas, adductor, gills and mantle. Maximal activity in all mussel tissue extracts was obtained in the presence of Ca(2+) and Mg(2+) at pH 7.0 with dsDNA as substrate. The neutral DNase activity in mussel tissue decreases in order hepatopancreas, mantle>gills>adductor. The enzyme activity displays interindividual variability in particular tissue as well as variability among tissues within one specimen. In the hepatopancreas one to three distinct proteins expressing neutral, Ca(2+), Mg(2+)-dependent, DNase activity were detected by denaturating SDS-PAGE zymogram. This heterogeneity of neutral nucleases involved in DNA hydrolysis in hepatopancreas could reflect interindividual variability in mussel food utilization and nutrient requirement.