Search

Recognizing multiple anthropogenic pressures in marine coastal zones, countries of the Mediterranean region have implemented several directives focused on sustainable management of the Mediterranean Sea coastal zones. One of the essential directives is the Marine Strategy Framework Directive (MSFD), challenging countries, including Croatia, to achieve and maintain Good Environmental Status of their marine environment. The health of the marine environment is undeniably influenced by microorganisms which play a key role in functioning of marine food webs and biogeochemical cycling. The MSFD neglects the importance of microbial communities and possible changes in their function and structure under the anthropogenic influence influencing stability of the whole marine ecosystem. Project MicroLink, funded by the Croatian Science Foundation, aims to highlight the significance of microbial communities in monitoring and consequently preserving the quality of marine environment with the final goal to propose incorporation of the selected microbial attributes among MSFD Quality Descriptors. Sampling of 67 sediments was performed in seven harbors along the eastern Adriatic coast, which were previously identified as pollution “hot spots”. Sediments were comprehensively analyzed and parameters such as metal concentrations, Hg, TP, TN, TC, TBT, toxicity testing etc. were measured. To find the correlation between anthropogenic pollution and changes in the microbial communities, the project MicroLink integrates different approaches: i) multi-domain approach (analyzing changes in Bacteria, Archaea, Protists, and Fungi) and ii) multi-layer approach (changes in structure, microbial networks, abundance of functional genes and biodegradation potential). Due to continuous accumulation of pollutants, sediments and benthic microbial communities could be reliable indicators of the persisting exposure of the marine environment to anthropogenic pressures. Microbial communities should not be neglected in future strategies and measures for conservation of coastal and marine environments.

The aim of the study was to explore the relationship between membrane-regulating functional lipids of three fucacean species and their associated epiphytic bacterial communities. The analyzed algae Treptacantha barbata, Carpodesmia crinita and Cystoseira compressa, formerly classified under the Cystoseira genus, are indigenous to the Adriatic Sea. The thalli of sampled Fucales species were divided into perennial and annual parts. T. barbata and C. crinita show high contents of mono- and polyunsaturated fatty acids but, however, show a clear distinction between branches, cauloids and apical parts. Along with the highest unsaturation degree (4.32), the C. crinita branches and apical parts demonstrated high proportion of both C18 and C20 polyunsaturated fatty acids, with eicosapentaenoic (C20:5 n-3) and arachidonic (C20:4 n-6) fatty acids up to 5.46% and 13.82%, respectively. The highest proportion of saturated fatty acids was found in thalli of C. compressa, particularly on cauloids and old branches (&ge, 90%). The similarity profile routine analysis of fatty acids and microbial community structure has shown clear separation of the cauloids and apical parts from the branches of C. crinita and T. barbata. Based on the nonmetric multidimensional scaling analysis several representatives of the Gammaproteobacteria class, identified as Vibrionales (Vibrio), Cellvibrionales and Xanthomonadales, which contributed strongly to the separation of T. barbata apical parts and branches, C. compressa receptacles and C. crinita branches from the T. barbata cauloids and C. crinita apical parts and cauloids. The highest richness in polyunsaturated fatty acids, in particular C18:3(n-3), C20:4(n-6) and C20:5(n-3), was observed on branches, accompanied by a distinctive epiphytic microbial structure dominated by numerous representatives with potentially beneficial biological activity. The results showed a strong relationship between fatty acid profiles of the analyzed species and phycosphere community structure, underlining the host physiological condition in shaping the biological interactions and maintaining a healthy microbiome, as well as compiling the ecophysiological and molecular research in order to better assess the resilience of the ecosystem.

Harmonization of monitoring protocols and analytical methods is a crucial issue for transnational marine environmental status assessment, yet not the only one. Coherent data management and quality control become very relevant when environmental status is assessed at regional or subregional scale (e.g., for the Mediterranean or the Adriatic Sea), thus requiring data from different sources. Heavy metals are among the main targets of monitoring activities. Significant efforts have been dedicated to share best practices for monitoring and assessment of ecosystem status and to strengthen the network of national, regional and European large data infrastructures in order to facilitate the access to data among countries. Data comparability and interoperability depend not only on sampling and analytical protocols but also on how data and metadata are managed, quality controlled and made accessible. Interoperability is guaranteed by using common metadata and data formats, and standard vocabularies to assure homogeneous syntax and semantics. Data management of contaminants is complex and challenging due to the high number of information required on sampling and analytical procedures, high heterogeneity in matrix characteristics, but also to the large and increasing number of pollutants. Procedures for quality control on heterogeneous datasets provided by multiple sources are not yet uniform and consolidated. Additional knowledge and reliable long time-series of data are needed to evaluate typical ranges of contaminant concentration. The analysis of a coherent and harmonized regional dataset can provide the basis for a multi-step quality control procedure, which can be further improved as knowledge increases during data validation process.

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, Ploče) 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.

Environmental contamination includes a mixture of organic substances that can have detrimental effects on marine organisms and should be evaluated in the quality and risk assessment of investigated marine areas. Marine areas selected for this study are a protected area, a mariculture area, a shipyard and an industrial area. Based on the toxicity of the organic seawater extracts these areas were classified as an undisturbed reference area (S1), an area with the low anthropogenic impact (S2), a potentially endangered area (S3) and an area with high anthropogenic impact (S4) respectively. The organic mixtures present in seawater samples collected at the above-defined areas were tested for the induction of DNA damage and cell cycle alterations in the mussel Mytilus galloprovincialis hemocytes. Flow cytometric analyses were performed to detect changes in hemocytes DNA content distribution throughout the cell cycle. Organic seawater extracts from sampling sites S2, S3 and S4 induced an increase in the coefficient of variation of the G0/G1 peak and an increase in the number of cells in the G2/M phase reflecting the extent of DNA damage and (G_2/M) arrest, respectively. The (G_2/M) arrest in mussel hemocytes was concentration-dependent upon injection with organic seawater extracts from the S3 site and time dependant for S2, S3 and S4 sampling sites. The time dependence of the induction of the G2/M arrest showed a characteristic pattern for each site due to the different quantitative and qualitative composition of the organic seawater extracts. The (G_2/M) arrest was reversible 24 or 72 hours after treatment with organic seawater extracts from S2 or S3, and S4 sites, respectively. This reversibility was time- and site-specific indicating that such DNA damage is repairable to a certain degree according to the organic seawater extract composition. Thus, the hemocytes cell cycle alterations in the mussel Mytilus galloprovincialis caused by organic seawater extracts reliably reflect the extent of organic contamination effects for selected marine areas., Onečišćenje okoliša čini mješavina organskih tvari s mogućim učincima na morske organizme koje je potrebno procijeniti u sklopu istraživanja vezanih za utvrđivanje kvalitete okolišnog rizika za određeno morsko područje. Morska područja odabrana za svrhu ove studije uključuju zaštićeno morsko područje, područje marikulture, brodogradilište i industrijsko područje. Na temelju profila toksičnosti organskih ekstrakata uzoraka morske vode, navedena su područja razvrstana na nedirnuto morsko područje (S1), područje niskog antropogenog utjecaja (S2), potencijalno ugroženo područje (S3), odnosno područje visokog antropogenog utjecaja (S4). Mješavine organskih tvari prisutne u uzorcima morske vode prikupljenim na utvrđenim područjima testirane su na sposobnost indukcije oštećenja DNA te učinak na stanični ciklus hemocita dagnje Mytilus galloprovincialis. Kako bi se utvrdile promjene u distribuciji sadržaja DNA tijekom staničnog ciklusa hemocite su analizirane protočnom citometrijom. Organski ekstrakti morske vode sa S2, S3 i S4 inducirali su povećanje koeficijenta varijacije (G_2/M) maksimuma kao i porast u broju stanica u (G_2/M) fazi, odražavajući zaustavljanje ciklusa tj. “(G_2/M) arrest” uslijed oštećenja DNA. Povećanje u količini oštećenja DNA bilo je popraćeno porastom broja hemocita u (G_2/M) fazi. Zaustavljanje staničnog ciklusa u (G_2/M) fazi u hemocitima dagnje pokazalo se ovisnim o primijenjenoj dozi organskog ekstrakta morske vode sa mjesta uzorkovanja S3, a ovisnim o vremenu djelovanja ekstakta morske vode sa mjesta S2, S3 i S4. Utvrđena ovisnost o trajanju induciranog zaustavljanje ciklusa u (G_2/M) fazi pokazala se karakterističnom za pojedina mjesta uzorkovanja što je posljedica različitih kvantitativnih i kvalitativnih karakteristika samog sastava pojedinih organskih ekstrakata morske vode. Ponovno uspostavljanje zaustavljenog ciklusa u (G_2/M) fazi utvrđen je 24 sata nakon primjene organskih ekstrakata morske vode s mjesta S2, odnosno 72 sata nakon primjene organskih ekstrakata morske sa mjesta S3 i S4. Ustanovljen oporavak staničnog ciklusa nakon zaustavljanja pokazao se specifično vezanim uz vrijeme djelovanja i mjesto uzorkovanja, što upućuje na to da je popravak oštećenja DNA moguć do određene razine, ovisno o samome sastavu pojedinih organskih ekstrakata morske vode. Stoga se zaključuje da promjene u staničnom ciklusu hemocita dagnje Mytilus galloprovincialis uzrokovane djelovanjem pojedinih organskih ekstrakata vjerno odražavaju razinu štetnog djelovanja organskih zagađivala u odabranim morskim područjima.

The aim of this study was to compare the capability of "passive" and "active" biomonitoring to determine the environmental pressure. For this purpose, PAHs content and several biological responses in resident and caged mussels (Mytilus galloprovincialis) at five sampling sites (Rijeka Bay, Adriatic Sea) were analsed. Resident mussels were found better in reflecting the level of PAH loads at particular sites while only caged mussels could detect input of HMW PAHs. When data of each investigated parameter were compared separately, the majority of differences between resident and caged mussels' results were site-specific. Integration of biological response patterns expressed as Index of Biological Response (IBR) resulted with different sampling sites ranking for resident and caged mussels. Multiple Factor Analysis (MFA) based on integration of tissue PAH concentration and biological response revealed resident mussels as more powerful for detection of environmental pressure. The use of resident mussels is recommended as appropriate and less costly approach for monitoring the effect of pollution.

The presence and activity of deoxyribonucleases in the cortex and endosome sections from a sponge, the sea orange Tethya aurantium, were investigated. The maximal enzyme activity in sponge homogenate was detected at pH 4.27, pH 7.0 and pH 8.5–8.75. Among different specimens, several distinct patterns of neutral DNase isozymes were observed in the cortex section. In each investigated specimen the highest neutral DNase activity belonged to high molecular weight proteins (up to75 kDa). The acid DNases showed a low level of enzyme activity. In the endosome section the acid DNase activity was up to ten times higher than in the cortex and the presence of DNase II-like protein was detected. Neutral DNase, which expressed the highest enzyme activity in all the investigated specimens, has a molecular weight of 20 kDa and belongs to the DNase I-like family. The results indicate that the activity of neutral and acid DNases is related to sponge sections and their biological functions. The cortex, as the sponge section that communicates with the environment, expresses high interindividual variability and heterogeneity of neutral DNases, while the endosome section, where the intracellular digestion is localized, is a site of high acid DNase activity.

The hemocytes/coelomocytes DNA content in five selected marine invertebrates (sea mouse Aphrodita aculeata, spiny crab Maja crispata, sea star Echinaster sepositus, sea urchin Paracentrotus lividus, and tunicate Phallusia mammillata) was investigated by flow cytometry. The cell cycle analyses identified sea mouse coelomocytes as proliferating cells and revealed that spiny crab hemocytes and sea urchin coelomocytes complete their division in the hemolymph and coelom, respectively. The genome sizes of sea mouse and spiny crab are reported for the first time. The diploid DNA content (2C) in sea mouse A. aculeate was 1.24 pg, spiny crab M. crispata 7.76 pg, red starfish E. sepositus 1.52 pg and sea urchin P. lividus 1.08 pg. The mean diploid DNA content in tunicate P. mammillata was 0.11 pg with a high interindividual variability (45%). The presented results provide a useful database for future studies in the field of invertebrate physiology, ecotoxicology, biodiversity, species conservation and phylogeny.

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, mantlegillsadductor. 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.

Cytochrome P450 enzymes (CYPs) are essential components of cellular detoxification system. We identified and characterized seven new cytochrome P450 gene transcript clusters in the populations of bivalve mollusc Mytilus galloprovincialis from three different locations. The phylogenetic analysis identified all transcripts as clusters within the CYP4 branch. Identified clusters, each comprising a number of transcript variants, were designated CYP4Y1, Y2, Y3, Y4, Y5, Y6 and Y7. Transcript clusters CYP4Y2 and Y7, and CYP4Y5 and Y6 showed site specificity, while the transcript clusters CYP4Y1, Y3 and Y4 were present at all investigated locations. The comparison of transcripts deduced amino acid sequences with CYP4s from vertebrate and invertebrate species showed high conservation of the residues and domains essential to the putative function of the enzyme, as terminal ω-hydroxylation and prostaglandin hydroxylation. Our results suggest the great expansion of the CYP4Y cDNAs indicative of CYP4 proteins in the mussel M. galloprovincialis presumably as a response to different environmental conditions.

In view of the importance of Mytilus species in marine ecology and environmental quality assessment, all aspects of mussel enzymes that are related to environmental factors are of great relevance. A lysosomal enzyme, deoxyribonuclease II, a member of the acid deoxyribonucleases, has a major role in cellular DNA degradation. The presence and seasonal variations of the acid deoxyribonuclease (DNase) activity in the digestive gland and gills of the mussel Mytilus galloprovincialis were investigated. The enzyme was localized in the lysosomal vacuolar system of both gill and digestive gland tissues. Two active acid DNase isoforms were detected in the digestive gland (48 and 37 kDa) and one in the gills (48 kDa). All active enzyme isoforms coincided with protein bands immunoreactive to antibodies raised against human DNase II. Post-translational lysosomal processing of acid DNase in the digestive gland but not in the gills indicated tissue-specific enzyme maturation. The activity of acid DNase determined across the annual cycle showed a tissue-specific pattern that could be related to the seasonal activity of mussel organs and their biological function. In the digestive gland, high DNase activity was observed in relation to food availability and spawning. Higher enzyme activity in the gills observed in the autumn/winter period could be associated with feeding and cellular adaptation to low seawater temperatures.

Marine sponges as sessile filter feeders are inevitably under a constant influence of changes in their environment. Mediation of extracellular signals and regulation of cellular response to environmental stress is a key function of cellular protein kinases. Expression, proteolytical cleavage and phosphorylation of stress-responsive KRS_SD protein kinase, in control and tributyl-tin (TBT) treated sponges were investigated. In control sponge, two KRS_SD proteins were expressed: KRS_SD1 (54 kDa) corresponding to KRS_SD calculated molecular weight, and KRS_SD2 (50 kDa). Exposure of sponges to TBT resulted in alteration of KRS_SD1 and KRS_SD2 expression levels and their phosphorylation state. KRS_SD1 band disappearance after 4 h coincides with the appearance of additional 35 kDa immunoreactive protein band (p35) and decrease in number of viable cells. Incubation of recombinant KRS_SD with protein extracts from TBT stressed sponges resulted in cleavage of recombinant KRS_SD protein. The size of the resulting protein fragment suggests caspase-3 recognition site, present in both recombinant KRS_SD and KRS_SD, as a possible site of cleavage. Phosphorylated KRS_SD bands appear at the same time as p35 and remain after p35 band intensity reached control level. KRS_SD induction, its phosphorylation and proteolytical cleavage during TBT stress suggest that in sponge cells exist mechanism similar to one present in human cells where KRS/MST protein kinase is involved in promotion of apoptosis following oxidative stress.

The pattern of spatial distribution of polycyclic aromatic hydrocarbons (PAHs) in seawater, sediment, and mussels, potential toxicity of different matrices, and mussel anoxic survival from six sampling sites of the Gulf of Rijeka, the Adriatic Sea, Croatia was examined. The total concentrations of 10 PAHs vary from below detection limit to 305 ng/L in seawater, from 213 to 695 microg/kg dry weight in sediment and from 49.2 to 134 ng/g wet weight in mussel tissue. Combustion is the principal source of PAH contamination in seawater and sediment samples. Sediment samples are distinguished from the majority of seawater and mussel samples by the presence of high molecular weight PAHs, whereas mussels from majority of sampling sites tend to accumulate PAHs of lower molecular weight. The PAH dynamic between different matrices is complex and site specific. Toxicity of seawater and sediment organic extract is correlated with PAH content, indicating that PAHs are the predominant toxic compounds. There is no correlation between toxicity of mussel biological fluids and toxicity of seawater and sediment, or between toxicity of mussel biological fluid and PAH content in mussel, seawater, or sediment. There is a positive correlation between potential toxicity of mussel biological fluids and reduction of anoxic survival time. Mussel anoxic survival is influenced by the presence of complex mixture of toxic contaminants, not only PAHs. The relationship between PAH contents in different marine matrices and their ability to affect mussels revealed specific interactions between an organism and complex mixture of toxic contaminants present in the marine environment.

The effect of artificial and natural UV irradiation on DNA in marine phytoplankton Isochrysis galbana monoculture was investigated. The presence of cyclobutane pyrimidine dimers (CPDs) in unlabelled I. galbana DNA was detected by a non-radiometric alkaline filter elution assay after T4-endonuclease V digestion. The quantity of CPDs was estimated by alkaline agarose gel electrophoresis. Precise determination of the amount of DNA in the presence of I. galbana pigments was achieved by oxazole yellow homodimer (YOYO) dye. T4-endonuclease V-sensitive sites frequency (ESS/kb), measured after exposure to 2-40 kJ m(-2) of artificial UV light, increased in a dose-dependent manner. Twelve hours after irradiation cell culture growth was disrupted, and 50% of initial DNA damage in the cells was observed. After 1 h of sunlight exposure, the incidence of CPDs increase significantly. Prolonged exposition to sunlight decrease CPDs incidence due to efficiency of I. galbana DNA repair mechanisms. The presence of water-soluble crude oil fraction (WSOF) affected DNA repair efficiency resulting in accumulation of CPDs in I. galbana DNA.