Pan-regional marine benthic cryptobiome biodiversity patterns revealed by metabarcoding Autonomous Reef Monitoring Structures.

Autonomous Reef Monitoring Structures (ARMS) have been applied worldwide to characterize the critical yet frequently overlooked biodiversity patterns of marine benthic organisms. In order to disentangle the relevance of environmental factors in benthic patterns, here, through standardized metabarcoding protocols, we analyse sessile and mobile (<2 mm) organisms collected using ARMS deployed across six regions with different environmental conditions (3 sites × 3 replicates per region): Baltic, Western Mediterranean, Adriatic, Black and Red Seas, and the Bay of Biscay. A total of 27,473 Amplicon Sequence Variants (ASVs) were observed ranging from 1,404 in the Black Sea to 9,958 in the Red Sea. No ASVs were shared among all regions. The highest number of shared ASVs was between the Western Mediterranean and the Adriatic Sea (116) and Bay of Biscay (115). Relatively high numbers of ASVs (103), mostly associated with the genus Amphibalanus, were also shared between the lower salinity seas (Baltic and Black Seas). We found that compositional differences in spatial patterns of rocky-shore benthos are determined slightly more by dispersal limitation than environmental filtering. Dispersal limitation was similar between sessile and mobile groups, while the sessile group had a larger environmental niche breadth than the mobile group. Further, our study can provide a foundation for future evaluations of biodiversity patterns in the cryptobiome, which can contribute up to 70% of the local biodiversity.

Characterization of Rapana thomasiana as an indicator of environmental quality of the Black Sea coast of Bulgaria.

The aim of this investigation was to determine the contents of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), minerals, trace elements and bioactivity in the gastropod Rapana thomasiana, which can be used as an environmental bioindicator organism. The chemical differences between Rapana thomasiana from polluted (RapaPol) and non-polluted (RapaNPol) sites of the Black Sea coast in Bulgarian were investigated. Chromatography and high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) were used for evaluation of PAHs, PCBs, minerals and trace elements. Methanol extracts from RapaPol and RapaNPol (to a lesser degree) contained relatively high amounts of free phenolics (2.50 +/- 0.3 and 1.57 +/- 0.18 mg GAE/g DW, respectively) and exhibited the following respective levels of antioxidant activities determined by two radical-scavenging assays (microMTE/g DW): 1.8 +/- 0.2 and 0.98 +/- 0.08 by 1-diphenyl-2-picrylhydrazyl method (DPPH); 1.74 +/- 0.17 and 1.04 +/- 0.12 by cupric reducing antioxidant capacity (CUPRAC). The total amounts of elements, PAHs and PCBs were higher in RapaPol than in RapaNPol. The obtained indices of Rapana thomasiana can serve as a bioindicator of the environmental ecological quality.

Antibiotic resistance genes correlate with metal resistances and accumulate in the deep water layers of the Black Sea.

Seas and oceans are a global reservoir of antibiotic resistance genes (ARGs). Only a few studies investigated the dynamics of ARGs along the water column of the Black Sea, a unique environment, with a peculiar geology, biology and history of anthropogenic pollution. In this study, we analyzed metagenomic data from two sampling campaigns (2013 and 2019) collected across three different sites in the Western Black Sea at depths ranging from 5 to 2000 m. The data were processed to annotate ARGs, metal resistance genes (MRGs) and integron integrase genes. The ARG abundance was significantly higher in the deep water layers and depth was the main driver of beta-diversity both for ARGs and MRGs. Moreover, ARG and MRG abundances strongly correlated (r = 0.95). The integron integrase gene abundances and composition were not influenced by the water depth and did not correlate with ARGs. The analysis of the obtained MAGs showed that some of them harbored intI gene together with several ARGs and MRGs, suggesting the presence of multidrug resistant bacteria and that MRGs and integrons could be involved in the selection of ARGs. These results demonstrate that the Black Sea is not only an important reservoir of ARGs, but also that they accumulate in the deep water layers where co-selection with MRGs could be assumed as a relevant mechanism of their persistence.

New Knowledge on Distribution and Abundance of Toxic Microalgal Species and Related Toxins in the Northwestern Black Sea.

Numerous potentially toxic plankton species commonly occur in the Black Sea, and phycotoxins have been reported. However, the taxonomy, phycotoxin profiles, and distribution of harmful microalgae in the basin are still understudied. An integrated microscopic (light microscopy) and molecular (18S rRNA gene metabarcoding and qPCR) approach complemented with toxin analysis was applied at 41 stations in the northwestern part of the Black Sea for better taxonomic coverage and toxin profiling in natural populations. The combined dataset included 20 potentially toxic species, some of which (Dinophysis acuminata, Dinophysis acuta, Gonyaulax spinifera, and Karlodinium veneficum) were detected in over 95% of the stations. In parallel, pectenotoxins (PTX-2 as a major toxin) were registered in all samples, and yessotoxins were present at most of the sampling points. PTX-1 and PTX-13, as well as some YTX variants, were recorded for the first time in the basin. A positive correlation was found between the cell abundance of Dinophysis acuta and pectenotoxins, and between Lingulodinium polyedra and Protoceratium reticulatum and yessotoxins. Toxic microalgae and toxin variant abundance and spatial distribution was associated with environmental parameters. Despite the low levels of the identified phycotoxins and their low oral toxicity, chronic toxic exposure could represent an ecosystem and human health hazard.

The microbiome of the Black Sea water column analyzed by shotgun and genome centric metagenomics.

BACKGROUND: The Black Sea is the largest brackish water body in the world, although it is connected to the Mediterranean Sea and presents an upper water layer similar to some regions of the former, albeit with lower salinity and temperature. Despite its well-known hydrology and physicochemical features, this enormous water mass remains poorly studied at the microbial genomics level. RESULTS: We have sampled its different water masses and analyzed the microbiome by shotgun and genome-resolved metagenomics, generating a large number of metagenome-assembled genomes (MAGs) from them. We found various similarities with previously described Black Sea metagenomic datasets, that show remarkable stability in its microbiome. Our datasets are also comparable to other marine anoxic water columns like the Cariaco Basin. The oxic zone resembles to standard marine (e.g. Mediterranean) photic zones, with Cyanobacteria (Synechococcus but a conspicuously absent Prochlorococcus), and photoheterotrophs domination (largely again with marine relatives). The chemocline presents very different characteristics from the oxic surface with many examples of chemolithotrophic metabolism (Thioglobus) and facultatively anaerobic microbes. The euxinic anaerobic zone presents, as expected, features in common with the bottom of meromictic lakes with a massive dominance of sulfate reduction as energy-generating metabolism, a few (but detectable) methanogenesis marker genes, and a large number of "dark matter" streamlined genomes of largely unpredictable ecology. CONCLUSIONS: The Black Sea oxic zone presents many similarities to the global ocean while the redoxcline and euxinic water masses have similarities to other similar aquatic environments of marine (Cariaco Basin or other Black Sea regions) or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very well the different types of metabolisms expected in this kind of environment. We are adding critical information about this unique and important ecosystem and its microbiome.

Determination of PAHs, PCBs, minerals, trace elements, and fatty acids in Rapana thomasiana as an indication of pollution.

The aim of this investigation was to introduce several analytical methods for determination of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), minerals, trace elements, and fatty acids in Rapana thomasiana as a marine pollution indicator organism. The chemical differences of the gastropod Ra. thomasiana from polluted and nonpolluted sites of the Black Sea on the Bulgarian coast were investigated. Chromatography and high-resolution inductively coupled plasma/MS analyses were used for evaluation of PAHs, PCBs, fatty acids, minerals, and trace elements. These methods can be applied to other marine products.

Spatial distribution of antibiotic and heavy metal resistance genes in the Black Sea.

Antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) are worldwide considered as emerging contaminants of large interest, and a primary threat to human health. It is becoming clear that the environment plays a central role in the transmission, spread, and evolution of antibiotic resistance. Although marine systems have been largely investigated, only a few studies have considered the presence of ARGs in meso- and bathypelagic waters. To date, no molecular based studies have yet been made to investigate the occurrence of ARGs in the Black Sea, the largest meromictic basin in the world, receiving water from a number of important European rivers and their residues of anthropogenic activities in permanently stratified mesopelagic water masses. In this study, we determined the presence and the abundance of five ARGs (blaCTXM, ermB, qnrS, sul2, tetA) and of the heavy metal resistance gene (HMRG) czcA, in different sampling sites in the eastern and western Black Sea, at several depths (up to 1000 m) and various distances from the shoreline. Three ARGs (blaCTXM, sul2, and tetA) and czcA were present in at least 43% of the analysed samples, whereas ermB and qnrS were never detected. In particular, sul2 abundances increased significantly in coastal location, whereas tetA increased with sampling depth. These findings point out the Black Sea as a source of ARGs and HMRGs distributed along the whole water column.

Comparative analysis of morphological and molecular approaches integrated into the study of the dinoflagellate biodiversity within the recently deposited Black Sea sediments - benefits and drawbacks.

One of the assets, assigned to the phytoplankton resting stages, is that of serving as the "memory" of the aquatic ecosystems and preserved biodiversity in the course of time. However, an accurate cyst identification proves to be a more difficult and extremely challenging process, even today. In order to gain a better taxonomic coverage of cyst assemblages in the Black Sea, an integrated approach of the classical morphological identification with metabarcoding methods (MySeq sequencing of V7-V9 regions of the 18S rDNA) was applied on thirteen surface sediment samples collected from different sites. A total number of 112 dinoflagellate taxa was detected at the species level and ascribed to 51 genera. In general, it is the molecular analysis that yields a higher number of taxa as compared to those obtained through the morphological taxonomy (66 taxa based on the DNA sequences versus 56 morphologically-identified taxa). Besides, it should be pointed out that the integrated dataset includes 14 potentially toxic dinoflagellate species. Discerned, subsequently, was a good dataset consistency for ten species, followed by some discrepancies as to a number of taxa, identified with one of the methods only, due to specific methodological biases. On the whole, it could be concluded that the combination of morphological and molecular methods is likely to increase the potential for a more reliable taxonomic assessment of phytoplankton diversity in marine sediments which, in turn, proves conclusively the utmost importance of the integrated approach.

Genomic Comparison and Spatial Distribution of Different Synechococcus Phylotypes in the Black Sea.

Picocyanobacteria of the genus Synechococcus are major contributors to global primary production and nutrient cycles due to their oxygenic photoautotrophy, their abundance, and the extensive distribution made possible by their wide-ranging biochemical capabilities. The recent recovery and isolation of strains from the deep euxinic waters of the Black Sea encouraged us to expand our analysis of their adaptability also beyond the photic zone of aquatic environments. To this end, we quantified the total abundance and distribution of Synechococcus along the whole vertical profile of the Black Sea by flow cytometry, and analyzed the data obtained in light of key environmental factors. Furthermore, we designed phylotype-specific primers using the genomes of two new epipelagic coastal strains - first described here - and of two previously described mesopelagic strains, analyzed their presence/abundance by qPCR, and tested this parameter also in metagenomes from two stations at different depths. Together, whole genome sequencing, metagenomics and qPCR techniques provide us with a higher resolution of Synechococcus dynamics in the Black Sea. Both phylotypes analyzed are abundant and successful in epipelagic coastal waters; but while the newly described epipelagic strains are specifically adapted to this environment, the strains previously isolated in mesopelagic waters are able, in low numbers, to withstand the aphotic and oxygen depleted conditions of deep layers. This heterogeneity allows different Synechococcus phylotypes to occupy different niches and underscores the importance of a more detailed characterization of the abundance, distribution, and dynamics of individual populations of these picocyanobacteria.

The mesopelagic anoxic Black Sea as an unexpected habitat for Synechococcus challenges our understanding of global "deep red fluorescence".

The Black Sea is the largest meromictic sea with a reservoir of anoxic water extending from 100 to 1000 m depth. These deeper layers are characterised by a poorly understood fluorescence signal called "deep red fluorescence", a chlorophyll a- (Chl a) like signal found in deep dark oceanic waters. In two cruises, we repeatedly found up to 103 cells ml-1 of picocyanobacteria at 750 m depth in these waters and isolated two phycoerythrin-rich Synechococcus sp. strains (BS55D and BS56D). Tests on BS56D revealed its high adaptability, involving the accumulation of Chl a in anoxic/dark conditions and its capacity to photosynthesise when re-exposed to light. Whole-genome sequencing of the two strains showed the presence of genes that confirms the putative ability of our strains to survive in harsh mesopelagic environments. This discovery provides new evidence to support early speculations associating the "deep red fluorescence" signal to viable picocyanobacteria populations in the deep oxygen-depleted oceans, suggesting a reconsideration of the ecological role of a viable stock of Synechococcus in dark deep waters.

Concentration of bioactive compounds in mussels Mytilus galloprovincialis as an indicator of pollution.

The polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organotins were analyzed in mussels Mytilus galloprovincialis from polluted and unpolluted sites from Mokpo Bay, Korea. The total PAH's concentrations (10(-3)mgkg(-1)) measured by GC-MS were in the range from 31+/-23 to 1+/-1. Among the eight PAHs the predominant ones were fluoranthene, phenanthrene and pyrene and accounted approximately 63% of the total PAHs. Among the four detected PCBs the highest content was of PCB 153, which accounted about 47% of the total PCBs. The main organotin compounds were dibutyltindichloride (DBT) and tributyltinchloride (TBT) and their composition was approximately 33% and 24%. PAHs, PCBs and organotins were found only in the mussels from polluted site. The antioxidant activity by ABTS [2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] test was higher in mussels from polluted than from unpolluted sites (P<0.05). It was found a correlation between the determined compounds (PAHs, PCBs and organotins) and the antioxidant activity of the mussel tissue from polluted site and the correlation coefficients were 0.96, 0.92 and 0.80, respectively. Such correlation can be explained by the properties of mussels. Since the mussel cell wall and tissues are hydrophobic, they can concentrate a number of hydrophobic pollutants like PAHs and PCBs from the marine environment by solubility rules. On the other hand, proteins are lipophilic compounds having antioxidant properties. Certain amino acid residues and thiol (-SH) groups, contained in proteins, respond to the ABTS antioxidant activity assay. Thus there may be a correlation between the total antioxidant activity of the organism and the PAH-PCB pollutants which were concentrated from its environment. The studied properties of mussels from polluted site can be used as an additional indicator of pollution.

Applicability of massively parallel sequencing on monitoring harmful algae at Varna Bay in the Black Sea.

In this study the plankton diversity in 13 environmental samples from Varna Bay (in the western Black Sea) was analyzed using massively parallel sequencing (MPS). This preliminary study was undertaken to assess the potential of this technology for future implementation in monitoring programs in the Black Sea. Amplicon sequences of the 18S rRNA gene (V4-5 regions) were obtained using the Illumina MiSeq 250PE platform. A total of 1137 operational taxonomic units (OTUs) were obtained among which 242 OTUs with >0.990 BLAST top hit similarity (21.3% of all detected OTUs) closely related to sequences belonging to -protists. A large portion (175 OTUs=72.3%) was identified at the species levels, including species typical for the Bulgarian Black Sea plankton community, as well as many that haven't been reported earlier in the Bulgarian Black Sea coast (124 OTUs=51.2%). Dinoflagellates were represented by the highest species number (77 OTUs comprising 31.8% of protist species), with dominant genera Gyrodinium and Heterocapsa. The present survey revealed the presence of 12 species listed as harmful, some of which have been previously overlooked, such as Cochlodinium polykrikoides, Karenia bicuneiformis, and Karlodinium veneficum. Species identification was possible for 10.3-36.0% of the detected OTUs in the six major supergroups. The frequency in Rhizaria was significantly lower than that in other major groups (p<0.05-0.01), implying difficulties in the classification from morphology-based observations. The metagenetic data had an insufficient resolution of the 18S rRNA gene for species identification in many genera. These issues may hamper the implementation of MPS-based surveys for plankton monitoring, especially for detecting harmful algal blooms (HAB). The sequencing technology is steadily improving and it is expected that sequence length and quality issues will be resolved in the near future. The ongoing efforts to register taxonomic information and quality controls in the international nucleotide sequence databases (INSDs) will be essential for improving taxonomic identification power.

Changes in mussel Mytilus galloprovincialis protein profile as a reaction of water pollution.

In the present report the changes in mussels Mytilus galloprovincialis protein profile, as a reaction of water pollution, is presented. The antioxidant capacity was determined by the efficacy to scavenge the derived radical in mussel samples using the Trolox Equivalent Antioxidant Capacity (TEAC) assay. The highest percentage of inhibition was estimated in protein mussels from the dirty area (DM) in comparison with the clean samples (CM). The amounts of both zinc and copper determined by atomic spectroscopy were significantly higher in the protein mussels from the dirty than from the clean areas (P<0.05). Fourier Transform Infrared (FT-IR) spectroscopy and fluorescence revealed specific qualitative changes in secondary and tertiary structures of mussel proteins from the dirty area by the shift in the amides I and II positions and fluorescence intensity. Estimated temperature and enthalpy of denaturation in the protein mussels well correlated with fluorescence and spectroscopic measurements and showed the changes influenced by water pollution.

Relationship between seawater pollution and qualitative changes in the extracted proteins from mussels Mytilus galloprovincialis.

The aim of this study was to find a reliable biomarker of seawater pollution. For this purpose the contents of Zn and Cu, proteins and antioxidant activity in mussels Mytilus galloprovincialis collected from polluted and non-polluted sites of the Bulgarian Black Sea coast were compared. To determine the above-mentioned indices atomic spectroscopy, Fourier Transform Infrared (FT-IR) spectroscopy, fluorescence, differential scanning calorimetry (DSC), and two antioxidant tests were used. It was found that the amounts of Zn and Cu were significantly higher in the mussel proteins from the polluted than from the non-polluted sites (P<0.05). FT-IR spectroscopy and fluorescence revealed specific qualitative changes in secondary and tertiary structures in mussel proteins in the samples from polluted sites. The thermodynamic properties of proteins and the changes upon denaturation were correlated with the secondary structure of proteins and disappearance of alpha-helix. Purified protein scavenging activity against 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS(*+)) was significantly higher in mussel samples from polluted than from non-polluted sites. Therefore, the changes in Zn and Cu concentration, in protein's secondary and tertiary structures and antioxidant activity in mussels M. galloprovincialis from polluted sites can be a reliable biomarker of the level of the seawater pollution.

Rapana venosa consumption improves the lipid profiles and antioxidant capacities in serum of rats fed an atherogenic diet.

In the recent years, the consumption of seafood has increased. There are no results on the studies of Rapana venosa (Rv) as a supplementation to the diets. We hypothesized that Rv would increase antioxidant capacity and reduce blood lipids, based on the composition of bioactive compounds and fatty acids. Therefore, the aim of this investigation was to evaluate in vitro and in vivo actions of Rv from contaminated (C) and non-C (NC) regions of collection on lipid profiles, antioxidant capacity, and enzyme activities in serum of rats fed an atherogenic diet. Twenty-four male Wistar rats were divided into 4 groups of 6 each and named control, cholesterol (Chol), Chol/RvC and Chol/RvNC. Rats of all 4 groups were fed the basal diet, which included wheat starch, casein, soybean oil, cellulose, vitamin (American Institute of Nutrition for laboratory animals vitamin mixtures), and mineral mixtures (American Institute of Nutrition for laboratory animals mineral mixtures). During 28 days of the experiment, the rats of the control group received the basal diet only, and the diets of the other 3 groups were supplemented with 1% of Chol, 1% of Chol, and 5% of Rv dry matter from C and NC areas. Dry matter from C and NC areas supplemented diets slightly hindered the rise in serum lipids vs. Chol group: total Chol, 13.18% and 11.63% and low-density lipoprotein Chol, 13.57% and 15.08%, respectively. Cholesterol significantly decreased the value of total antioxidant capacity. The supplementation of Rv to the Chol diet significantly affected the increase of antioxidant capacity in serum of rats, expressed by the 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) method. The water extracts of Rv exhibited high binding properties with bovine serum albumin in comparison with quercetin. In conclusion, atherogenic diets supplemented with Rv from C and NC areas hindered both the rise in serum lipids levels and the decrease in the antioxidant capacity. Based on fluorescence and electrospray-ionization mass spectrometry profiles and in vivo studies, changes in the intensity of the found peaks were estimated in the serum samples after supplemented diets. These findings indicate that the supplementation of Rv to the atherogenic diets improve the lipid profiles and the antioxidant status in serum of rats.

PESI - a taxonomic backbone for Europe.

BACKGROUND: Reliable taxonomy underpins communication in all of biology, not least nature conservation and sustainable use of ecosystem resources. The flexibility of taxonomic interpretations, however, presents a serious challenge for end-users of taxonomic concepts. Users need standardised and continuously harmonised taxonomic reference systems, as well as high-quality and complete taxonomic data sets, but these are generally lacking for non-specialists. The solution is in dynamic, expertly curated web-based taxonomic tools. The Pan-European Species-directories Infrastructure (PESI) worked to solve this key issue by providing a taxonomic e-infrastructure for Europe. It strengthened the relevant social (expertise) and information (standards, data and technical) capacities of five major community networks on taxonomic indexing in Europe, which is essential for proper biodiversity assessment and monitoring activities. The key objectives of PESI were: 1) standardisation in taxonomic reference systems, 2) enhancement of the quality and completeness of taxonomic data sets and 3) creation of integrated access to taxonomic information. NEW INFORMATION: This paper describes the results of PESI and its future prospects, including the involvement in major European biodiversity informatics initiatives and programs.

Antioxidants in the black mussel (Mytilus galloprovincialis) as an indicator of Black Sea coastal pollution.

The biologically active compounds, antioxidant activities, and free radical scavenging effects of black mussel dry matter (Mytilus galloprovincialis)--(BMDM) were investigated. The extract from BMDM with absolute methanol (BMDMAMet) showed the strongest inhibition of lipid peroxidation as a function of its concentration, and was comparable to the antioxidant activity of butylated hydroxyanisole, at the same concentration of 0.2 mg/ml. The extract with 50%methanol/water (BMDM50%Met) had the weakest antioxidant activity, whereas other extracts such as 1.2 M HCl in 50%methanol/water (BMDM50%Met/HCl) exhibited an average inhibition of lipid peroxidation. BMDMAMet extract showed marked activities in free radical scavenging determined by beta-carotene bleaching (beta-carotene), nitric oxide (NO*) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS*+ methods. The antioxidant activities of BMDMAMet extract determined beta-carotene, NO* and ABTS*+ methods showed the highest correlation coefficients (R2) such as 0.998, 0.9976 and 0.6103, respectively, with the presence of total polyphenols. BMDM50%Met had lower antioxidant ability than BMDM50%Met/HCl in different scavenging methods, indicating that the major antioxidant components in these extracts must be derived from the polyphenols. Correlation between proteins and antioxidants was very low (0.0318 and 0.0433). The mussel extracts from polluted areas have shown by all using methods higher antioxidant activity than from the clean ones. The results presented in this report indicate that the antioxidant activities of mussel extracts could possibly be an additional index of pollution characterization.

Seasonal variability of phytoplankton at Varna Bay (Black Sea).

Intrinsic fluorescence and SDS-PAGE analysis were employed to study the seasonal qualitative and quantitative changes of phytoplankton composition at Varna Bay (Black Sea). Variation in the maximum emission wavelength (lambda(max)) of the phytoplankton proteins (398 nm in the summer and 340 nm in the spring) was observed. In addition, a decrease in fluorescence intensity, and a shift in lambda(max) as a result of changes in phytoplankton protein stability, according to the season, was noted. Similarly, SDS-PAGE analysis showed different protein patterns for each season, for example in summertime the major protein constituents were of 14, 37, 48 and 70 kDa, while in the springtime the sizes ranged between 38 and 48 kDa. In general, higher carbohydrate and protein contents correlated with larger phytoplankton biomass found during the summer. The dominant species, the Bacillariophyceae and Dinophyceae, were found to be present in the water body in an alternate pattern. All of these changes could be accounted for by the adaptation of the organism to seasonal variations that modify the sea environment at Varna Bay.