Ecology of aerobic anoxygenic phototrophs on a fine-scale taxonomic resolution in Adriatic Sea unravelled by unsupervised neural network.

BACKGROUND: Aerobic anoxygenic phototrophs are metabolically highly active, diverse and widespread polyphyletic members of bacterioplankton whose photoheterotrophic capabilities shifted the paradigm about simplicity of the microbial food chain. Despite their considerable contribution to the transformation of organic matter in marine environments, relatively little is still known about their community structure and ecology at fine-scale taxonomic resolution. Up to date, there is no comprehensive (i.e. qualitative and quantitative) analysis of their community composition in the Adriatic Sea. RESULTS: Analysis was based on pufM gene metabarcoding and quantitative FISH-IR approach with the use of artificial neural network. Significant seasonality was observed with regards to absolute abundances (maximum average abundances in spring 2.136 ± 0.081 × 104 cells mL-1, minimum in summer 0.86 × 104 cells mL-1), FISH-IR groups (Roseobacter clade prevalent in autumn, other Alpha- and Gammaproteobacteria in summer) and pufM sequencing data agglomerated at genus-level. FISH-IR results revealed heterogeneity with the highest average relative contribution of AAPs assigned to Roseobacter clade (37.66%), followed by Gammaproteobacteria (35.25%) and general Alphaproteobacteria (31.15%). Community composition obtained via pufM sequencing was dominated by Gammaproteobacteria clade NOR5/OM60, specifically genus Luminiphilus, with numerous rare genera present in relative abundances below 1%. The use of artificial neural network connected this community to biotic (heterotrophic bacteria, HNA and LNA bacteria, Synechococcus, Prochlorococcus, picoeukaryotes, heterotrophic nanoflagellates, bacterial production) and abiotic environmental factors (temperature, salinity, chlorophyll a and nitrate, nitrite, ammonia, total nitrogen, silicate, and orthophosphate concentration). A type of neural network, neural gas analysis at order-, genus- and ASV-level, resulted in five distinct best matching units (representing particular environments) and revealed that high diversity was generally independent of temperature, salinity, and trophic status of the environment, indicating a potentially dissimilar behaviour of aerobic anoxygenic phototrophs compared to the general bacterioplankton. CONCLUSION: This research represents the first comprehensive analysis of aerobic anoxygenic phototrophs in the Adriatic Sea on a trophic gradient during a year-round period. This study is also one of the first reports of their genus-level ecology linked to biotic and abiotic environmental factors revealed by unsupervised neural network algorithm, paving the way for further research of substantial contribution of this important bacterial functional group to marine ecosystems.

Artificial neural network analysis of microbial diversity in the central and southern Adriatic Sea.

Bacteria are an active and diverse component of pelagic communities. The identification of main factors governing microbial diversity and spatial distribution requires advanced mathematical analyses. Here, the bacterial community composition was analysed, along with a depth profile, in the open Adriatic Sea using amplicon sequencing of bacterial 16S rRNA and the Neural gas algorithm. The performed analysis classified the sample into four best matching units representing heterogenic patterns of the bacterial community composition. The observed parameters were more differentiated by depth than by area, with temperature and identified salinity as important environmental variables. The highest diversity was observed at the deep chlorophyll maximum, while bacterial abundance and production peaked in the upper layers. The most of the identified genera belonged to Proteobacteria, with uncultured AEGEAN-169 and SAR116 lineages being dominant Alphaproteobacteria, and OM60 (NOR5) and SAR86 being dominant Gammaproteobacteria. Marine Synechococcus and Cyanobium-related species were predominant in the shallow layer, while Prochlorococcus MIT 9313 formed a higher portion below 50 m depth. Bacteroidota were represented mostly by uncultured lineages (NS4, NS5 and NS9 marine lineages). In contrast, Actinobacteriota were dominated by a candidatus genus Ca. Actinomarina. A large contribution of Nitrospinae was evident at the deepest investigated layer. Our results document that neural network analysis of environmental data may provide a novel insight into factors affecting picoplankton in the open sea environment.

Changes in the Trophic Pathways within the Microbial Food Web in the Global Warming Scenario: An Experimental Study in the Adriatic Sea.

A recent analysis of the Mediterranean Sea surface temperature showed significant annual warming. Since small picoplankton microorganisms play an important role in all major biogeochemical cycles, fluxes and processes occurring in marine systems (the changes at the base of the food web) as a response to human-induced temperature increase, could be amplified through the trophic chains and could also significantly affect different aspects of the structure and functioning of marine ecosystems. In this study, manipulative laboratory growth/grazing experiments were performed under in situ simulated conditions to study the structural and functional changes within the microbial food web after a 3 °C increase in temperature. The results show that a rise in temperature affects the changes in: (1) the growth and grazing rates of picoplankton, (2) their growth efficiency, (3) carrying capacities, (4) sensitivity of their production and grazing mortality to temperature, (5) satisfying protistan grazer carbon demands, (6) their preference in the selection of prey, (7) predator niche breadth and their overlap, (8) apparent uptake rates of nutrients, and (9) carbon biomass flow through the microbial food web. Furthermore, temperature affects the autotrophic and heterotrophic components of picoplankton in different ways.

Temperature and phosphorus interacts in controlling the picoplankton carbon flux in the Adriatic Sea: an experimental versus field study.

Temperature and phosphorus positively interacted in controlling picoplankton biomass production and its transfer towards higher trophic levels. Two complementary approaches (experimental and field study) indicated several coherent patterns: (1) the impact of temperature on heterotrophic bacteria was high at temperatures lower than 16°C and levelled off at higher temperatures, whereas this impact on autotrophic picoplankton was linear along the entire range of the investigated temperatures; (2) the addition of phosphorus increased the values of picoplankton production and grazing, but did not change the nature of their relationships with temperature substantially; (3) the picoplankton carbon flux towards higher trophic levels was larger during the warmer months (grazing by HNF dominated during the warmer period and by ciliates during the colder period) and also strengthened in conditions without phosphorus limitation; (4) the hypothesis that the available phosphorus can be better utilized at higher temperatures was confirmed for both autotrophic and heterotrophic picoplankton; (5) the hypothesis that the rise in temperature stimulates growth only in conditions of sufficient phosphorus was confirmed only for heterotrophic bacteria. Therefore, in the global warming scenario, an increase of the picoplankton carbon flux towards higher trophic levels can be expected in the Adriatic Sea, particularly under unlimited phosphorus conditions.

Relations between mercury fractions and microbial community components in seawater under the presence and absence of probable phosphorus limitation conditions.

Microbial transformations of toxic monomethylmercury (MMHg) and dissolved gaseous mercury (DGM) at the lower levels of the marine food web are not well understood, especially in oligotrophic and phosphorus-limited seas. To examine the effects of probable phosphorus limitation (PP-limitation) on relations between mercury (Hg) fractions and microorganisms, we determined the total mercury (THg), total methylated mercury (MeHg), DGM, and microbiological and chemical parameters in the Central Adriatic Sea. Using statistical analysis, we assessed the potential microbial effects on Hg transformations and bioaccumulation. Only in the absence of PP-limitation conditions (NO-PP-limitation) is MeHg significantly related to most chemical and microbial parameters, indicating metabolism-dependent Hg transformations. The heterotrophic activity of low nucleic acid bacteria (abundant in oligotrophic regions) seems responsible for most of Hg methylation under NO-PP-limitation. Under these conditions, DGM is strongly related to microbial fractions and chlorophyll a, indicating biological DGM production, which is probably not metabolically induced, as most of these relations are also observed under PP-limitation. MMHg biomagnification was observed through an increased bioaccumulation factor from microseston to mesozooplankton. Our results indicate that Hg transformations and uptake might be enhanced under NO-PP-limitation conditions, emphasizing their impact on the transfer of Hg to higher trophic levels.

Oceanographic characteristics of the Adriatic Sea - Support to secondary HAOP spread through natural dispersal.

Vessels, specifically ballast water and hull fouling, are a major vector for the introduction of non-indigenous species (NIS) in European seas. The Mediterranean is one of the world's marine regions where their invasion is heaviest. The shallow Adriatic basin is a highly sensitive area that is already experiencing its consequences. The secondary spread of NIS over a wider area through natural dispersion is a complex process that depends on a wide range of oceanographic factors. This work analysed the dataset of the BALMAS project, in whose framework twelve ports in the Adriatic Sea were subjected to a Port Baseline Survey (PBS), to estimate the natural spread of NIS organisms from their port of arrival to the wider Adriatic basin. Its findings indicate that the prevailing water circulation patterns facilitate the natural dispersal of harmful aquatic organisms and pathogens (HAOP).

Polycyclic aromatic hydrocarbons in surface sediments of the mid-Adriatic and along the Croatian coast: Levels, distributions and sources.

This study provides contamination levels, distributions and source apportionment of PAHs in surface sediments in the mid-Adriatic and along the Croatian coast. Median summed concentrations of parent and alkyl-PAHs are circa 10 times lower in the off-shore transect stations of the mid-Adriatic (22.3 and 18.2 µg.kg-1 d.w.) than the ranges determined at the coastal stations, including those of Kastela bay (227-331 and 11.7-197 µg.kg-1 d.w., respectively). The highest levels, circa 20 times higher, were found in Sibenik bay (median 6603 and 3051 µg.kg-1). The overall range of PAH concentrations spans more than 2000 times between the lowest and the highest contamination level. The geographical distributions reflect the presence of strong gradients at local and regional scales. A major factor influencing sedimentary PAH distributions at local scale appears to be the distance from their known continental and coastal upstream emission sites (urban, industrial, harbour …), whereas at regional scale, this distribution depends more on the routes of entry of PAHs into the study area. Two combustion and one petroleum model source profiles of PAHs were determined by alternative least square analysis. Benzo[b+j]fluoranthenes and fluoranthene/pyrene are compounds characterizing two pyrogenic sources respectively, while signatures of alkyl-substituted homologues (phenanthrenes/anthracenes, fluranthenes/pyrenes, chrysenes and dibenzothiophenes) delineate a petrogenic source profile. The quantitative apportionment of source contributions shows significant geographical differences, with a dominant petrogenic source found along the mid-Adriatic transect (approximately 74%) and in Kastela bay (61%). In the coastal sediments about a fifty-fifty contamination mix is assigned to a petrogenic/pyrogenic source of PAHs (47% and 53% respectively), whereas in Sibenik bay a strong predominance is apportioned to the combustion compounds (81%).

Assessing the environmental quality of sediments from Split coastal area (Croatia) with a battery of cell-based bioassays.

A battery of cell-based bioassays, including PLHC-1 cells, zebrafish-Pxr-transfected COS-7 cells and estrogen receptor-recombinant yeast assay (ER-RYA), were applied to detect the presence of bioactive pollutants in sediments collected from Kastela Bay and Brac Channel (Croatia). Exposure of PLHC-1 cells to the sediment extracts evidenced significant cytotoxicity and presence of CYP1A inducers in sediments collected in Kastela Bay, near the industrial zone and cargo port of Split. Sediments from this area, which is highly contaminated with PCBs, HCB, DDTs and γ-HCH, also activated the zebrafish Pxr (zfPxr) reporter system. No evidence of estrogenicity was detected for any of the sediments extracts in the ER-RYA assay. Importantly, the battery of in vitro assays identified Kastela Bay as the area with the higher anthropogenic impact, where sediment-bound pollutants could pose a risk to aquatic organisms. In contrast, sediments from the Brac Channel showed rather low response in the different bioassays.

Distribution of aerobic anoxygenic phototrophs in the Eastern Adriatic Sea.

The spatial patterns of aerobic anoxygenic phototrophs abundances were investigated, for the first time, in the Adriatic Sea. Also, the spatial patterns of the whole picoplankton community as well as the environmental factors that potentially influence these patterns were highlighted. AAP abundances was in average 66.9 ± 66.8 × 103 cell mL-1, and their proportion in total bacteria was 7.3 ± 4.3%. These values are in the upper range of AAP abundances observed in marine environments. Multivariate analyses proved that environmental factors influenced the picoplankton community interdependently. Chl a was the main driving factor for the picoplankton community, accounting for 33.3% of picoplankton community variance, followed by NO2- (17.9% of variance explained) and temperature (14.2% of variance explained). Chl a showed stronger correlation with AAPs, non-pigmented bacteria and Picoeucaryotes than with cyanobacteria. Abundance of cyanobacteria was stronger correlated to salinity and the N:P ratio than to nutrient concentrations.

Bioaccumulation of trace metals in mussel (Mytilus galloprovincialis) from Mali Ston Bay during DSP toxicity episodes.

The Croatian National Monitoring Program revealed the presence of Diarrhetic Shellfish Poisoning (DSP) toxicity in Mediterranean blue mussel (Mytilus galloprovincialis) from breeding farms in southern Adriatic Sea through January to June 2011. The mouse bioassay tests (MBA; at the time the official method for DSP toxins) were accompanied by atypical symptomatology in the animals and this caused doubts about the assay results. Consequently, in parallel studies reported here, the concentration of Cd, Cr, Cu, Ni, Pb and Zn in soft tissue of DSP positive and negative mussels samples was determined. Cd, Cr, Zn and Ni show higher values in approximately 75% of the DSP positive samples, whereas for Pb and Cr the values were 26% and 34%, respectively. This trend was unchanged during the whole observation period.

Impact of fish farming on the distribution of phosphorus in sediments in the middle Adriatic area.

During the last decade, intensive fish farming developed along the central Croatian coast, creating a need to study and evaluate its potential influence on unaffected sites. We considered phosphorus as an indicator of the influence of fish farming and investigated the distribution of phosphorus forms in sediment from several fish farms and marine areas of different trophic status in the middle Adriatic. Analyses of samples were performed with modified SEDEX techniques. Our results indicated that authigenic apatite phosphorus showed no significant differences among the investigated stations, while organic phosphorus concentrations reflected the trophic status of the station area. Below-cage sediment was characterized by enhanced fish debris phosphorus and low detrital apatite phosphorus concentrations, while sediment from an anthropogenically influenced bay showed the highest values of iron bound phosphorus species. Among the different P fractions, fish debris phosphorus proved to be the most sensitive indicator of the influence of fish farming on marine sediment.