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.

The relationship between toxic phytoplankton species occurrence and environmental and meteorological factors along the Eastern Adriatic coast.

In this study, the time series of toxic phytoplankton species collected between 2004 and 2018 from the Northern Adriatic, Sibenik Bay, and Mali Ston Bay was analyzed in relation to environmental (temperature, salinity, water column stability, and river flow) and meteorological parameters (precipitation and wind). Because of the mostly non-linear relation between biotic and abiotic parameters, self-organizing maps (SOM) were used to identify these relationships. SOM analysis distinguished species of the genus Dinophysis from Gonyaulax spinifera and Lingulodinium polyedrum species, which better tolerate wind-induced disturbance. Among the Dinophysis species, Dinophysis fortii, Dinophysis tripos, and Dinophysis acuta preferred higher precipitation rate and river flow in addition to optimal temperatures. The abundances of Alexandrium species, which occurred more frequently in estuarine areas, were associated with river flow and maximum stable water column. Regardless of the ecological preferences of individual harmful algae, freshwater inflow-caused stratification is present in all clusters of environmental conditions associated with increased abundances of harmful algae in the SOM analysis. It is highly likely that stratification represents an important factor for the development and maintenance of HABs. The non-linear relationship between the NAO index and rainfall was noted, of which the most important for the development of harmful algae is the proportional correlation between the positive phase of the NAO index and higher rainfall, especially in winter and spring. Such conditions are conducive to the development of harmful algae because, with the increase in temperature accompanying the positive phase of the NAO index, increased rainfall further stimulates their growth. This can be achieved either through nutrient yields or through higher freshwater inflow that further stabilizes the water column.

Massive Occurrence of the Harmful Benthic Dinoflagellate Ostreopsis cf. ovata in the Eastern Adriatic Sea.

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Kastela Bay. In order to taxonomically identify the Ostreopsis species responsible for this event and determine their toxin profile, cells collected in seawater and from benthic macroalgae were analyzed. Conservative taxonomic methods (light microscopy and SEM) and molecular methods (PCR-based assay) allowed the identification of the species Ostreopsis cf. ovata associated with Coolia monotis. The abundance of O. cf. ovata reached 2.9 × 104 cells L-1 in seawater, while on macroalgae, it was estimated to be up to 2.67 × 106 cells g-1 of macroalgae fresh weight and 14.4 × 106 cells g-1 of macroalgae dry weight. An indirect sandwich immunoenzymatic assay (ELISA) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) were used to determine the toxin profile. The ELISA assay revealed the presence of 5.6 pg palytoxin (PLTX) equivalents per O. cf. ovata cell. LC-HRMS was used for further characterization of the toxin profile, which showed that there were 6.3 pg of the sum of ovatoxins (OVTXs) and isobaric PLTX per O. cf. ovata cell, with a prevalence of OVTXs (6.2 pg cell-1), while the isobaric PLTX concentration was very low (0.1 pg cell-1). Among OVTXs, the highest concentration was recorded for OVTX-a (3.6 pg cell-1), followed by OVTX-b (1.3 pg cell-1), OVTX-d (1.1 pg cell-1), and OVTX-c (0.2 pg cell-1).

Dinoflagellate resting cysts from surface sediments of the Adriatic Ports: Distribution and potential spreading patterns.

The ability of microalgae to preserve viable in coastal sediments as resting forms provides a reservoir of biodiversity and a useful tool to determine species spreadings. This study represents the first port baseline survey on dinoflagellate cysts, investigated in nine Adriatic ports during a cross border project. 40 dinoflagellate taxa were detected. The assemblages resulted in all ports dominated by Lingulodinium polyedra and Alexandrium minutum/affine/tamutum group. General separation to the western and eastern side of the Adriatic regarding cysts assemblage composition, partially abundance, was observed. Six taxa were detected as non-indigenous species for the Adriatic. Two taxa are included in the list of harmful aquatic organisms, indicating the potential threat of ballast waters in the Adriatic. Potential spreading of taxa by general circulation and ballast waters, intra- and extra-Adriatic was investigated. The entering in to force of the ballast waters management regulations should enhance prospects to minimize future harmful impacts.

Phytoplankton diversity in Adriatic ports: Lessons from the port baseline survey for the management of harmful algal species.

An inventory of phytoplankton diversity in 12 Adriatic ports was performed with the port baseline survey. Particular emphasis was put on the detection of harmful aquatic organisms and pathogens (HAOP) because of their negative impact on ecosystem, human health, and the economy. Phytoplanktonic HAOP are identified as species, either native or non-indigenous (NIS), which can trigger harmful algal blooms (HAB). A list of 691 taxa was prepared, and among them 52 were classified as HAB and five as NIS. Records of toxigenic NIS (Pseudo-nitzschia multistriata, Ostreopsis species including O. cf. ovata) indicate that the intrusion of non-native invasive phytoplankton species has already occurred in some Adriatic ports. The seasonal occurrence and abundance of HAOP offers a solid baseline for a monitoring design in ports in order to prevent ballast water uptake and possible expansion of HAOP outside their native region.

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).

Strategy of port baseline surveys (PBS) in the Adriatic Sea.

Port baseline surveys (PBS) provide species inventories in and around ports, with a focus on non-indigenous species that may have been introduced by vessels, primarily via ballast water. PBS are an essential tool to support effective management strategies for non-indigenous as well as native harmful aquatic organisms and pathogens (HAOP). This paper describes the methodology of PBS that were conducted in 12 Adriatic ports. The PBS employed existing protocols that were adapted to meet the characteristics of the Adriatic sites. Their results are reported in several papers included in this special issue, each of which is devoted to a specific community. An overview of existing surveys protocols - which provide valuable support to decision-making and to design effective monitoring of non-indigenous species - is also supplied.

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.

Lipophilic toxin profile in Mytilus galloprovincialis during episodes of diarrhetic shellfish poisoning (DSP) in the N.E. Adriatic Sea in 2006.

Dinophysis spp. blooms and related shellfish toxicity events of diarrhetic shellfish poisoning (DSP) have been the most reported toxicity event through the Croatian National monitoring program. With the aim to characterize the DSP toxin profile in shellfish farmed in Croatia, for the first time a complete analysis of the toxin profile of Croatian mussels has been carried out using the LC-MS/MS technique. The obtained results showed okadaic acid (OA) as the main toxin contaminating Croatian mussels at that time. The maximum concentration of OA in shellfish tissue was recorded 12 days after the Dinophysis fortii bloom, thus suggesting that rapid growth of the toxin level in the shellfish occurred in the first week after the bloom while it was slower in the second week. Furthermore, the presence of only OA at concentrations which could endanger human health suggests D. fortii as the main organism responsible for the toxic event that occurred in Lim Bay. The presence of gymnodimine and spirolides in Croatian mussel has been detected for the first time, while the presence of yessotoxin and pectenotoxin-2 is confirmed.

Domoic acid--a new toxin in the Croatian Adriatic shellfish toxin profile.

This is the first study that presents concentrations of domoic acid detected in the whole shellfish tissue from breeding and harvesting areas along the Croatian coast of the Adriatic Sea during the period 2006 to 2008. Shellfish sample analyses after SAX cleaning procedures, using a UV-DAD-HPLC system, showed the presence of domoic acid in four species. The most prevalent of those species were the blue mussel (Mytilus galloprovincialis), followed by European flat oyster (Ostrea edulis), Mediterranean scallop (Pecten jacobaeus) and proteus scallop (Flexopecten proteus). Domoic acid, a potentially lethal phycotoxin that causes amnesic shellfish poisoning (ASP), was detected for the first time in January 2006 with the highest value of 6.5486 µg g⁻¹ in whole shellfish tissue. Pseudo-nitzschia spp. bloom events preceded these high domoic acid concentrations. According to this study, retention of domoic acid in the blue mussel M. galloprovincialis is more than 42 days. This investigation indicates the first presence of domoic acid in Croatian shellfish, but in concentrations under the regulatory limit (20 µg g⁻¹), therefore shellfish consumption was not found to endanger human health.