Effects of extreme temperatures and recovery potential of Gongolaria barbata from a coastal lagoon in the northern Adriatic Sea: An ex situ approach.

BACKGROUND AND AIMS: Globally, rising seawater temperatures contribute to the regression of marine macroalgal forests. Along the Istrian coastline (northern Adriatic), an isolated population of Gongolaria barbata persists in a coastal lagoon, representing one of the last marine macroalgal forests in the region. Our objective was to examine the impact of extreme temperatures on morphology and physiology of G. barbata, and test its potential for recovery after simulating marine heatwave (MHW) conditions. METHODS: We explored the occurrence of marine heatwaves in southern Istria, adjacent to the study area, as well as extreme temperatures inside the area itself. Subsequently, we performed a thermotolerance experiment, consisting of a stress and recovery phase where we exposed G. barbata thalli to four extreme (28 °C, 30 °C, 32 °C, 34 °C) and one favourable (18 °C) temperature. We then monitored morphological and physiological responses. KEY RESULTS: Our findings indicate a significant rise in frequency, duration, and intensity of MHWs over decades on the southern Istrian coast. Experimental results show that G. barbata demonstrates both morphological and physiological recovery potential after exposure to temperatures as high as 32 °C. However, exposure to 34 °C led to thallus decay, with limited regeneration ability. CONCLUSIONS: Our results show that G. barbata has a remarkable resilience to long term exposure to extreme temperatures up to 32 °C and suggests that short term exposure to temperatures beyond this, as currently recorded inside the lagoon, do not notably affect the physiology or morphology of local G. barbata. With more MHWs expected in the future, such an adapted population may represent an important donor suitable for future restoration activities along the Istrian coast. These results emphasize the resilience of this unique population, but also warns of the vulnerability of marine macroalgal forests to rising seawater temperatures under rapidly changing climate conditions.

First Restoration Experiment for Endemic Fucus virsoides on the Western Istrian Coast-Is It Feasible?

Fucus virsoides is an endemic species of the Mediterranean limited to the Adriatic Sea. In recent decades, it has undergone a severe regression, which is well documented in the northern Adriatic. To develop a tool for mitigating this problem, we tested the feasibility of F. virsoides restoration and designed a very simple yet effective method for ex situ cultivation and planting. We also tested the effect of positioning in the upper vs. lower intertidal on the growth of F. virsoides. After planting, the algae reached fertility in nine months, which was followed by a period of stagnation and reduction in size due to grazing and fouling. There were some differences in growth of the algae according to positioning in the intertidal at different measurement times, but that had little impact on the overall success of the restoration experiment. This represents, to our knowledge, the first successful F. virsoides ex situ cultivation and restoration attempt.

Temporal variation in the prokaryotic community of a nearshore marine environment.

Prokaryotic communities inhabiting surface waters of temperate areas exhibit patterns of seasonal succession. Generally, studies describing these temporal changes are not performed in the proximity to the coast. In the present study, temporal variation of these communities was determined in surface waters at two stations located in the close proximity to the eastern shore of the northern Adriatic Sea. Sequencing of the V4 region of the 16S rRNA gene identified the highest community richness in December with distinct shifts in community structure between periods from April to May, June to October, and November to March. Temperature was shown to be the main environmental force explaining community temporal variation. The NS5 marine group, uncultured Cryomorphaceae, SAR86 clade, and Synechococcus were present throughout the year. Members without know relatives within Rhodobacteraceae and the NS4 marine group were more pronounced in the period from April to May, the AEGEAN-169 marine group, SAR11 subclade III, and HIMB11 in the period from June to October, and SAR11 subclade Ia and Archaea in the period from November to March. Litoricola and OM60 (NOR5) clade were characteristic for both the community sampled from April to May and November to March. Taken together, prokaryotic communities inhabiting nearshore surface waters exhibit a general pattern in community structure similar to other surface associated assemblages of temperate areas. However, the identified specific community composition and temporal patterns differ from other coastal areas.

Seasonal Dynamics of Epiphytic Microbial Communities on Marine Macrophyte Surfaces.

Surfaces of marine macrophytes are inhabited by diverse microbial communities. Most studies focusing on epiphytic communities of macrophytes did not take into account temporal changes or applied low sampling frequency approaches. The seasonal dynamics of epiphytic microbial communities was determined in a meadow of Cymodocea nodosa invaded by Caulerpa cylindracea and in a monospecific settlement of C. cylindracea at monthly intervals. For comparison the ambient prokaryotic picoplankton community was also characterized. At the OTU level, the microbial community composition differed between the ambient water and the epiphytic communities exhibiting host-specificity. Also, successional changes were observed connected to the macrophyte growth cycle. Taxonomic analysis, however, showed similar high rank taxa (phyla and classes) in the ambient water and the epiphytic communities, with the exception of Desulfobacterota, which were only found on C. cylindracea. Cyanobacteria showed seasonal changes while other high rank taxa were present throughout the year. In months of high Cyanobacteria presence the majority of cyanobacterial sequences were classified as Pleurocapsa. Phylogenetic groups present throughout the year (e.g., Saprospiraceae, Rhodobacteraceae, members without known relatives within Gammaproteobacteria, Desulfatitalea, and members without known relatives within Desulfocapsaceae) constituted most of the sequences, while less abundant taxa showed seasonal patterns connected to the macrophyte growth cycle. Taken together, epiphytic microbial communities of the seagrass C. nodosa and the macroalga C. cylindracea appear to be host-specific and contain taxa that undergo successional changes.

Selective DNA and Protein Isolation From Marine Macrophyte Surfaces.

Studies of unculturable microbes often combine methods, such as 16S rRNA sequencing, metagenomics, and metaproteomics. To apply these techniques to the microbial community inhabiting the surfaces of marine macrophytes, it is advisable to perform a selective DNA and protein isolation prior to the analysis to avoid biases due to the host material being present in high quantities. Two protocols for DNA and protein isolation were adapted for selective extractions of DNA and proteins from epiphytic communities inhabiting the surfaces of two marine macrophytes, the seagrass Cymodocea nodosa and the macroalga Caulerpa cylindracea. Protocols showed an almost complete removal of the epiphytic community regardless of the sampling season, station, settlement, or host species. The obtained DNA was suitable for metagenomic and 16S rRNA sequencing, while isolated proteins could be identified by mass spectrometry. Low presence of host DNA and proteins in the samples indicated a high specificity of the protocols. The procedures are based on universally available laboratory chemicals making the protocols widely applicable. Taken together, the adapted protocols ensure an almost complete removal of the macrophyte epiphytic community. The procedures are selective for microbes inhabiting macrophyte surfaces and provide DNA and proteins applicable in 16S rRNA sequencing, metagenomics, and metaproteomics.

Mesozooplankton taurine production and prokaryotic uptake in the northern Adriatic Sea.

Dissolved free taurine, an important osmolyte in phytoplankton and metazoans, has been shown to be a significant carbon and energy source for prokaryotes in the North Atlantic throughout the water column. However, the extent of the coupling between taurine production and consumption over a seasonal cycle has not been examined yet. We determined taurine production by abundant crustacean zooplankton and its role as a carbon and energy source for several prokaryotic taxa in the northern Adriatic Sea over a seasonal cycle. Taurine concentrations were generally in the low nanomolar range, reaching a maximum of 22 nmol L-1 in fall during a Pseudonitzschia bloom and coinciding with the highest zooplankton taurine release rates. Taurine accounted for up to 5% of the carbon, 11% of the nitrogen, and up to 71% of the sulfur requirements of heterotrophic prokaryotes. Members of the Roseobacter clade, Alteromonas, Thaumarchaeota, and Euryarchaeota exhibited higher cell-specific taurine assimilation rates than SAR11 cells. However, cell-specific taurine and leucine assimilation were highly variable in all taxa, suggesting species and/or ecotype specific utilization patterns of taurine and dissolved free amino acids. Copepods were able to cover the bulk taurine requirements of the prokaryotic communities in fall and winter and partly in the spring-summer period. Overall, our study emphasizes the significance of taurine as a carbon and energy source for the prokaryotic community in the northern Adriatic Sea and the importance of crustacean zooplankton as a significant source of taurine and other organic compounds for the heterotrophic prokaryotic community.

Seasonal dynamics of marine snow-associated and free-living demethylating bacterial communities in the coastal northern Adriatic Sea.

The extent of DMSP demethylation has been hypothesized to depend on DMSP availability and bacterial sulfur demand, which might lead to niche differentiation of the demethylating bacterial community. In this study, we determined DMSP concentrations in marine snow and the ambient water over a seasonal cycle and linked DMSP concentrations to the abundance of bacteria harbouring the demethylation dmdA gene in the Adriatic Sea. In marine snow, DMSP concentrations were up to four times higher than in the ambient water and three times higher in marine snow in summer than in winter. The average dmdA:recA gene ratio over the sampling period was 0.40 ± 0.24 in marine snow and 0.48 ± 0.21 in the ambient water. However, at the subclade level, differences in the demethylating bacterial community of marine snow and the ambient water were apparent. Seasonal patterns of potentially demethylating bacteria were best visible at the oligotype level. In the ambient water, the SAR116 and the OM60/NOR5 clade were composed of oligotypes that correlated to high DMSP concentrations, while oligotypes of the Rhodospirillales correlated to low DMSP concentrations. Our results revealed a pronounced seasonal variability and spatial heterogeneity in DMSP concentrations and the associated demethylating bacterial community.

Bivalve trophic ecology in the Mediterranean: Spatio-temporal variations and feeding behavior.

The trophic ecology of two bivalves, the clam Callista chione and the cockle Glycymeris bimaculata was studied using environmental and biochemical variables of the suspended particulate matter and the sediment. Samples were collected from two shallow sites, Pag and Cetina, in the coastal oligotrophic Mediterranean Sea, during a 17 month period. The temporal variation of the particulate matter reflected a mixture between marine and terrestrial sources throughout the year, with a clear marine influence during summer and fall, and predominance of terrestrial inputs during spring and winter. The digestive gland was a useful rapid turnover tissue, where the carbon isotope signal was species-specific and the nitrogen isotope one was site-specific. FA markers in the digestive gland revealed a mixed diet where Callista chione fed more upon fresh material than G. bimaculata which relied largely on bacteria-derived detritus. Overall, little feeding niche overlap was observed between the two species during the year, indicating resource partitioning, expected for a food-limited system. The present trophic ecology study in co-occurring species allowed identifying species-specific feeding adaptations to environmental variability.

Seasonal variations in extracellular enzymatic activity in marine snow-associated microbial communities and their impact on the surrounding water.

Seasonal changes of microbial abundance and associated extracellular enzymatic activity in marine snow and in seawater were studied in the northern Adriatic during a three-year period. Marine snow was present during the entire period of investigation, although in higher concentrations during summer than during winter. Microorganisms densely colonized marine snow and aggregate-associated enzymatic activity was substantially higher (up to 105 times) than in seawater. Alkaline phosphatase activity (APA) and aminopeptidase activity in marine snow showed seasonal variations with higher values in late spring-summer than in autumn-winter, probably in response to changes in the quantity and quality of organic matter. The highest cell-specific bacterial activity was found for phosphatase, followed by peptidase, and the lowest was for glucosidases. Differential hydrolysis of marine snow-derived organic matter points to the well-known phosphorus limitation of the northern Adriatic and indicates preferential utilization of phosphorus- and nitrogen-rich organic compounds by microbes, while hydrolysis of polysaccharides seemed to be less important. In oligotrophic conditions during summer, organic matter released from marine snow might represent a significant source of substrate for free-living bacteria in seawater. For the first time microorganisms producing APA in marine snow were identified, revealing that dense populations of bacteria expressed APA, while cyanobacteria did not. Cyanobacteria proliferating in marine snow could benefit from phosphorus release by bacteria and nanoflagellates.

Bioprospecting for Genes Encoding Hydrocarbon-Degrading Enzymes from Metagenomic Samples Isolated from Northern Adriatic Sea Sediments.

Three metagenomic libraries were constructed using surface sediment samples from the northern Adriatic Sea. Two of the samples were taken from a highly polluted and an unpolluted site respectively. The third sample from a polluted site had been enriched using crude oil. The results of the metagenome analyses were incorporated in the REDPET relational database (http://redpet.bioinfo.pbf.hr/REDPET), which was generated using the previously developed MEGGASENSE platform. The database includes taxonomic data to allow the assessment of the biodiversity of metagenomic libraries and a general functional analysis of genes using hidden Markov model (HMM) profiles based on the KEGG database. A set of 22 specialised HMM profiles was developed to detect putative genes for hydrocarbon-degrading enzymes. Use of these profiles showed that the metagenomic library generated after selection on crude oil had enriched genes for aerobic n-alkane degradation. The use of this system for bioprospecting was exemplified using potential alkB and almA genes from this library.

Limited influence of primary treated sewage waters on bacterial abundance, production and community composition in coastal seawaters.

The response of bacteria in terms of abundance, production and community structure to changes induced by the discharge of primary treated sewage waters was investigated combining microbiological, chemical and molecular tools. The primary treatment did not affect substantially the bacterial community structure in wastewaters and did not reduce the concentrations of fecal indicators. The spatial distribution of the sewage plume was governed by vertical stratification and currents. Bacterial abundance and production in the sea receiving waste waters depended predominantly on environmental conditions. In the waters with the highest concentration of fecal pollution indicators the bacterial community was characterized by allochthonous bacteria belonging to Epsilonproteobacteria, Firmicutes, Gammaproteobacteria and Bacteroidetes. The latter two taxa were also present in unpolluted waters but had a different structure, typical for oligotrophic environments. Although the impact of primary treated sewage waters was limited, a sanitary risk persisted due to the relevant presence of potentially pathogenic bacteria.

Organic aggregates formed by benthopleustophyte brown alga Acinetospora crinita (Acinetosporaceae, Ectocarpales).

This work presents the elemental, polysaccharide, and fatty acid compositions of benthic aggregates formed by the filamentous brown alga Acinetospora crinita, which are widely spread on the rocky bottoms of the Mediterranean Sea. The aggregates can be characterized as mineralized centers in which regeneration of nutrients and recycling of dissolved organic matter actively occur and favor the development of an abundant phytoplankton community. Analyses of the stable isotopes of C and N display their marine origin and could provide evidence of the processes that occur inside/outside of the aggregates. The monosaccharide compositions of Adriatic and Tyrrhenian mucilages produced by brown alga A. crinita were quite similar. In particular, the Adriatic sample compositions resembled the average composition of the Tyrrhenian high molecular weight exopolymers, and the observed differences could be ascribed to different degradation stages. The fatty acid patterns found for the aggregates were similar to those observed in the isolated A. crinita algae with variable contributions from embedded diatom species. The bacterial contribution to the fatty acid pool was quite low, most likely due to the known poor conditions for their heterotrophic growth.

Relationships between heterotrophic bacteria and cyanobacteria in the northern Adriatic in relation to the mucilage phenomenon.

High variability of heterotrophic bacterial (HB; 0.1.10(9)-6.10(9) cells L(-1)), nanoflagellates (HNF; 0.02.10(6)-2.4.10(6) cells L(-1)) and cyanobacterial (CB; 10(6)-700.10(6) cells L(-1)) abundances were observed during approximately monthly measurements at six stations along the transect Po Delta-Rovinj from March 1999 to August 2002. Substantially higher values were observed during the stratification period (June-September) in the surface layer of the western stations that were under more direct influence of Po River discharges. Changes of the HB abundance were significantly correlated with temperature in the entire water column and chlorophyll a (Chla) concentration in the surface layer. The nutrients did not look to have directly influenced the HB growth, except orthophosphate in the eastern, more oligotrophic part of transect, where probably HB efficiently competed with phytoplankton for this nutrient. Temperature was also important for CB growth that, however, appeared to occur more intensively in waters with low nutrient concentrations. Probably, in these conditions CB can still develop, while larger autotrophs are strongly nutrient limited. In the upper water column of reduced salinity (35-37), the contributions of CB carbon biomass to the total picoplankton biomass (CB+HB) were mostly larger than 30% (up to 80%) in years with mucilage events (1991, 2000-2002) than in other years. For example, in 1999 only a few values were higher than 30% (up to 50%). In abundant presence of mucilaginous aggregates (e.g. in June 2000 and late June 2002) the chlorophyll a ascribed to CB accounted for much larger portions of total measured Chla. From these results it was concluded that cyanobacteria during mucilage events may play a substantially increased role both as primary producers and prey within the microbial loop of the northern Adriatic.

Fatty acid profiling of microbial community during aging of mucilaginous aggregates in the northern Adriatic.

Aggregates differing in size and phytoplankton community composition were sampled in winter/spring 2001 and summer 1997 and 2002 (during mucilaginous event) in the northern Adriatic Sea. The fatty acid profiles (FAME) were determined in aggregates, bacterial population was grown from each aggregate and each bacterial isolate from aggregate's plated cultures. All aggregates irrespective of the season, aggregate size or phytoplankton community composition contained isolates from three distinct groups, with fatty acid profiles corresponding to alpha-Proteobacteria, gamma-Proteobacteria and Cytophaga-Flavobacter (CF) complex but in different relative proportions. Fatty acid profiles of the bacterial population grown from each aggregate revealed either the domination of one group (gamma, alpha or CF) or contribution of more groups (gamma and CF; alpha and CF). Specifically, as the aggregates age a shift in favor of bacteria belonging to CF-complex and corresponding decrease in bacteria similar to that of Proteobacteria occur. During the aggregate aging process and degradation of the mucous matrix, besides bacterial succession, the phytoplankton growth took place inside the aggregates. Such an aging pattern was confirmed through laboratory experiments with aggregates inhabited by the diatom Cylindrotheca closterium.