Atlas of phytoplankton phenology indices in selected Eastern Mediterranean marine ecosystems.

Phytoplankton is a fundamental component of marine food webs and play a crucial role in marine ecosystem functioning. The phenology (timing of growth) of these microscopic algae is an important ecological indicator that can be utilized to observe its seasonal dynamics, and assess its response to environmental perturbations. Ocean colour remote sensing is currently the only means of obtaining synoptic estimates of chlorophyll-a (a proxy of phytoplankton biomass) at high temporal and spatial resolution, enabling the calculation of phenology metrics. However, ocean colour observations have acknowledged weaknesses compromising its reliability, while the scarcity of long-term in situ data has impeded the validation of satellite-derived phenology estimates. To address this issue, we compared one of the longest available in situ time series (20 years) of chlorophyll-a concentrations in the Eastern Mediterranean Sea (EMS), along with concurrent remotely-sensed observations. The comparison revealed a marked coherence between the two datasets, indicating the capability of satellite-based measurements in accurately capturing the phytoplankton seasonality and phenology metrics (i.e., timing of initiation, duration, peak and termination) in the studied area. Furthermore, by studying and validating these metrics we constructed a satellite-derived phytoplankton phenology atlas, reporting in detail the seasonal patterns in several sub-regions in coastal and open seas over the EMS. The open waters host higher concentrations from late October to April, with maximum levels recorded during February and lowest during the summer period. The phytoplankton growth over the Northern Aegean Sea appeared to initiate at least a month later than the rest of the EMS (initiating in late November and terminating in late May). The coastal waters and enclosed gulfs (such as Amvrakikos and Maliakos), exhibit a distinct seasonal pattern with consistently higher levels of chlorophyll-a and prolonged growth period compared to the open seas. The proposed phenology atlas represents a useful resource for monitoring phytoplankton growth periods in the EMS, supporting water quality management practices, while enhancing our current comprehension on the relationships between phytoplankton biomass and higher trophic levels (as a food source).

Depth-driven patterns in lytic viral diversity, auxiliary metabolic gene content, and productivity in offshore oligotrophic waters.

Introduction: Marine viruses regulate microbial population dynamics and biogeochemical cycling in the oceans. The ability of viruses to manipulate hosts' metabolism through the expression of viral auxiliary metabolic genes (AMGs) was recently highlighted, having important implications in energy production and flow in various aquatic environments. Up to now, the presence and diversity of viral AMGs is studied using -omics data, and rarely using quantitative measures of viral activity alongside. Methods: In the present study, four depth layers (5, 50, 75, and 1,000 m) with discrete hydrographic features were sampled in the Eastern Mediterranean Sea; we studied lytic viral community composition and AMG content through metagenomics, and lytic production rates through the viral reduction approach in the ultra-oligotrophic Levantine basin where knowledge regarding viral actions is rather limited. Results and Discussion: Our results demonstrate depth-dependent patterns in viral diversity and AMG content, related to differences in temperature, nutrients availability, and host bacterial productivity and abundance. Although lytic viral production rates were similar along the water column, the virus-to-bacteria ratio was higher and the particular set of AMGs was more diverse in the bathypelagic (1,000 m) than the shallow epipelagic (5, 50, and 75 m) layers, revealing that the quantitative effect of viruses on their hosts may be the same along the water column through the intervention of different AMGs. In the resource- and energy-limited bathypelagic waters of the Eastern Mediterranean, the detected AMGs could divert hosts' metabolism toward energy production, through a boost in gluconeogenesis, fatty-acid and glycan biosynthesis and metabolism, and sulfur relay. Near the deep-chlorophyll maximum depth, an exceptionally high percentage of AMGs related to photosynthesis was noticed. Taken together our findings suggest that the roles of viruses in the deep sea might be even more important than previously thought as they seem to orchestrate energy acquisition and microbial community dynamics, and thus, biogeochemical turnover in the oceans.

Metal and metallothionein concentrations in mesozooplankton from an oligotrophic offshore area in the eastern Mediterranean Sea (Cretan Passage/Levantine Sea).

Spatial variability of Cd, Cu, Cr, Ni, Zn, Fe, Mn, Pb and metallothionein (MT) concentrations were determined in mesozooplankton samples along the west-east axis of the Cretan Passage in the western Levantine Sea (Eastern Mediterranean). Metal and MT values from the present study are proposed as background levels, due to the lack of substantial anthropogenic activities in the area, where only maritime traffic and atmospheric deposition could be potential sources. Higher concentrations, of both metals and MTs, were recorded mainly at the western stations indicating higher metal bioavailability than in the eastern part. An inverse relationship of the metal zooplankton levels with zooplankton biomass, abundance and vital rates (production, respiration and ingestion rates), as well as salinity, was evident. We discuss the hypothesis that physical and biological characteristics of the marine environment, affecting growth dynamics of phytoplankton and zooplankton communities, may also act on metal uptake in oligotrophic marine systems.

Phytoplankton community structure changes during autumn and spring in response to environmental variables in Methana, Saronikos Gulf, Greece.

Phytoplankton community was investigated during two contrasting periods using offshore plankton samples in the volcanic area of Methana peninsula (Saronikos Gulf): the first at early autumn (warm period, September 2016) and the second one at early spring (cold period, March 2017). In order to investigate the phytoplankton community structure in the complex geo-biochemical conditions of the area, samples were collected from stations near the CO2 hydrothermal vents, at the hydrothermal sulfur and radioactive springs and at a fishery nearby Methana town. Three major phytoplankton groups, Bacillariophyceae, Dinophyceae, and Prymnesiophyceae, were studied, using inverted microscopy. In early autumn, Dinophyceae were dominant in the majority of the stations with cell concentrations of Prorocentrum spp. up to ~ 35.5 × 103 cells l-1. In early spring, the dominant class was Bacillariophyceae with dominant genus Nitzschia/Pseudo-nitzschia presenting cell concentrations up to ~ 33.9 × 103 cells l-1. Furthermore, Prymnesiophyceae appeared in both spring and autumn samples with small fluctuations. Total phytoplankton cell concentrations followed a seasonal trend, presenting slightly lower values in the hydrothermal-effected area in comparison with the broader Saronikos Gulf, confirming the prevalence of oligotrophic conditions. Seasonal variation was very strong, revealing an association with water temperature and nutrient content. Those environmental variables proved to have a strong effect that was reflected in the phytoplankton community structure.

Grazing effect of flagellates on bacteria in response to phosphate addition in the oligotrophic Cretan Sea, NE Mediterranean.

The planktonic food web in the oligotrophic Mediterranean Sea is dominated by small-sized (<20 µm) microbes, with nanoflagellates being the major bacterial grazers and the main participants in nutrient cycling. Phosphate is a key nutrient in the P-limited Cretan Sea (NE Mediterranean) and P-availability can affect its trophic dynamics. Here, we examined the grazing potential of heterotrophic (HF) and pigmented (PF) nanoflagellates as a response mechanism to phosphate amendment. Flagellate grazing effect on bacteria was quantified in P-amended nutrient-depleted water from the Cretan Sea over the course of 4 days using microcosm experiments. P-addition positively affected HF abundance, while PF abundance remained unchanged. At the community level, P-addition had a negative effect on PF bacterial removal rates. In the control, PF-grazing rate was significantly higher than that of HF throughout the experiment. Pigment analysis showed no changes in phytoplankton community composition as a result of P-addition, indicating that PF grazing rate declined as a physiological response of the cells. The present study emphasizes the dominant grazing role of PF under P-depleted conditions and reveals that during the late stratified season PF respond to P-addition by lowering their grazing rates, enhancing the relative importance of bacterial removal by HF.

Coccolithophore community response along a natural CO2 gradient off Methana (SW Saronikos Gulf, Greece, NE Mediterranean).

A natural pH gradient caused by marine CO2 seeps off the Methana peninsula (Saronikos Gulf, eastern Peloponnese peninsula) was used as a natural laboratory to assess potential effects of ocean acidification on coccolithophores. Coccolithophore communities were therefore investigated in plankton samples collected during September 2011, September 2016 and March 2017. The recorded cell concentrations were up to ~50 x103 cells/l, with a high Shannon index of up to 2.8, along a pH gradient from 7.61 to 8.18, with values being occasionally <7. Numerous holococcolithophore species represented 60-90% of the surface water assemblages in most samples during September samplings. Emiliania huxleyi was present only in low relative abundances in September samples, but it dominated in March assemblages. Neither malformed nor corroded coccolithophores were documented. Changes in the community structure can possibly be related to increased temperatures, while the overall trend associates low pH values with high cell densities. Our preliminary results indicate that in long-termed acidified, warm and stratified conditions, the study of the total coccolithophore assemblage may prove useful to recognize the intercommunity variability, which favors the increment of lightly calcified species such as holococcolithophores.

Ultrasensitive and high-throughput analysis of chlorophyll a in marine phytoplankton extracts using a fluorescence microplate reader.

Chlorophyll a (Chl a) is the predominant pigment in every single photosynthesizing organism including phytoplankton and one of the most commonly measured water quality parameters. Various methods are available for Chl a analysis, but the majority of them are of limited throughput and require considerable effort and time from the operator. The present study describes a high-throughput, microplate-based fluorometric assay for rapid quantification of Chl a in phytoplankton extracts. Microplate sealing combined with ice cooling was proved an effective means for diminishing solvent evaporation during sample loading and minimized the analytical errors involved in Chl a measurements with a fluorescence microplate reader. A set of operating parameters (settling time, detector gain, sample volume) were also optimized to further improve the intensity and reproducibility of Chl a fluorescence signal. A quadratic regression model provided the best fit (r 2 = 0.9998) across the entire calibration range (0.05-240 pg µL-1). The method offered excellent intra- and interday precision (% RSD 2.2 to 11.2%) and accuracy (% relative error -3.8 to 13.8%), while it presented particularly low limits of detection (0.044 pg µL-1) and quantification (0.132 pg µL-1). The present assay was successfully applied on marine phytoplankton extracts, and the overall results were consistent (average % relative error -14.8%) with Chl a concentrations (including divinyl Chl a) measured by high-performance liquid chromatography (HPLC). More importantly, the microplate-based method allowed the analysis of 96 samples/standards within a few minutes, instead of hours or days, when using a traditional cuvette-based fluorometer or an HPLC system. Graphical abstract TChl a concentrations (i.e. sum of Chl a and divinyl Chl a in ng L-1) measured in seawater samples by HPLC and fluorescence microplate reader.