Increased contribution of parasites in microbial eukaryotic communities of different Aegean Sea coastal systems.

Background-Aim: Protistan communities have a major contribution to biochemical processes and food webs in coastal ecosystems. However, related studies are scarce and usually limited in specific groups and/or sites. The present study examined the spatial structure of the entire protistan community in seven different gulfs and three different depths in a regional Mediterranean Sea, aiming to define taxa that are important for differences detected in the marine microbial network across the different gulfs studied as well as their trophic interactions. Methods: Protistan community structure analysis was based on the diversity of the V2-V3 hypervariable region of the 18S rRNA gene. Operational taxonomic units (OTUs) were identified using a 97% sequence identity threshold and were characterized based on their taxonomy, trophic role, abundance and niche specialization level. The differentially abundant, between gulfs, OTUs were considered for all depths and interactions amongst them were calculated, with statistic and network analysis. Results: It was shown that Dinophyceae, Bacillariophyta and Syndiniales were the most abundant groups, prevalent in all sites and depths. Gulfs separation was more striking at surface corroborating with changes in environmental factors, while it was less pronounced in higher depths. The study of differentially abundant, between gulfs, OTUs revealed that the strongest biotic interactions in all depths occurred between parasite species (mainly Syndiniales) and other trophic groups. Most of these species were generalists but not abundant highlighting the importance of rare species in protistan community assemblage. Conclusion: Overall this study revealed the emergence of parasites as important contributors in protistan network regulation regardless of depth.

Sources of major elements and nutrients in the water cycle of an undisturbed river basin - Samothraki Island, Greece.

We studied the origin of elements of an undisturbed stream basin during the dry season as derived by atmospheric inputs and lithological processes. Α mass balance model was applied taking into account atmospheric (rain and vapor) inputs and their origin from marine aerosol and dust, as well as the contribution of rock mineral weathering and dissolution of soluble salts. The model results were enhanced using element enrichment factors, element ratios and water stable isotopes. Weathering and dissolution of bedrock and soil minerals contributed the main element portions, besides sodium and sulfate that chiefly derived from wet deposition. Vapor was shown to contribute water to inland waters of the basin. However, rain was the main source of elements compared to vapor, with marine aerosol being the only atmospheric chloride source, contributing also over 60 % of atmospheric sodium and magnesium. Silicate derived from mineral weathering (mainly plagioclase and amorphous silica), while soluble salt dissolution contributed the main portions of the rest of major elements. In headwater springs and streams, element concentrations were more affected by atmospheric inputs and silicate mineral weathering was more intense, contrary to lowland waters that were more affected by soluble salt dissolution. Effective self-purification processes were mirrored in low nutrient levels, despite the significant inputs from wet deposition, with rain being more important contributor than vapor for the majority of nutrient species. Relatively high nitrate concentrations in headwaters were attributed to increased mineralization and nitrification, while the downstream nitrate diminishing was due to prevailing denitrification processes. The ultimate goal of this study is to contribute in establishing stream elements' reference conditions using mass balance modeling approaches.

Spatial insurance against a heatwave differs between trophic levels in experimental aquatic communities.

Climate change-related heatwaves are major threats to biodiversity and ecosystem functioning. However, our current understanding of the mechanisms governing community resistance to and recovery from extreme temperature events is still rudimentary. The spatial insurance hypothesis postulates that diverse regional species pools can buffer ecosystem functioning against local disturbances through the immigration of better-adapted taxa. Yet, experimental evidence for such predictions from multi-trophic communities and pulse-type disturbances, like heatwaves, is largely missing. We performed an experimental mesocosm study to test whether species dispersal from natural lakes prior to a simulated heatwave could increase the resistance and recovery of plankton communities. As the buffering effect of dispersal may differ among trophic groups, we independently manipulated the dispersal of organisms from lower (phytoplankton) and higher (zooplankton) trophic levels. The experimental heatwave suppressed total community biomass by having a strong negative effect on zooplankton biomass, probably due to a heat-induced increase in metabolic costs, resulting in weaker top-down control on phytoplankton. While zooplankton dispersal did not alleviate the negative heatwave effects on zooplankton biomass, phytoplankton dispersal enhanced biomass recovery at the level of primary producers, providing partial evidence for spatial insurance. The differential responses to dispersal may be linked to the much larger regional species pool of phytoplankton than of zooplankton. Our results suggest high recovery capacity of community biomass independent of dispersal. However, community composition and trophic structure remained altered due to the heatwave, implying longer-lasting changes in ecosystem functioning.

Geology Can Drive the Diversity-Ecosystem Functioning Relationship in River Benthic Diatoms by Selecting for Species Functional Traits.

The biodiversity-ecosystem functioning (BEF) relationship has been studied extensively for the past 30 years, mainly in terrestrial plant ecosystems using experimental approaches. Field studies in aquatic systems are scarce, and considering primary producers, they mainly focus on phytoplankton assemblages, whereas benthic diatoms in rivers are considerably understudied in this regard. We performed a field study across nine rivers in Greece, and we coupled the observed field results with model simulations. We tested the hypothesis that the diversity-biomass (as a surrogate of ecosystem functioning) relationship in benthic diatoms would be affected by abiotic factors and would be time-dependent due to the highly dynamic nature of rivers. Indeed, geology played an important role in the form of the BEF relationship that was positive in siliceous and absent in calcareous substrates. Geology was responsible for nutrient concentrations, which, in turn, were responsible for the dominance of specific functional traits. Furthermore, model simulations showed the time dependence of the BEF form, as less mature assemblages tend to present a positive BEF. This was the first large-scale field study on the BEF relationship of benthic diatom assemblages, offering useful insights into the function and diversity of these overlooked ecosystems and assemblages.

REVIVE: A feasibility assessment tool for freshwater fish conservation translocations in Mediterranean rivers.

Conservation translocation is a management action applied for population recovery of threatened freshwater fishes, often however with partially successful outcome, mainly due to inadequate feasibility assessment prior to the translocation. Up to date, feasibility assessments have been mainly focused on economically important species (e.g., salmonids) inhabiting perennial rivers, while little attention has been given to fish translocations in rivers in Mediterranean climate areas. In this study, we developed a robust feasibility assessment tool for freshwater fish translocations in Mediterranean-type riverine ecosystems within an interdisciplinary, multispecies approach. The REVIVE tool integrates quantitative and semi-quantitative data, incorporates uncertainty and consists of two main components. The first component is the evaluation of the potential release water bodies (R-WBs) for their suitability for the planned translocation, incorporating a number of essential criteria for Mediterranean rivers, with emphasis on flow regime and habitat quantity. Additional criteria include the current and historical presence of the target species, water and biological quality, habitat suitability in terms of the ecological requirements of the target species, alien invasive species' pressure, and hydromorphological pressures, including their mitigation potential. The second component is the evaluation of the potential source water bodies (S-WBs) in terms of genetic compatibility and provision of a sufficient number of propagules. A trial application in a Mediterranean basin (Vassilopotamos River, Southern Greece) for the potential translocation of two threatened cyprinids in five R-WBs indicated the robustness of the tool. This integrative, flexible tool combines several elements identified as essential in reintroduction biology and can have wider applications, for a multitude of freshwater fish taxa and riverine systems, maximizing the success of planned translocation actions by natural resources' managers. Modifications to enable its transferability to other river types or fish taxa are also discussed.

Microalgae show a range of responses to exometabolites of foreign species.

Studies on microalgae interspecific interactions have so far focused either on nutrient competition or allelopathic effects due to excreted substances from Harmful Algal Bloom (HAB) species. Evidence from plants, bacteria and specific microalgae groups, point to a range of responses mediated by sensing or direct chemical impact of exometabolites from foreign species. Such processes remain under-investigated, especially in non-HAB microalgae, despite the importance of such knowledge in ecology and industrial applications. Here, we study the directional effect of exometabolites of 4 "foreign" species Heterosigma akashiwo, Phaeocystis sp., Tetraselmis sp. and Thalassiosira sp. to each of three "target" species across a total of 12 treatments. We disentangle these effects from nutrient competition by adding cell free medium of each "foreign" species into our treatment cultures. We measured the biomass response, to the foreign exometabolites, as cell number and photosynthetic biomass (Chla), whereas nutrient use was measured as residual phosphorus (PO4) and intracellular phosphorus (P). Exometabolites from filtrate of foreign species were putatively annotated by untargeted metabolomics analysis and were discussed in association to observed responses of target species. Among others, these metabolites included L-histidinal, Tiliacorine and dimethylsulfoniopropionate (DMSP). Our findings show that species show a range of responses with the most common being biomass suppression, and less frequent biomass enhancement and intracellular P storage. Filtrate from the green microalgae Tetraselmis caused the most pronounced negative effects suggesting that non-HAB species can also cause negative chemical interference. A candidate metabolite inducing this response is L-histidinal which was measured in high abundance uniquely in Tetraselmis and its L-histidine form derived from bacteria was previously confirmed as a microalgal algicidal. H. akashiwo also induced biomass suppression on other microalgae and a candidate metabolite for this response is Tiliacorine, a plant-derived alkaloid with confirmed cytotoxic activity.

Gut Microbiota of Five Sympatrically Farmed Marine Fish Species in the Aegean Sea.

In this study, we hypothesized that sympatrically grown farmed fish, i.e. fish which experience similar environmental conditions and nutritionally similar diets, would have more convergent gut microbiota. Using a "common garden" approach, we identified the core microbiota and bacterial community structure differences between five fish species farmed in the same aquaculture site on the west coast of the Aegean Sea, Greece. The investigated individuals were at similar developmental stages and reared in adjacent (< 50 m) aquaculture cages; each cage had 15 kg fish m-3. The diets were nutritionally similar to support optimal growth for each fish species. DNA from the midgut of 3-6 individuals per fish species was extracted and sequenced for the V3-V4 region of the bacterial 16S rRNA. Only 3.9% of the total 181 operational taxonomic units (OTUs) were shared among all fish. Between 5 and 74 OTUs were unique to each fish species. Each of the investigated fish species had a distinct profile of dominant OTUs, i.e. cumulative relative abundance of ≥ 80%. Co-occurrence network analysis for each fish species showed that all networks were strongly dominated by positive correlations between the abundances of their OTUs. However, each fish species had different network characteristics suggesting the differential significance of the OTUs in each of the five fish species midgut. The results of the present study may provide evidence that adult fish farmed in the Mediterranean Sea have a rather divergent and species-specific gut microbiota profile, which are shaped independently of the similar environmental conditions under which they grow.

Nitrogen as the main driver of benthic diatom composition and diversity in oligotrophic coastal systems.

Phytoplankton is the main indicator group for eutrophication in coastal ecosystems, however its high dispersal potential does not enable the assessment of localized effects of coastal nutrient enrichment. Benthic diatoms are sessile microalgae associated with sandy substrates and have the potential to reflect more localized pollution impacts. Although benthic diatoms are widely used bioindicators in freshwater systems, they have rarely been used for assessing the eutrophication status of oligotrophic environments such as the eastern Mediterranean Sea. In the present study, we assess the efficiency of benthic diatoms as bioindicators of nutrient enrichment in oligotrophic coastal systems, by investigating the effect of different physicochemical conditions and nutrient concentrations on the assemblage composition, diversity and individual species populations. To do this, we sampled along a eutrophication gradient formed by anthropogenic nutrient inputs from a metropolitan area. The main driver of assemblage composition, diversity and biomass of diatoms was nitrogen concentration and its temporal and spatial changes. Nitrogen loadings were positively correlated with increased biomass of Cocconeis spp. and negatively correlated with Mastogloia spp. Our findings suggest that in coastal ecosystems of oligotrophic marine ecoregions, benthic diatom assemblage structure and specific taxonomic groups can be reliable predictors of coastal eutrophication offering higher spatial resolution compared to phytoplankton.

Everything is not everywhere: can marine compartments shape phytoplankton assemblages?

The idea that 'everything is everywhere, but the environment selects' has been seminal in microbial biogeography, and marine phytoplankton is one of the prototypical groups used to illustrate this. The typical argument has been that phytoplankton is ubiquitous, but that distinct assemblages form under environmental selection. It is well established that phytoplankton assemblages vary considerably between coastal ecosystems. However, the relative roles of compartmentalization of regional seas and site-specific environmental conditions in shaping assemblage structures have not been specifically examined. We collected data from coastal embayments that fall within two different water compartments within the same regional sea and are characterized by highly localized environmental pressures. We used principal coordinates of neighbour matrices (PCNM) and asymmetric eigenvector maps (AEM) models to partition the effects that spatial structures, environmental conditions and their overlap had on the variation in assemblage composition. Our models explained a high percentage of variation in assemblage composition (59-65%) and showed that spatial structure consistent with marine compartmentalization played a more important role than local environmental conditions. At least during the study period, surface currents connecting sites within the two compartments failed to generate sufficient dispersal to offset the impact of differences due to compartmentalization. In other words, our findings suggest that, even for a prototypical cosmopolitan group, everything is not everywhere.

Effects of olive mill wastewater discharge on benthic biota in Mediterranean streams.

Olive mill wastewaters (OMW) discharging in river ecosystems cause significant adverse effects on their water chemistry and biological communities. We here examined the effects of OMW loads in four streams of a Mediterranean basin characterized by changing flow. The diatom and macroinvertebrate community structures were compared between upstream (control) and downstream (impacted) sites receiving OMW discharge. We also tested if effects occurred at the organism level, i.e. the occurrence of deformities in diatom valves, and the sediment toxicity on the midge Chironomus riparius. We evaluated these effects through a two-year analysis, at various levels of chemical pollution and dilution capacity. The impacted sites had high phenol concentrations and organic carbon loads during and after olive mill (OM) operation, and were characterized by higher abundances of pollution-tolerant diatom and macroinvertebrate taxa. Diatom valve deformities occurred more frequently at the impacted sites. The development of C. riparius was affected by phenolic compounds and organic carbon concentrations in the sediments. The similarity in the diatom and macroinvertebrate assemblages between control and impacted sites decreased at lower flows. Diatoms were more sensitive in detecting deterioration in the biological status of OMW receiving waterways than macroinvertebrates. Our results indicate that the negative effects of OMW extended to the whole benthic community, at both assemblage and organism level.

Temporal phosphate gradients reveal diverse acclimation responses in phytoplankton phosphate uptake.

Phytoplankton face environmental nutrient variations that occur in the dynamic upper layers of the ocean. Phytoplankton cells are able to rapidly acclimate to nutrient fluctuations by adjusting their nutrient-uptake system and metabolism. Disentangling these acclimation responses is a critical step in bridging the gap between phytoplankton cellular physiology and community ecology. Here, we analyzed the dynamics of phosphate (P) uptake acclimation responses along different P temporal gradients by using batch cultures of the diatom Phaeodactylum tricornutum. We employed a multidisciplinary approach that combined nutrient-uptake bioassays, transcriptomic analysis, and mathematical models. Our results indicated that cells increase their maximum nutrient-uptake rate (Vmax) both in response to P pulses and strong phosphorus limitation. The upregulation of three genes coding for different P transporters in cells experiencing low intracellular phosphorus levels supported some of the observed Vmax variations. In addition, our mathematical model reproduced the empirical Vmax patterns by including two types of P transporters upregulated at medium-high environmental and low intracellular phosphorus levels, respectively. Our results highlight the existence of a sequence of acclimation stages along the phosphate continuum that can be understood as a succession of acclimation responses. We provide a novel conceptual framework that can contribute to integrating and understanding the dynamics and wide diversity of acclimation responses developed by phytoplankton.

Acute and sub-chronic toxicity bioassays of Olive Mill Wastewater on the Eastern mosquitofish Gambusia holbrooki.

Olive oil production generates large volumes of wastewaters mostly in peri-Mediterranean countries with adverse impacts on the biota of the receiving aquatic systems. Few studies have however documented its toxicity on aquatic species, with an almost total lack of relative studies on fish. We assessed the acute and sub-chronic OMW toxicity, as well as the acute and sub-chronic behavioural, morphological and biochemical effects of OMW exposure on the mosquitofish Gambusia holbrooki. LC50 values of the acute bioassays ranged from 7.31% (24 h) to 6.38% (96 h). Behavioural symptoms of toxicity included hypoactivity and a shift away from the water surface, coupled with a range of morphological alterations, such as skin damage, excessive mucus secretion, hemorrhages, fin rot and exophhalmia, with indications also of gill swelling and anemia. Biochemical assays showed that OMW toxicity resulted in induction of catalase (CAT) and inhibition of acetylcholinesterase (AChE) activities. The implications of our results at the level of environmental policy for the sustainable management of the olive mill industry, i.e. the effective restriction of untreated OMW disposal of in adjacent waterways, as well as the implementation of new technologies that reduce their impact (detoxification and/or revalorization of its residues) are discussed.

Multiple stressor effects on biodiversity and ecosystem functioning in a Mediterranean temporary river.

The hydrological and biological complexity of temporary rivers as well as their importance in providing goods and services is increasingly recognized, as much as it is the vulnerability of the biotic communities in view of climate change and increased anthropogenic pressures. However, the effects of flow intermittency (resulting from both seasonal variations and rising hydrological pressure) and pollution on biodiversity and ecosystem functioning have been overlooked in these ecosystems. We explore the way multiple stressors affect biodiversity and ecosystem functioning, as well as the biodiversity-ecosystem functioning (B-EF) relationship in a Mediterranean temporary river. We measured diversity of benthic communities (i.e. diatoms and macroinvertebrates) and related ecosystem processes (i.e. resource use efficiency-RUE and organic matter breakdown-OMB) across a pollution and flow intermittency gradient. Our results showed decreases in macroinvertebrate diversity and the opposite trend in diatom assemblages, whereas ecosystem functioning was negatively affected by both pollution and flow intermittency. The explored B-EF relationships showed contrasting results: RUE decreased with higher diatom diversity, whereas OMB increased with increased macroinvertebrate diversity. The different responses suggest contrasting operating mechanisms, selection effects possibly driving the B-EF relationship in diatoms and complementarity effects driving the B-EF relationship in macroinvertebrates. The understanding of multiple stressor effects on diversity and ecosystem functioning, as well as the B-EF relationship in temporary rivers could provide insights on the risks affecting ecosystem functioning under global change.

Potentially toxic elements in water, sediments and fish of the Evrotas River under variable water discharges.

Among different stressors like drought, hydro-morphological alterations, and pollution from agricultural activities, nutrients, organic compounds and discharges from wastewater treatment plants (WWTPs), potentially toxic elements (PTE) may also contribute to the overall pollution of the Evrotas River, Greece. Nevertheless, information on pollution of elements in water and sediments in this river is scarcely documented. There is also no information available on the impact of elemental pollution from the aquatic environmental compartments on biota. To fill these gaps, in this study, water, sediment and fish samples were collected from four sampling sites along the Evrotas River under variable flow regimes (July 2015, higher discharge; June 2016, low discharge and September 2016, minimum discharge). Total and dissolved element concentrations in water samples, total and acetic acid extractable contents in sediments, and element concentrations in fish samples were determined by inductively coupled plasma mass spectrometry and significant relationships between samples were established using correlation analysis. The concentrations of PTE (Ni, Cr, Cd, As, Pb, Zn and Cu) in water were generally low, while elevated Ni and Cr contents were found in sediments (up to 150 and 300 mg/kg, respectively), with total Cr concentration in water and sediment being positively correlated. The ecological risk posed by the simultaneous presence of PTE in sediments evaluated by calculating the Probable Effect Concentration Coefficient (PEC-Q), demonstrated that PEC-Qs, which were above the critical value of 0.34, derived mostly from Cr and Ni inputs. Since their mobile sediment fraction was extremely low, Cr and Ni origin is most probably geogenic. The analysis of elements in the target fish species, the Evrotas chub, showed low to moderate PTE concentrations, with Pb being positively correlated with total Pb concentration in water. Moderate Zn concentrations found in fish samples from the Evrotas are possibly derived from pesticides and fertilizers.

Interplay between r- and K-strategists leads to phytoplankton underyielding under pulsed resource supply.

Fluctuations in nutrient ratios over seasonal scales in aquatic ecosystems can result in overyielding, a condition arising when complementary life-history traits of coexisting phytoplankton species enables more complete use of resources. However, when nutrient concentrations fluctuate under short-period pulsed resource supply, the role of complementarity is less understood. We explore this using the framework of Resource Saturation Limitation Theory (r-strategists vs. K-strategists) to interpret findings from laboratory experiments. For these experiments, we isolated dominant species from a natural assemblage, stabilized to a state of coexistence in the laboratory and determined life-history traits for each species, important to categorize its competition strategy. Then, using monocultures we determined maximum biomass density under pulsed resource supply. These same conditions of resource supply were used with polycultures comprised of combinations of the isolated species. Our focal species were consistent of either r- or K-strategies and the biomass production achieved in monocultures depended on their efficiency to convert resources to biomass. For these species, the K-strategists were less efficient resource users. This affected biomass production in polycultures, which were characteristic of underyielding. In polycultures, K-strategists sequestered more resources than the r-strategists. This likely occurred because the intermittent periods of nutrient limitation that would have occurred just prior to the next nutrient supply pulse would have favored the K-strategists, leading to overall less efficient use of resources by the polyculture. This study provides evidence that fluctuation in resource concentrations resulting from pulsed resource supplies in aquatic ecosystems can result in phytoplankton assemblages' underyielding.

Assessing the ecological effects of water stress and pollution in a temporary river - Implications for water management.

Temporary rivers are dynamic and complex ecosystems that are widespread in arid and semi-arid regions, such as the Mediterranean. Biotic communities adapted in their intermittent nature could withstand recurrent drought events. However, anthropogenic disturbances in the form of water stress and chemical pollution challenge biota with unpredictable outcomes, especially in view of climate change. In this study we assess the response of the biotic community of a temporary river to environmental stressors, focusing on water stress and pollution. Towards this aim, several metrics of four biotic groups (diatoms, macrophytes, macroinvertebrates and fish) were applied. All biotic groups responded to a pollution gradient mainly driven by land use, distinct functional groups of all biota responded to water stress (a response related to the rheophilic nature of the species and their resistance to shear stress), while the combined effects of water stress and pollution were apparent in fish. Biotic groups presented a differential temporal response to water stress, where diatom temporal assemblage patterns were explained by water stress variables of short-time response (15days), while the responses of the other biota were associated to longer time periods. There were two time periods of fish response, a short (15days) and a long-time response (60-75days). When considering management decisions, our results indicate that, given the known response of river biota to pollution, biomonitoring of temporary rivers should also involve metrics that can be utilized as early warnings of water stress.