Microbial functional changes mark irreversible course of Tibetan grassland degradation.

The Tibetan Plateau's Kobresia pastures store 2.5% of the world's soil organic carbon (SOC). Climate change and overgrazing render their topsoils vulnerable to degradation, with SOC stocks declining by 42% and nitrogen (N) by 33% at severely degraded sites. We resolved these losses into erosion accounting for two-thirds, and decreased carbon (C) input and increased SOC mineralization accounting for the other third, and confirmed these results by comparison with a meta-analysis of 594 observations. The microbial community responded to the degradation through altered taxonomic composition and enzymatic activities. Hydrolytic enzyme activities were reduced, while degradation of the remaining recalcitrant soil organic matter by oxidative enzymes was accelerated, demonstrating a severe shift in microbial functioning. This may irreversibly alter the world´s largest alpine pastoral ecosystem by diminishing its C sink function and nutrient cycling dynamics, negatively impacting local food security, regional water quality and climate.

Disentangling multiple chemical and non-chemical stressors in a lotic ecosystem using a longitudinal approach.

Meeting ecological and water quality standards in lotic ecosystems is often failed due to multiple stressors. However, disentangling stressor effects and identifying relevant stressor-effect-relationships in complex environmental settings remain major challenges. By combining state-of-the-art methods from ecotoxicology and aquatic ecosystem analysis, we aimed here to disentangle the effects of multiple chemical and non-chemical stressors along a longitudinal land use gradient in a third-order river in Germany. We distinguished and evaluated four dominant stressor categories along this gradient: (1) Hydromorphological alterations: Flow diversity and substrate diversity correlated with the EU-Water Framework Directive based indicators for the quality element macroinvertebrates, which deteriorated at the transition from near-natural reference sites to urban sites. (2) Elevated nutrient levels and eutrophication: Low to moderate nutrient concentrations together with complete canopy cover at the reference sites correlated with low densities of benthic algae (biofilms). We found no more systematic relation of algal density with nutrient concentrations at the downstream sites, suggesting that limiting concentrations are exceeded already at moderate nutrient concentrations and reduced shading by riparian vegetation. (3) Elevated organic matter levels: Wastewater treatment plants (WWTP) and stormwater drainage systems were the primary sources of bioavailable dissolved organic carbon. Consequently, planktonic bacterial production and especially extracellular enzyme activity increased downstream of those effluents showing local peaks. (4) Micropollutants and toxicity-related stress: WWTPs were the predominant source of toxic stress, resulting in a rapid increase of the toxicity for invertebrates and algae with only one order of magnitude below the acute toxic levels. This toxicity correlates negatively with the contribution of invertebrate species being sensitive towards pesticides (SPEARpesticides index), probably contributing to the loss of biodiversity recorded in response to WWTP effluents. Our longitudinal approach highlights the potential of coordinated community efforts in supplementing established monitoring methods to tackle the complex phenomenon of multiple stress.

Spatial, temporal, and inter-compartmental environmental monitoring of lipophilic pollutants by virtual organisms.

Sampling points belonging to the Harz National Park river system, Germany, were selected between the period of 2014 and 2017 for monitoring polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in water, air, and sediment. Triolein-containing Virtual Organisms (VO) were employed to assess the levels of chemicals in water, air and triolein as surrogate for natural fat. To avoid overestimation of the concentrations 20 performance reference compounds (PRCs)-16 PRCs-PAHs and 4 PRCs-PCBs were covering the range of properties of native compounds. Results manifested the highest concentration of individual PAH as follows: 31 ng fluoranthene/L water, 3600 ng pyrene/g fat, 62 ng phenanthrene/m3 air and 2800 ng fluoranthene/g dw sediment. All PCBs and OCPs values were below above mentioned PAH concentrations and far below EU-limit levels. Environmental partition of chemicals was investigated by calculating fugacity, suggesting a mass transport from water to air. Only quite volatile compounds such as hexachlorobutadiene showed higher fugacity in air. Ratios of sediment/water concentrations and log Kow within individual sampling periods at Holtemme River exhibited strong linear relationships. Interestingly, during summer months of the years water and fat contents well correlate to the flow rates of Holtemme River. Our results show that VO can be successfully used as a tool for ongoing exposure assessment studies and predictions of worst case levels in food and nutrition.

Isolation and characterization of eleven novel microsatellite markers for fine-scale population genetic analyses of Gammarus pulex (Crustacea: Amphipoda).

The freshwater amphipod species Gammarus pulex (Linnaeus, 1758) is widespread across Europe and Asia and is able to live in a broad range of environmental conditions. Yet, it is of great interest to which degree it is able to tolerate and adapt to the current rapid anthropogenic environmental changes affecting its habitat, such as pollution, changes in river morphology, and invasions of alien gammarid species. Microsatellite primers for genetic population studies with G. pulex have been developed but due to the existence of several genetically different lineages within the species, the application of these primers is not always successful. In order to investigate the impacts of anthropogenic environmental changes on the spatio-genetic patterns of G. pulex lineage E in streams in the Saale river catchment in Germany, we designed eleven novel polymorphic microsatellites for this lineage using a high-throughput sequencing approach. These microsatellites enabled highly specific characterization of three closely related populations. The results show genetically distinct populations reflected by both a principal coordinates analysis and an analysis of molecular variance. Several of the newly designed microsatellite primers also enabled successful cross-amplification of the respective microsatellites in specimens of G. pulex lineage C, while only two microsatellites were amplified successfully and showed polymorphisms for all of the analyzed specimens of G. fossarum Koch, 1836. The microsatellites identified here are suitable for future assessments of micro-evolutionary dynamics of G. pulex from central Germany.

Quantitative ecotoxicological impacts of sewage treatment plant effluents on plankton productivity and assimilative capacity of rivers.

Sewage treatment plants are sources of inorganic and organic matter as well as contaminants for the receiving watercourses. We analyzed the ecological consequences of such effluents by following a holistic and synecological ecotoxicological approach based on quantifying extracellular enzyme activities (EEA), primary production and bacterial cell, and biomass production rates. Samples were obtained at three locations at the Rivers Holtemme and Elbe, Germany and Lower Jordan River, Israel and West Bank, as well as from their adjacent sewage treatment plants. Blending river samples with sewage treatment plant effluents mainly resulted in a stimulation of EEAs, which was diminished in blends with 0.2-µm filtered sewage treatment plant effluents. Stimulation for primary production and bacterial cell and biomass production of River Holtemme and Elbe samples was observed, and inhibition of these rates for Lower Jordan River samples probably linked to generally high turbidity. The quantified bacterial biomass versus cell production rates showed almost unbalanced (≫ 1) growth. Very high biomass to cell production ratios were found for sewage and sewage-containing samples, which provides a semi-quantitative indicator function for high quantities of microbial easy utilizable dissolved organic matter as nutrition source. The presented approach enables the simultaneous quantification of inhibitory and stimulating toxic responses as well as supplying ecosystem-based data for policy decision-making, and for direct incorporation in models to derive management and remediation strategies.

Tandem Action of Natural and Chemical Stressors in Stream Ecosystems: Insights from a Population Genetic Perspective.

Agricultural and urban land use has dramatically increased over the last century and one consequence is the release of anthropogenic chemicals into aquatic ecosystems. One of the rarely studied consequences is the effect of land use change on internal concentrations of organic micropollutants (OMPs) in aquatic invertebrates and its effects on their genotype diversity. Here, we applied population genetic and internal concentrations of OMPs analyses to determine evolutionary implications of chemical pollution on Gammarus pulex populations from a natural and two agricultural streams. Along 14 consecutive months sampled, 26 different OMPs were quantified in G. pulex extracts with the highest number, concentration, and toxic pressure in the anthropogenically stressed stream ecosystems. Our results indicate distinct internal OMP profiles and changes in both genetic variation and genetic structure in streams affected by anthropogenic activity. Genetic variation was attributed to chemical pollution whereas changes in the genetic structure were attributed to environmental disturbances, such as changes in discharge in the impacted stream ecosystems, which worked both independently and in tandem. Finally, we conclude that human-impacted streams are subjected to severe alterations in their population genetic patterns compared to nonimpacted stream ecosystems.

A new approach for evaluating transformations of dissolved organic matter (DOM) via high-resolution mass spectrometry and relating it to bacterial activity.

Streams are important sites of transformation of dissolved organic matter (DOM). The molecular characterization of DOM-quality changes requires sophisticated analytical evaluation techniques. The goal of our study was to link molecular DOM transformation with bacterial activity. We measured the degradation of leaf leachate over a gradient of bacterial production obtained by different rates of percolation of sediments in seven experimental flumes on five sampling dates. We developed a new strategy for evaluating molecular formula data sets obtained by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS), in which the time-dependent change of component abundance was fitted by a linear regression model after normalization of mass peak intensities. All components were categorized by calculating the slope (change of percent intensity per day) in each of the seven flumes. These slopes were then related to cumulative bacterial production. The concentration of DOM decreased quickly in all flumes. Bacterial activity was higher in flumes with percolated sediment than in those without percolation, whereas plankton bacterial activity was higher in flumes without percolation or without sediment. There were no differences in molecular-DOM characteristics between flumes, but there were distinct changes over time. Positive slopes, i.e. increasing intensities over time, were found for small molecules (MW < 450 Da) and high O/C ratios, whereas decreasing intensities were observed less often and only for large molecules and low O/C ratios. The positive slopes of produced components showed a positive relationship to bacterial production for small and for oxygen-rich components. The negative slopes of degraded components were negatively related to bacterial production for large and for oxygen-deficient molecules. Overall, the approach provided new insights into the transformation of specific molecular DOM components.

Contrasting habitats but comparable microbial decomposition in the benthic and hyporheic zone.

Input of allochthonous leaf litter is the main carbon source for heterotrophic metabolism in low-order forested streams. A major part of this leaf litter is accumulated at benthic retention structures or buried in the hyporheic zone. As a result of hyporheic sediment characteristics, hyporheic physicochemistry differs from that of the benthic zone selecting the microbial community. The present study aimed at understanding the influence of the hydrological and physiochemical differences between the benthic and hyporheic zone on microbial leaf litter decomposition and on the structure and function of the associated microbial community. Leached leaves of Alnus glutinosa were exposed for 62days in 250-µm mesh bags in the benthic zone and buried in the hyporheic zone at a depth of 2-3cm. Decomposition rates were comparable for both zones. In contrast, respiration, bacterial abundance, ergosterol content, fungal spore production and richness of fungal morphotypes were lower associated with hyporheic than with benthic leaves. Microbial community structure displayed zone-dependent temporal dynamics. Thus, the microbial community carried out leaf litter decomposition independently of its structure. These results suggest that carbon processing is not necessarily impaired by environmental constraints because the community structure may compensate those constraints (i.e. functional redundancy).

Anthropogenic Stressors Shape Genetic Structure: Insights from a Model Freshwater Population along a Land Use Gradient.

Environmental pollution including mutagens from wastewater effluents and discontinuity by man-made barriers are considered typical anthropogenic pressures on microevolutionary processes that are responsible for the loss of biodiversity in aquatic ecosystems. Here, we tested for the effects of wastewater treatment plants (WWTPs), weirs and other stressors on the invertebrate species Gammarus pulex at the population genetic level combining evolutionary ecotoxicology, body burden analysis and testing for exposure to mutagens. Exposure to chemical pollution alone and in combination with the presence of weirs resulted in a depression of allelic richness in native G. pulex populations. Our results suggest that the input of a mutagenic effluent from a WWTP resulted in a strong increase in private alleles over the affected populations. In addition, the presence of weirs along the river disrupted the migration across the river and thus the gene flow between G. pulex upstream and downstream. This study provides strong evidence that the assessment of genetic variation including private alleles together with the contamination of mutagenic and nonmutagenic chemical pollution offers new insights into the regulation of genetic population structure and highlights the relevance of emerging anthropogenic pressures at the genetic level.

Biogeochemical patterns in a river network along a land use gradient.

The Bode catchment (Germany) shows strong land use gradients from forested parts of the National Park (23% of total land cover) to agricultural (70%) and urbanised areas (7%). It is part of the Terrestrial Environmental Observatories of the German Helmholtz association. We performed a biogeochemical analysis of the entire river network. Surface water was sampled at 21 headwaters and at ten downstream sites, before (in early spring) and during the growing season (in late summer). Many parameters showed lower concentrations in headwaters than in downstream reaches, among them nutrients (ammonium, nitrate and phosphorus), dissolved copper and seston dry mass. Nitrate and phosphorus concentrations were positively related to the proportion of agricultural area within the catchment. Punctual anthropogenic loads affected some parameters such as chloride and arsenic. Chlorophyll a concentration and total phosphorus in surface waters were positively related. The concentration of dissolved organic carbon (DOC) was higher in summer than in spring, whereas the molecular size of DOC was lower in summer. The specific UV absorption at 254 nm, indicating the content of humic substances, was higher in headwaters than in downstream reaches and was positively related to the proportion of forest within the catchment. CO2 oversaturation of the water was higher downstream compared with headwaters and was higher in summer than in spring. It was correlated negatively with oxygen saturation and positively with DOC concentration but negatively with DOC quality (molecular size and humic content). A principle component analysis clearly separated the effects of site (44%) and season (15%), demonstrating the strong effect of land use on biogeochemical parameters.

Resource quantity and seasonal background alter warming effects on communities of biofilm ciliates.

The impacts of experimental warming on field-related communities of biofilm ciliates were studied in contrasting seasons (winter vs. summer), which incorporated both different species sets and environmental background conditions. The biofilms for the experiments were cultivated in river bypass systems that were exposed to increasing temperatures based on the ambient river temperature. Opposing effects of warming were observed for ciliate 'summer' and 'winter' communities. While winter warming resulted in both stimulation (abundance and biomass) of the ciliate communities and significant shifts in the community structure, summer warming induced a significant decline in the ciliate biomass, but did not affect the relative community composition. By the simultaneous manipulation of temperature and resource density in summer, it was demonstrated that negative warming effects on the ciliate quantity during summer could be compensated by increasing the availability of food. Taken together, our results indicate that the responses of ciliate communities towards warming are strongly coupled to the availability of resources, and that the strongest impacts of environmental warming should thus be expected in resource-rich environments.

A new flagship peritrich (Ciliophora, Peritrichida) from the River Rhine, Germany: Apocarchesium arndti n. sp.

We discovered a free-living peritrich ciliate with outstanding features in the River Rhine. Its morphology and 18S rRNA gene sequence were studied with standard methods. Apocarchesium arndti n. sp. has several peculiarities. (i) There are ordinary zooids, macrozooids, and microzooids, which form a hemispherical rosette on a discoidal base, the stalk dish, locking the approximately 18 microm wide and up to 2 mm long, spirally contracting colony stalk. (ii) The stalk myoneme is connected only to the microzooids. (iii) A rosette contains up to 50 zooids not connected to each other but individually attached to the stalk dish with the scopula. (iv) The ordinary zooids are epistylidid, trumpet-shaped (approximately 6:1 length:width), about 180 x 30 microm in size, and have an ellipsoidal macronucleus subapically between oral cavity and dorsal side. (v) The myoneme system of the zooids, which can contract individually, forms a tube-like structure in the narrow posterior half of the cell. (vi) The silverline pattern belongs to the transverse-striate type. (vii) The oral apparatus is of usual structure, with kinety 1 of peniculus 3 distinctly shortened proximally. (viii) The 18S rRNA places A. arndti n. sp. as a distinct lineage near Vorticella and Carchesium. These data are used to provide an improved diagnosis of the genus Apocarchesium. Features (i)-(iii) and the molecular data indicate that Apocarchesium could be the type genus of a new peritrich family.

Responses of biofilm-dwelling ciliate communities to planktonic and benthic resource enrichment.

Four experiments covering different seasons were performed to test the impact of increased benthic and planktonic resource availability on the structure of biofilm-dwelling ciliate communities which were cultivated in river bypass systems. The growth of benthic bacteria was stimulated by the addition of dissolved organic carbon. The enrichment of the planktonic resource was achieved by supplementation with suspended bacteria. It was shown that both resource enrichments can differentially influence abundance and taxonomic structure of ciliate communities. Furthermore, both resources can influence different stages during biofilm colonization. Increased benthic bacterial growth mainly resulted in both an accumulation of primarily grazing-resistant bacterial filaments and in an increase in the number of vagile heterotrophic flagellates. This can stimulate nanophagous ciliates (feeding on flagellates) in addition to the direct stimulation of bacteriovorous ciliates. The effects of the planktonic bacteria enrichments were twofold: They could have been utilized either directly by suspension-feeding ciliates or indirectly through an enhanced growth of suspension-feeding attached heterotrophic flagellates, which were then in turn grazed upon by ciliates. The magnitude of responses of the total ciliate abundance to the two resource enrichments further depended on the background conditions, thereby showing temporarily variable limitations of these resources. Furthermore, the particular taxonomic groups stimulated by one resource type sometimes differed between the experiments, an observation which demonstrates that the response depends on different environmental factors and is not easily predictable based simply on resource type. Taken together, our results emphasize the need of a differentiated view on the effects of resources on complex biofilm-dwelling consumer communities with respect to both the origin of carbon source as well as the particular environmental conditions.

Effects of resource supplements on mature ciliate biofilms: an empirical test using a new type of flow cell.

Biofilm-dwelling consumer communities play an important role in the matter flux of many aquatic ecosystems. Due to their poor accessibility, little is as yet known about the regulation of natural biofilms. Here, a new type of flow cell is presented which facilitates both experimental manipulation and live observation of natural, pre-grown biofilms. These flow cells were used to study the dynamics of mature ciliate biofilms in response to supplementation of planktonic bacteria. The results suggest that enhanced ciliate productivity could be quickly transferred to micrometazoans (ciliate grazers), making the effects on the standing stock of the ciliates detectable only for a short time. Likewise, no effect on ciliates appeared when micrometazoan consumers were ab initio abundant. This indicates the importance of 'top-down' control of natural ciliate biofilms. The flow cells used here offer great potential for experimentally testing such control mechanisms within naturally cultivated biofilms.

Impact of local temperature increase on the early development of biofilm-associated ciliate communities.

Indications of global climate change and associated unusual temperature fluctuations have become increasingly obvious over the past few decades. Consequently, the relevance of temperature increases for ecological communities and for whole ecosystems is one of the major challenges of current ecological research. One approach to investigating the effects of increasing temperatures on communities is the use of fast-growing microbial communities. Here we introduce a river bypass system in which we tested the effect of temperature increases (0, 2, 4, 6 degrees C above the long-term average) on both the colonization speed and the carrying capacity of biofilm-associated ciliate communities under different seasonal scenarios. We further investigated interactions of temperature and resource availability by cross-manipulations in order to test the hypothesis that temperature-mediated effects will be strongest in environments that are not resource-limited. Strong seasonal differences in both tested parameters occurred under natural conditions (no resource addition), and the effects of temperature increase at a given time were relatively low. However, increasing temperature can significantly accelerate the colonization speed and reduce the carrying capacity in particular seasons. These effects were strongest in winter. Simultaneous manipulation of temperature and of resource availability amplified the response to temperature increase, adumbrating strong interactive control of populations by temperature and resource availability. Our results show that the response of communities to local temperature increases strongly depends on the seasonal setting, the resource availability and the stage of succession (early colonization speed vs. carrying capacity).

Control of microbial communities by the macrofauna: a sensitive interaction in the context of extreme summer temperatures?

Climate models predict an increasing frequency of extremely hot summer events in the northern hemisphere for the near future. We hypothesised that microbial grazing by the metazoan macrofauna is an interaction that becomes unbalanced at high temperatures due to the different development of the grazing rates of the metazoans and the growth rates of the microbial community with increasing temperature. In order to test this hypothesis, we performed grazing experiments in which we measured the impact of increasing temperatures on the development of the grazing rates of riverine mussels in relation to the growth rates of a unicellular prey community (a natural heterotrophic flagellate community from a large river). In a first experimental series using Corbicula fluminea as a grazer and under the addition of a carbon source (yeast extract), the increase of the prey's growth rates was considerably stronger than that of the predator's grazing rates when temperatures were increased from 19 to over 25 degrees C. This was also the outcome when the mussels had been acclimatized to warm temperatures. Hereafter, specific experiments with natural river water at temperatures of 25 and 30 degrees C were performed. Again, a strong decrease of the mussels' grazing rates in relation to the flagellate growth rates with increasing temperature occurred for two mussel species (C. fluminea and Dreissena polymorpha). When performing the same experiment using a benthic microbial predator community (biofilms dominated by ciliates) instead of the benthic mussels, an increase of the grazing rates relative to the growth rates with temperature could be observed. Our data suggest that predator-prey interactions (between metazoans and microbes) that are balanced at moderate temperatures could become unbalanced at high temperatures. This could have significant effects on the structure and function of microbial communities in light of the predicted increasing frequency of summer heat waves.