The assessment of restoration success often neglects trophic interactions within food webs, focusing instead on biodiversity and community structure. Here, we analysed the long-term recovery of food web structure based on stable isotopes (δ¹³C and δ¹⁵N) of benthic invertebrates and quantified responses of food web metrics to time since restoration. The samples derived from twelve restored sites with different restoration ages, sampled annually from 2012 to 2021, and covering an investigation period of up to 28 years after restoration for the whole catchment. Temporal developments of the restored sites were compared to the development of two near-natural sites. The restoration measures consisted of the cessation of sewage inflow and morphological restoration of the channels. As a clear and consistent result over almost all sites, trophic similarity (proportion of co-existing species occupying similar trophic niches) increased with time since restoration, and reached values of near-natural sites, suggesting an increase in the stability and resilience of the food webs. Surprisingly, resource diversity decreased at most restored sites within 10 years after restoration, probably due to the removal of wastewater-derived resources, and a shift towards leaf litter as the dominant resource following the regrowth of the riparian vegetation. Food chain length showed no consistent pattern over time at the different sites both increasing and decreasing with time since restoration. Overall, restoration had clear effects on the food web structure of stream ecosystems. While some effects such as the increase in trophic similarity were consistent at almost all sites, others such as response of the food chain length were context dependent. The study demonstrates the potential of utilizing food web metrics, particularly trophic similarity, in restoration research to achieve a more holistic understanding of ecosystem recovery.

Human activities, past and present, have a big impact on nature, affecting ecosystems in profound ways. Scientists are working hard to figure out the best methods to restore damaged ecosystems. But ecosystem restoration often does not go as planned, resulting in very different ecosystems than before. For example, some animals that used to live in an ecosystem can take a long time to return or do not come back at all. To understand the complexities of ecosystem recovery, scientists have come up with a theory called the asymmetric response concept (ARC), to understand how ecosystems recover. The ARC helps us describe the various responses that can happen after ecosystem damage and why the responses happen that way. Once we understand these responses, we can help ecosystems become healthy again. By learning how organisms rejoin damaged ecosystems, we can better protect our environment for the future.

Urban streams are exposed to a variety of anthropogenic stressors. Freshwater salinization is a key stressor in these ecosystems that is predicted to be further exacerbated by climate change, which causes simultaneous changes in flow parameters, potentially resulting in non-additive effects on aquatic ecosystems. However, the effects of salinization and flow velocity on urban streams are still poorly understood as multiple-stressor experiments are often conducted at pristine rather than urban sites. Therefore, we conducted a mesocosm experiment at the Boye River, a recently restored stream located in a highly urbanized area in Western Germany, and applied recurrent pulses of salinity along a gradient (NaCl, 9 h daily of +0 to +2.5 mS/cm) in combination with normal and reduced current velocities (20 cm/s vs. 10 cm/s). Using a comprehensive assessment across multiple organism groups (macroinvertebrates, eukaryotic algae, fungi, parasites) and ecosystem functions (primary production, organic-matter decomposition), we show that flow velocity reduction has a pervasive impact, causing community shifts for almost all assessed organism groups (except fungi) and inhibiting organic-matter decomposition. Salinization affected only dynamic components of community assembly by enhancing invertebrate emigration via drift and reducing fungal reproduction. We caution that the comparatively small impact of salt in our study can be due to legacy effects from past salt pollution by coal mining activities >30 years ago. Nevertheless, our results suggest that urban stream management should prioritize the continuity of a minimum discharge to maintain ecosystem integrity. Our study exemplifies a holistic approach for the assessment of multiple-stressor impacts on streams, which is needed to inform the establishment of a salinity threshold above which mitigation actions must be taken.

Freshwater salinization poses global challenges for aquatic organisms inhabiting urban streams, impacting their physiology and ecology. However, current salinization research predominantly focuses on mortality endpoints in limited model species, overlooking the sublethal effects on a broader spectrum of organisms and the exploration of adaptive mechanisms and pathways under natural field conditions. To address these gaps, we conducted high-throughput sequencing transcriptomic analysis on the gill tissue of the euryhaline fish Gasterosteus aculeatus, investigating its molecular response to salinity stress in the highly urbanized river Boye, Germany. We found that in stream sections with sublethal concentrations of chloride costly osmoregulatory systems were activated, evidenced by the differential expression of genes related to osmoregulation. Our enrichment analysis revealed differentially expressed genes (DEGs) related to transmembrane transport and regulation of transport and other osmoregulation pathways, which aligns with the crucial role of these pathways in maintaining biological homeostasis. Notably, we identified candidate genes involved in increased osmoregulatory activity under salinity stress, including those responsible for moving ions across membranes: ion channels, ion pumps, and ion transporters. Particularly, genes from the solute carrier family SLC, aquaporin AQP1, chloride channel CLC7, ATP-binding cassette transporter ABCE1, and ATPases member ATAD2 exhibited prominent differential expression. These findings provide insights into the potential molecular mechanisms underlying the adaptive response of euryhaline fish to salinity stress and have implications for their conservation and management in the face of freshwater salinization.

1. Intensive agriculture drives an ongoing deterioration of stream biodiversity and ecosystem functioning across the planet. Key agricultural stressors include increased deposited fine sediment and insecticides flushed from adjacent land into streams. The individual and combined effects on aquatic biota are increasingly studied, but the functional consequences of biodiversity loss associated with agricultural stressors remain poorly understood. We addressed this knowledge gap by examining the effects of fine sediment and different concentrations of the insecticide chlorantraniliprole on organic-matter decomposition.
2. We conducted an outdoor stream mesocosm experiment. Mesocosms contained a standardised organic-matter assay (the cotton-strip assay), which was used to assess organic-matter decomposition rates (as tensile-strength loss of the fabric) and microbial respiration of the cotton strips.
3. The decomposition rate of strips buried under fine sediment was inhibited, a result we attribute to the limited accessibility for invertebrate feeding and microbial activities, as well as the limited nutrient and dissolved oxygen exchange. The insecticide also inhibited decay rates, a finding we relate to reduced invertebrate grazing and associated excessive algal growth. In contrast to decomposition rates, we did not observe stressor effects on microbial respiration. An interaction between fine sediment and chlorantraniliprole was not identified.
4. Our results suggest that stressors induced by agriculture affect functions of stream ecosystems through a variety of pathways and operate by modifying habitats and biotic interactions.
5. By examining a combination of stressors and responses that have not been addressed before, this study gives important insights into the effects of agricultural practices on streams. Understanding the effects of chlorantraniliprole is especially important since it is likely to become more widely used in future agricultural practice due to the increasing ban on neonicotinoid insecticides. Furthermore, most experimental studies address multiple stressor effects on biota. For a comprehensive understanding of complex stressor effects on ecosystems, ecosystem functions also need to be studied, such as the organic-matter decomposition within streams.

To overcome resistance of degraded ecological communities to restorative interventions, we need to understand windows of opportunity—limited time frames when species reintroduction attempts are still successful. More specifically, we need to understand what makes these windows close, as this may enable us to stretch or reopen them. We investigated this using models of simple food web modules. We show how joint changes of bottom–up and top–down control may be applied to change windows of opportunity and increase reintroduction success. Which reintroduction densities were most effective seemed system-specific. A more general result is that reintroduction success was strongly enhanced by low to intermediate carrying capacities of basal species (e.g. periphyton and other algae in streams). This can be seen as equivalent to low to intermediate nutrient levels. When these were too high, almost any combination of restorative measures was rendered ineffective. Interestingly, reintroducing primarily and secondarily lost species at the same time was more effective than sequential reintroductions that first attempted to fix secondary extinctions. We could further enhance the success of species reintroductions by reducing the carrying capacities of basal species before the reintroduction of primarily and secondarily lost species. We discuss our results in the light of empirical work on macro-invertebrates in streams. This serves to exemplify how our results can be applied in the practice of ecological restoration.

Agriculture has been identified as a main cause for more than 90% of Germany´s rivers still not meeting good ecological status in 2021. While many large-scale studies observed a negative effect of catchment agricultural land use on river biota, they rarely considered differences in cultivation intensities, although small-scale studies highlight clear differences between the effects of agricultural crops. Here we used Germany-wide and spatially explicit information on crop types to calculate agricultural intensity indices for nutrients and pesticides, weighting different crop types based on average pesticide treatment and nutrient application rates. These indices were then used as explanatory variables for the ecological status of n = 7677 biological sampling sites. Pesticides were more important than nutrient pollution for macroinvertebrates and macrophytes, while diatoms were more sensitive to nutrients. Considering the most relevant intensity index (pesticide or nutrient) slightly increased the correlative strength with ecological status, as compared to the correlation with agricultural land or cropland cover by up to R² = 0.14 for diatoms. Correlative strength of agricultural intensity indices was substantially larger in small mountain and (pre)-alpine streams compared to lowland streams, with an R² up to 0.43 for macroinvertebrates. These results not only confirm previous large-scale studies by demonstrating the detrimental effects of present-day agriculture on river biota, but also shed light on the main pathways involved, particularly highlighting the adverse impacts of agrochemicals. Consequently, to protect river biota, a shift to more sustainable agricultural practices, like reducing pesticide application, is urgently required.

Urbanization is a key factor driving water quality and biological communities in freshwater systems. While urbanization impacts on water resources were well-studied in urban areas, few studies have examined the growing impacts in non-urban areas. Here we investigate the spatiotemporal trends of urbanization using high-resolution impervious surface data of both urban and non-urban areas across Germany, and evaluate the urbanization impacts on changes of run-off, nutrient emissions, and macroinvertebrate communities. The emission changes were modelled using the MONERIS-PCRaster tool, while the macroinvertebrate community responses to urbanization were determined using Generalized Least Square models. We find that urbanization expanded by 3.2% from 2006 to 2015 nationwide, and primarily occurred in less-populated regions. The non-urban areas contributed 19.3–19.6% of the imperviousness expansion nationwide, which were equivalent to the ‘unaccounted’ increases of 15.5–15.9% of run-off, 10.6–11.0% of total nitrogen, and 12.5–12.9% of total phosphorus when only emissions from urban areas were modelled. Incorporating the emissions from both urban and non-urban areas revealed significant impacts of urbanization on macroinvertebrate community composition changes, with increases in more pollution-resistant and non-native species (particularly in large rivers), and reductions of more sensitive taxa. The community responses were jointly determined by local catchment characteristics and urbanization stressors. Our results suggest neglected effects of non-urban impervious surfaces and the importance of using impervious surface data instead of urban land classes to better represent urbanization processes and impacts in long-term planning and management of freshwater systems.

Context

Improving our understanding of how riverine communities respond to anthropogenic change requires spatial comparisons across multiple sites, high-resolution temporal analyses, and examination of both taxa and trait responses. However, studies that encompass all these aspects remain scarce.

Objectives

We used 10 years of annual monitoring data from 14 sampling sites in the Rhine-Main-Observatory (a Long-Term Ecological Research—LTER—site in Germany) to investigate spatiotemporal responses of stream macroinvertebrate communities along anthropogenic disturbance gradients (measured as ‘ecological quality’).

Methods

We examined spatiotemporal changes in various community components, including taxa, traits, metrics summarizing community responses (e.g., richness), and community composition.

Results

Spatially, consistent patterns over a decade of sampling revealed that less-disturbed communities were characterized by higher taxonomic and trait diversity and occurrence of pollution-sensitive taxa. Anthropogenic disturbance tended to become less severe through time, particularly in more upstream sites, likely driven by improvements in land-use and water quality. Conversely, more downstream sites exhibited a lesser degree of improvement (or none at all) likely owing to persistent or cumulative stressors. Overall, taxonomic/trait metrics consistently reflected the magnitude of the environmental improvement, while community composition did not, suggesting a weaker link between community changes and anthropogenic impacts severity.

Conclusion

Our results emphasize the importance of accounting for the variability in community responses to anthropogenic changes, and identifying optimal monitoring strategies to track such responses. In heterogeneous catchments, choosing which community component to focus and where to locate monitoring sites (e.g., monitoring ecological quality for the EU Water Framework Directive) can determine a timely detection of anthropogenic impacts.

Background

Freshwater ecosystem degradation and biodiversity decline are strongly associated with intensive agricultural practices. Simultaneously occurring agricultural stressors can interact in complex ways, preventing an accurate prediction of their combined effects on aquatic biota. Here, we address the limited mechanistic understanding of multiple stressor effects of two globally important stressors, an insecticide (chlorantraniliprole), and increased fine sediment load and assessed their impact on the transcriptomic profile of two stream macroinvertebrates: the amphipod Gammarus pulex and the caddisfly Lepidostoma basale.

Results

We identified mainly antagonistic stressor interactions at the transcriptional level, presumably because the insecticide adsorbed to fine sediment particles. L. basale, which is phylogenetically more closely related to the insecticide’s target taxon Lepidoptera, exhibited strong transcriptional changes when the insecticide stressor was applied, whereas no clear response patterns were observed in the amphipod G. pulex. These differences in species vulnerability can presumably be attributed to molecular mechanisms determining the cellular affinity toward a stressor as well as differential exposure patterns resulting from varying ecological requirements between L. basale and G. pulex. Interestingly, the transcriptional response induced by insecticide exposure in L. basale was not associated with a disruption of the calcium homeostasis, which is the described mode of action for chlorantraniliprole. Instead, immune responses and alterations of the developmental program appear to play a more significant role.

Conclusions

Our study shows how transcriptomic data can be used to identify multiple stressor effects and to explore the molecular mechanisms underlying stressor-induced physiological responses. As such, stressor effects assessed at the molecular level can inform about modes of action of chemicals and their interplay with non-chemical stressors. We demonstrated that stressor effects vary between different organismic groups and that insecticide effects are not necessarily covered by their described mode of action, which has important implications for environmental risk assessment of insecticides in non-target organisms.

A shift in phototrophic organisms occurs along lake trophic gradients characterized by a change from macrophyte- to phytoplankton-dominated states. Before lakes reach a turbid and phytoplankton-dominated state, shifts from meadow-forming Characeae to canopy-forming macrophyte species can occur where Characea are present, especially in oligotrophic to mesotrophic lakes with sand or gravel substrate. However, eutrophication intensity causing this shift has not previously been examined. We analysed data from 132 lakes located in Mecklenburg-Vorpommern (Germany) with a generalized linear model (GLM) and random forest (RF) models complemented with grid approximation to determine if (1) species richness of macrophytes declines along eutrophication gradients, (2) the chlorophyll a (Chl-a) concentrations above which the abundance of Characeae declines, and (3) the Chl-a concentrations above which the abundance of canopy-forming species declines. The number of macrophyte taxa declined gradually following a log-linear trend and with increasing Chl-a concentrations. Based on the RF models, the abundance of Characeae decreased abruptly at 5–13 µg L⁻¹ Chl-a, whereas canopy-forming species showed a monotonous and slight unimodal decreasing response at 35–103 µg L⁻¹ Chl-a. The results support the theory of shifts in growth forms along eutrophication gradients in lakes and provide, for the first time, estimates of Chl-a concentrations required for these shifts. Changes in growth forms are obvious indicators for eutrophication and can serve as an additional incentive to improve lake trophic status.

Water temperature is a key environmental variable in stream ecosystems determining species distribution ranges, community composition, and ecological processes. In addition to global warming, direct anthropogenic impacts, for example through the influx of power plant cooling water or due to sun exposure after the removal of riparian vegetation, result in elevated water temperatures. However, temperature effects in stream ecosystems have mostly been tested in recirculating experimental systems, which can neither capture diurnal and seasonal variability in other environmental variables nor allow for entrainment of stream organisms. In contrast, open flow-through systems, which are constantly supplied with stream water, offer a more realistic setting for stream ecological experiments, yet are difficult to implement. Here, we outline a heating module for the purpose of differential temperature regulation in a flow-through mesocosm system by automatic control of warm water supply. We validated the functionality of the module in indoor trials as well as in an outdoor ExStream experimental mesocosm system. Furthermore, we tested the implications of different warm water temperatures for the survival of invertebrates drifting through the heating module to derive recommendations for the maximum warm water temperature for mixing with the natural water inflow. The module allows for controlled open flow-through experiments in the field and the key components are flexible and scalable. Therefore, the module can be easily integrated into existing experimental flow-through setups.

Background

Recent studies indicate a partial recovery of European stream macroinvertebrate diversity. However, the key determinants shaping the overall community trends are only partly explored, owing to insufficient long-term environmental data collected in parallel with community responses. We investigate long-term trends in stream macroinvertebrate communities (i.e., taxonomic and trait composition and metrics), and explore their relationships to diverse environmental drivers (i.e., land-use, runoff, water temperature, and in-stream chemicals). We use macroinvertebrate data collected annually in spring and summer between 2007 and 2021 at four sampling sites within the Rhine-Main-Observatory Long-Term Ecological Research site. These sampling sites encompass a gradient from less-disturbed to disturbed conditions.

Results

Over time, shifts in taxonomic and trait composition and metrics indicated an improvement in environmental conditions. Long-term trends of biological trait metrics mirrored those for taxonomic metrics; for example, increases over time in taxonomic richness were paralleled by increases in functional richness and functional dispersion. Meanwhile, trends of ecological trait metrics were particularly driven by changes in environmental drivers. Land-use, water temperature, and runoff explained around 20% of the overall variance in long-term trends of macroinvertebrate communities. Water temperature and land-use played relatively equal roles in shaping taxonomic and trait composition and metric responses in spring, while water temperature emerged as the most influential driver in summer. However, when incorporating long-term chemical data as a more direct measurement of changes in land-use, the overall variance explained in macroinvertebrate community trends increased to c.a. 50% in both seasons.

Conclusions

Examining more relevant driver variables beyond land-use and climate improves insights into why biodiversity exhibits long-term trends. We call for an increase in initiatives to link biodiversity monitoring with parallel sampling of relevant environmental drivers.

Metabarcoding is a powerful tool to detect classical, and well-known “long-branch” Microsporidia in environmental samples. Several primer pairs were developed to target these unique microbial parasites, the majority of which remain undetected when using general metabarcoding primers. Most of these Microsporidia-targeting primer pairs amplify fragments of different length of the small subunit ribosomal RNA (SSU-rRNA) gene. However, we lack a broad comparison of the efficacy of those primers. Here, we conducted in silico PCRs with three short-read (which amplify a few-hundred base pairs) and two long-read (which amplify over a thousand base pairs) metabarcoding primer pairs on a variety of publicly available Microsporidia sensu lato SSU-rRNA gene sequences to test which primers capture most of the Microsporidia diversity. Our results indicate that the primer pairs do result in slight differences in inferred richness. Furthermore, some of the reverse primers are also able to bind to microsporidian subtaxa beyond the classical Microsporidia, which include the metchnikovellidan Amphiamblys spp., the chytridiopsid Chytridiopsis typographi and the “short-branch” microsporidian Mitosporidium daphniae.

In lotic freshwater ecosystems, the drift or downstream movement of animals (e.g., macroinvertebrates) constitutes a key dispersal pathway, thus shaping ecological and evolutionary patterns. There is evidence that macroinvertebrate drift may be modulated by parasites. However, most studies on parasite modulation of host drifting behavior have focused on acanthocephalans, whereas other parasites, such as microsporidians, have been largely neglected. This study provides new insight into possible seasonal and diurnal modulation of amphipod (Crustacea: Gammaridae) drift by microsporidian parasites. Three 72 h drift experiments were deployed in a German lowland stream in October 2021, April, and July 2022. The prevalence and composition of ten microsporidian parasites in Gammarus pulex clade E varied seasonally, diurnally, and between drifting and stationary specimens of G. pulex. Prevalence was generally higher in drifting amphipods than in stationary ones, mainly due to differences in host size. However, for two parasites, the prevalence in drift samples was highest during daytime suggesting changes in host phototaxis likely related to the parasite’s mode of transmission and site of infection. Alterations in drifting behavior may have important implications for G. pulex population dynamics and microsporidians’ dispersal. The underlying mechanisms are more complex than previously thought.

Sequencing of amplified DNA is the first step towards the generation of Amplicon Sequence Variants (ASVs) or Operational Taxonomic Units (OTUs) for biodiversity assessment and comparative analyses of environmental communities and microbiomes. Notably, the rapid advancements in sequencing technologies have paved the way for the growing utilization of third-generation long-read approaches in recent years. These sequence data imply increasing read lengths, higher error rates, and altered sequencing chemistry. Likewise, methods for amplicon classification and reference databases have progressed, leading to the expansion of taxonomic application areas and higher classification accuracy. With Natrix, a user-friendly and reducible workflow solution, processing of prokaryotic and eukaryotic environmental Illumina sequences using 16S or 18S is possible. Here, we present an updated version of the pipeline, Natrix2, which incorporates VSEARCH as an alternative clustering method with better performance for 16S metabarcoding approaches and mothur for taxonomic classification on further databases, including PR ² , UNITE and SILVA. Additionally, Natrix2 includes the handling of Nanopore reads, which entails initial error correction and refinement of reads using Medaka and Racon to subsequently determine their taxonomic classification.

Rivers are the lifelines of our planet. We depend on them for drinking water and food production, and they are home to many plants and animals. Unfortunately, rivers are under pressure from stressors like increasing temperatures, pollution, and habitat destruction. We often know how a single stressor impacts a river, but when more than one stressor is present at the same time, the consequences are often unpredictable. To protect our rivers now and in the future, we must understand what happens in the rivers when multiple stressors are present at the same time. However, it is difficult to understand what multiple stressors are doing to aquatic life by just observing a river or doing a laboratory experiment. In this article, you will learn how experimental miniature streams can help us to investigate the consequences of multiple stressors on rivers.

Wastewater treatment processes can eliminate many pollutants, yet remainder pollutants contain organic compounds and microorganisms released into ecosystems. These remainder pollutants have the potential to adversely impact downstream ecosystem processes, but their presence is currently not being monitored. This study was set out with the aim of investigating the effectiveness and sensitivity of non-target screening of chemical compounds, 18S V9 rRNA gene, and full-length 16S rRNA gene metabarcoding techniques for detecting treated wastewater in receiving waters. We aimed at assessing the impact of introducing 33 % treated wastewater into a triplicated large-scale mesocosm setup during a 10-day exposure period. Discharge of treated wastewater significantly altered the chemical signature as well as the microeukaryotic and prokaryotic diversity of the mesocosms. Non-target screening, 18S V9 rRNA gene, and full-length 16S rRNA gene metabarcoding detected these changes with significant covariation of the detected pattern between methods.
The 18S V9 rRNA gene metabarcoding exhibited superior sensitivity immediately following the introduction of treated wastewater and remained one of the top-performing methods throughout the study. Full-length 16S rRNA gene metabarcoding demonstrated sensitivity only in the initial hour, but became insignificant thereafter. The non-target screening approach was effective throughout the experiment and in contrast to the metabarcoding methods the signal to noise ratio remained similar during the experiment resulting in an increasing relative strength of this method. Based on our findings, we conclude that all methods employed for monitoring environmental disturbances from various sources are suitable. The distinguishing factor of these methods is their ability to detect unknown pollutants and organisms, which sets them apart from previously utilized approaches and allows for a more comprehensive perspective. Given their diverse strengths, particularly in terms of temporal resolution, these methods are best suited as complementary approaches.

Morphologically similar flagellate taxa comprise a high cryptic diversity. This diversity evolved partly through parallel evolution in several phylogenetically distinct taxa. Here we investigate the effects of heat waves and salinization on growth and competition between cryptic taxa. For this purpose, we developed specific FISH probes targeting the phylogenetic clades comprising Pedospumella encystans, Spumella rivalis, and Poteriospumella lacustris, respectively.

Exposure to salt resulted in a decreasing growth rate for all three taxa. In contrast, a sudden increase in temperature to 27 °C stimulated particularly the growth of P. lacustris. This species showed a high competitive strength and the taxon–specific responses to stressors lead to a shift of community composition. This turn–over of differently adapted cryptic species with presumably similar feeding preferences and predator–prey interactions may stabilize microbial food webs facing environmental change.

Salinization and warming are of increasing concern for freshwater ecosystems. Interactive effects of stressors are often studied in bifactorial, two-level experimental setups. The shape of environmental reaction norms and the position of the “control” conditions along them, however, can influence the sign and magnitude of individual responses as well as interactive effects. We empirically model binary-stressor effects in the form of three-dimensional reaction norm surfaces. We monitored the growth of clonal cultures of six freshwater diatoms, Cymbella cf. incurvata, Nitzschia linearis, Cyclotella meneghiniana, Melosira varians, Ulnaria acus, and Navicula gregaria at various temperature (up to 28 ◦C) and salinity (until the growth ceased) shock treatments.

Fitting a broad range of models and comparing them using the Akaike information criterion revealed a large heterogeneity of effects. A bell-shaped curve was often observed in the response of the diatoms to temperature changes, while their growth tended to decrease with increasing electrical conductivity. C. meneghiniana was more tolerant to temperature, whilst C. incurvata and C. meneghiniana were the most sensitive to salinity changes. Empirical modelling revealed interactive effects of temperature and salinity on the slope and the breadth of response curves. Contrasting types of interactions indicates uncertainties in the estimation by empirical modelling.

Recent decades have witnessed a sharp biodiversity decline in freshwaters due to multiple stressors. The presence of multiple stressors is expected to affect community structure and interactions in freshwater ecosystems, with subsequent functional consequences. We synthesized the state of experimental, manipulative multiple‐stressor studies that focused on multispecies assemblages in freshwaters. Compared to rivers and lakes, wetland and groundwater ecosystems have received much less attention in identified multiple‐stressor research.

Most of the identified studies investigated combinations of abiotic stressors (e.g., nutrients, pesticides, heavy metals, warming, altered flow and sedimentation) on microbes and invertebrates while biotic stressors and vertebrates have been largely overlooked. The responses of community structure (e.g., alpha diversity, biomass, and abundance), some community/ecosystem functions (e.g., photosynthesis and autotrophic activity, leaf litter degradation), and morphological traits like body size and growth forms were frequently investigated. We observed a clear gap in biotic interactions under multiple‐stressor conditions, which, although difficult to study, could impede a deeper mechanistic understanding of how multiple stressors affect freshwater assemblages and associated ecological processes.

Although information on ecosystem recovery pathways following restoration is critical for freshwater management, few studies were designed to provide such information, signifying the disconnections between multiple‐stressor research and environmental practice. To bridge these gaps, researchers and environmental practitioners need to work together to identify key stressors and interactions at different spatial and temporal scales and prioritize stressor management. Such collaborations will enhance the translation of multiple‐stressor research into efficient management strategies to protect and restore freshwater ecosystems. This article is categorized under: Water and Life > Stresses and Pressures on Ecosystems Water and Life > Conservation, Management, and Awareness The frequency of combinations between organisms, stressors, and ecological responses used in identified multiple‐stressor experimental studies. Only the stressor‐response linkage nodes with ≥10 counts and organism‐stressor linkage nodes with ≥5 counts were included in this diagram for purposes of clarity.

Temporary rivers are widespread in the Mediterranean region and impose a challenge for the implementation of the Water Framework Directive (WFD) and other environmental regulations. Surprisingly, an overarching analysis of their ecological status and the stressors affecting them is yet missing. We compiled data on the ecological status of 1504 temporary rivers in seven European Mediterranean region countries and related their ecological status (1) to publicly available data on pressures from the European WISE-WFD dataset, and (2) to seven more specific stressors modelled on a sub-catchment scale. More than 50 % of the temporary water bodies in the Mediterranean countries reached good or even high ecological status. In general, status classes derived from phytobenthos and macrophyte assessment were higher than those derived from the assessment of benthic invertebrates or fish.

Of the more generally defined pressures reported to the WISE-WFD database, the most relevant for temporary rivers were 'diffuse agricultural' and 'point urban waste water'. Of the modelled more specific stressors, agricultural land use best explained overall ecological status, followed by total nitrogen load, and urban land use, while toxic substances, total phosphorus load and hydrological stressors were less relevant. However, stressors differed in relevance, with total nitrogen being most important for macrophytes, and agricultural land use for phytobenthos, benthic invertebrates and fish.

For macrophytes, ecological quality increases with stressor intensity. The results underline the overarching effect of land use intensity for the ecological status of temporary water bodies. However, assessment results do not sufficiently reflect hydrological stress, most likely as the biological indicators used to evaluate these systems were designed for perennial water bodies and thus mainly target land use and nutrient impacts. We conclude that biomonitoring systems need to be adapted or newly developed to better account for the specific situation of temporary water bodies.

Parasites can affect their hosts in various ways, and this implies that parasites may act as additional biotic stressors in a multiple-stressor scenario, resembling conditions often found in the field if, for example, pollutants and parasites occur simultaneously. Therefore, parasites represent important modulators of host reactions in ecotoxicological studies when measuring the response of organisms to stressors such as pollutants. In the present article, we introduce the most important groups of parasites occurring in organisms commonly used in ecotoxicological studies ranging from laboratory to field investigations.

After briefly explaining their life cycles, we focus on parasite stages affecting selected ecotoxicologically relevant target species belonging to crustaceans, molluscs, and fish. We included ecotoxicological studies that consider the combination of effects of parasites and pollutants on the respective model organism with respect to aquatic host-parasite systems. We show that parasites from different taxonomic groups (e.g. Microsporidia, Monogenea, Trematoda, Cestoda, Acanthocephala, Nematoda) clearly modulate the response to stressors in their hosts.

The combined effects of environmental stressors and parasites can range from additive, antagonistic to synergistic. This review points to potential drawbacks of ecotoxicological tests if parasite infections of test organisms, especially from the field, remain undetected and unaddressed. If these parasites are not detected and quantified, their physiological effects on the host cannot be separated from the ecotoxicological effects. This may render this type of ecotoxicological test erroneous. In laboratory tests, for example to determine effect or lethal concentrations, the presence of a parasite can also have a direct effect on the concentrations to be determined and thus, on the subsequently determined security levels such as predicted no effect concentrations.

Stable isotope analysis of individual compounds is emerging as a powerful tool to study nutrient origin and conversion in host-parasite systems. We measured the carbon isotope composition of amino acids and glucose in the cestode Schistocephalus solidus and in liver and muscle tissues of its second intermediate host, the three-spined stickleback (Gasterosteus aculeatus), over the course of 90 days in a controlled infection experiment.

Similar linear regressions of δ13C values over time and low trophic fractionation of essential amino acids indicate that the parasite assimilates nutrients from sources closely connected to the liver metabolism of its host. Biosynthesis of glucose in the parasite might occur from the glucogenic precursors alanine, asparagine and glutamine and with an isotope fractionation of − 2 to – 3 ‰ from enzymatic reactions, while trophic fractionation of glycine, serine and threonine could be interpreted as extensive nutrient conversion to fuel parasitic growth through one-carbon metabolism.

Trophic fractionation of amino acids between sticklebacks and their diets was slightly increased in infected compared to uninfected individuals, which could be caused by increased (immune-) metabolic activities due to parasitic infection. Our results show that compound-specific stable isotope analysis has unique opportunities to study host and parasite physiology.

Background

Urban streams are characterised by species-poor and frequently disturbed communities. The recovery of heavily polluted urban streams is challenging but the simple community structure makes recolonisation patterns more transparent. Therefore, they are generally applicable model systems for recolonisation of restored streams. Principal questions of stream restoration concern the drivers and patterns of recolonisation processes. Rarely, recolonisation of restored streams is recorded for a sufficient time to observe patterns of habitat and community development in detail. Over 10 years, we monitored benthic habitat changes and macroinvertebrate communities of eight restored sites in an urban stream network that was formerly used as an open sewer and thus, almost uninhabitable for macroinvertebrates prior to restoration. We analysed changes in environmental variables and communities with a selection of multi-variate analyses and identified indicator species in successional stages.

Results

Proportions of stony substrate and conductivity decreased over time since restoration, while the riparian vegetation cover increased along with the amount of sandy substrate. The communities fluctuated strongly after restoration but began to stabilise after around eight years. TITAN analysis identified 9 species, (e.g. the mayfly Cloeon dipterum and the beetle Agabus didymus), whose abundances decreased with time since restoration, and 19 species with an increasing abundance trend (e.g. several Trichopteran species, which colonised once specific habitats developed). Woody riparian vegetation cover and related variables were identified as major driver for changes in species abundance. In the last phase of the observation period, a dry episode resulted in complete dewatering of some sites. These temporarily dried sections were recolonised much more rapidly compared to the recolonisation following restoration.

Conclusions

Our results underline that community changes following urban stream restoration are closely linked to the evolving environmental conditions of restored streams, in particular habitat availability initialised by riparian vegetation. It takes about a decade for the development of a rich and stable community. Even in streams that were almost completely lacking benthic invertebrates before restoration, the establishment of a diverse macroinvertebrate community is possible, underlining the potential for habitat restoration in formerly heavily polluted urban areas.

Environmental parasitology: stressor effects on aquatic parasites

Anthropogenic stressors are causing fundamental changes in aquatic habitats and to the organisms inhabiting these ecosystems. Yet, we are still far from understanding the diverse responses of parasites and their hosts to these environmental stressors and predicting how these stressors will affect host–parasite communities. Here, we provide an overview of the impacts of major stressors affecting aquatic ecosystems in the Anthropocene (habitat alteration, global warming, and pollution) and highlight their consequences for aquatic parasites at multiple levels of organisation, from the individual to the community level. We provide directions and ideas for future research to better understand responses to stressors in aquatic host–parasite systems.

Temporal disturbance of a model stream ecosystem by high microbial diversity from treated wastewater

Microbial communities in freshwater streams play an essential role in ecosystem functioning via biogeochemical cycling. Yet, the impacts of treated wastewater influx into stream ecosystems on microbial strain diversity remain mostly unexplored. Here, we coupled full-length 16S ribosomal RNA gene Nanopore sequencing and strain-resolved metagenomics to investigate the impact of treated wastewater on a mesocosm system (AquaFlow) run with restored river water. Over 10 days, community Bray–Curtis dissimilarities between treated and control mesocosm decreased (0.57 ± 0.058 to 0.26 ± 0.046) based on ribosomal protein S3 gene clustering, finally converging to nearly identical communities. Similarly, strain-resolved metagenomics revealed a high diversity of bacteria and viruses after the introduction of treated wastewater; these microbes also decreased over time resulting in the same strain clusters in control and treatment at the end of the experiment. Specifically, 39.2% of viral strains detected in all samples were present after the introduction of treated wastewater only. Although bacteria present at low abundance in the treated wastewater introduced additional antibiotic resistance genes, signals of naturally occurring ARG-encoding organisms resembled the resistome at the endpoint. Our results suggest that the previously stressed freshwater stream and its microbial community are resilient to a substantial introduction of treated wastewater.

The Asymmetric Response Concept explains ecological consequences of multiple stressor exposure and release

Our capacity to predict trajectories of ecosystem degradation and recovery is limited, especially when impairments are caused by multiple stressors. Recovery may be fast or slow and either complete or partial, sometimes result in novel ecosystem states or even fail completely. Here, we introduce the Asymmetric Response Concept (ARC) that provides a basis for exploring and predicting the pace and magnitude of ecological responses to, and release from, multiple stressors. The ARC holds that three key mechanisms govern population, community and ecosystem trajectories. Stress tolerance is the main mechanism determining responses to increasing stressor intensity, whereas dispersal and biotic interactions predominantly govern responses to the release from stressors. The shifting importance of these mechanisms creates asymmetries between the ecological trajectories that follow increasing and decreasing stressor intensities. This recognition helps to understand multiple stressor impacts and to predict which measures will restore communities that are resistant to restoration.

Perceived multiple stressor effects depend on sample size and stressor gradient length

Multiple stressors are continuously deteriorating surface waters worldwide, posing many challenges for their conservation and restoration. Combined effect types of multiple stressors range from single-stressor dominance to complex interactions. Identifying prevalent combined effect types is critical for environmental management, as it helps to prioritise key stressors for mitigation. However, it remains unclear whether observed single and combined stressor effects reflect true ecological processes unbiased by sample size and length of stressor gradients. Therefore, we examined the role of sample size and stressor gradient lengths in 158 paired-stressor response cases with over 120,000 samples from rivers, lakes, transitional and marine ecosystems around the world. For each case, we split the overall stressor gradient into two partial gradients (lower and upper) and investigated associated changes in single and combined stressor effects. Sample size influenced the identified combined effect types, and stressor interactions were less likely for cases with fewer samples. After splitting gradients, 40 % of cases showed a change in combined effect type, 30 % no change, and 31 % showed a loss in stressor effects. These findings suggest that identified combined effect types may often be statistical artefacts rather than representing ecological processes. In 58 % of cases, we observed changes in stressor effect directions after the gradient split, suggesting unimodal stressor effects. In general, such non-linear responses were more pronounced for organisms at higher trophic levels. We conclude that observed multiple stressor effects are not solely determined by ecological processes, but also strongly depend on sampling design. Observed effects are likely to change when sample size and/or gradient length are modified. Our study highlights the need for improved monitoring programmes with sufficient sample size and stressor gradient coverage. Our findings emphasize the importance of adaptive management, as stress reduction measures or further ecosystem degradation may change multiple stressor-effect relationships, which will then require associated changes in management strategies.

Interest in local environmental conditions and the occurrence and behaviour of parasites has increased over the last 3 decades, leading to the discipline of Environmental Parasitology. The aim of this discipline is to investigate how anthropogenically altered environmental factors influence the occurrence of parasites and how the combined effects of pollutants and parasites affect the health of their hosts. Accordingly, in this paper, we provide an overview of the direct and indirect effects of pollutants on the occurrence and distribution of fish parasites.

However, based on current knowledge, it is difficult to draw general conclusions about these interdependencies, as the effects of pollutants on free-living (larval) parasite stages, as well as their effects on ectoparasites, depend on the pollutant–host–parasite combination as well as on other environmental factors that can modulate the harmful effects of pollutants. Furthermore, the question of the combined effects of the simultaneous occurrence of parasites and pollutants on the physiology and health of the fish hosts is of interest.

For this purpose, we differentiate between the dominance effects of individual stressors over other, additive or synergistically reinforcing effects as well as combined antagonistic effects. For the latter, there are only very few studies, most of which were also carried out on invertebrates, so that this field of research presents itself as very promising for future investigations.

Interpretation of stable isotope data is of upmost importance in ecology to build sound models for the study of animal diets, migration patterns and physiology. However, our understanding of stable isotope fractionation and incorporation into consumer tissues is still limited. We therefore measured the δ¹³C values of individual amino acids over time from muscle and liver tissue of three-spined sticklebacks (Gasterosteus aculeatus) on a high protein diet.

The δ¹³C values of amino acids in the liver quickly responded to small shifts of under ± 2.0‰ in dietary stable isotope compositions on 30-day intervals. We found on average no trophic fractionation in pooled essential (muscle, liver) and non-essential (muscle) amino acids. Negative Δδ¹³C values of − 0.7 ± 1.3‰ were observed for pooled non-essential (liver) amino acids and might indicate biosynthesis from small amounts of dietary lipids. Trophic fractionation of individual amino acids is reported and discussed, including unusual Δδ¹³C values of over + 4.9 ± 1.4‰ for histidine.

Arginine and lysine showed the lowest trophic fractionation on individual sampling days and might be useful proxies for dietary sources on short time scales. We suggest further investigations using isotopically enriched materials to facilitate the correct interpretation of ecological field data.

SeqCode: a nomenclatural code for prokaryotes described from sequence data

Most prokaryotes are not available as pure cultures and therefore ineligible for naming under the rules and recommendations of the International Code of Nomenclature of Prokaryotes (ICNP). Here we summarize the development of the SeqCode, a code of nomenclature under which genome sequences serve as nomenclatural types. This code enables valid publication of names of prokaryotes based upon isolate genome, metagenome-assembled genome or single-amplified genome sequences. Otherwise, it is similar to the ICNP with regard to the formation of names and rules of priority. It operates through the SeqCode Registry (https://seqco.de/), a registration portal through which names and nomenclatural types are registered, validated and linked to metadata. We describe the two paths currently available within SeqCode to register and validate names, including Candidatus names, and provide examples for both. Recommendations on minimal standards for DNA sequences are provided. Thus, the SeqCode provides a reproducible and objective framework for the nomenclature of all prokaryotes regardless of cultivability and facilitates communication across microbiological disciplines.

First report of microsporidians in the non-native shrimp Neocaridina davidi from a temperate European stream

The release of ornamental pets outside their native range can directly or indirectly impact the recipient community, e.g. via the co-introduction of associated pathogens. However, studies on parasites associated with non-native species, in particular freshwater decapods, have focused mainly on a limited set of pathogens. Here we provide data for the first time on microsporidian parasites of the non-native ornamental shrimp Neocaridina davidi, collected in a stream in Germany. Furthermore, we confirm an ongoing range expansion of the warm-adapted N. davidi from thermally polluted colder water. In the investigated shrimps, the microsporidian parasite Enterocytozoon hepatopenaei and an unknown microsporidian isolate were detected, raising concerns about their transmission potential and pathogenicity on native crustacean species.

Development of the SeqCode: a proposed nomenclatural code for uncultivated prokaryotes with DNA sequences as type

Over the last fifteen years, genomics has become fully integrated into prokaryotic systematics. The genomes of most type strains have been sequenced, genome sequence similarity is widely used for delineation of species, and phylogenomic methods are commonly used for classification of higher taxonomic ranks. Additionally, environmental genomics has revealed a vast diversity of as-yetuncultivated taxa. In response to these developments, a new code of nomenclature, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), has been developed over the last two years to allow naming of Archaea and Bacteria using DNA sequences as the nomenclatural types. The SeqCode also allows naming of cultured organisms, including fastidious prokaryotes that cannot be deposited into culture collections. Several simplifications relative to the International Code of Nomenclature of Prokaryotes (ICNP) are implemented to make nomenclature more accessible, easier to apply and more readily communicated. By simplifying nomenclature with the goal of a unified classification, inclusive of both cultured and uncultured taxa, the SeqCode will facilitate the naming of taxa in every biome on Earth, encourage the isolation and characterization of as-yet-uncultivated taxa, and promote synergies between the ecological, environmental, physiological, biochemical, and molecular biological disciplines to more fully describe prokaryotes.

Generalist parasites persist in degraded environments: a lesson learned from microsporidian diversity in amphipod

The present study provides new insight into suitable microsporidian-host associations. It relates regional and continental-wide host specialization in microsporidians infecting amphipods to degraded and recovering habitats across 2 German river catchments. It provides a unique opportunity to infer the persistence of parasites following anthropogenic disturbance and their establishment in restored rivers. Amphipods were collected in 31 sampling sites with differing degradation and restoration gradients. Specimens were morphologically (hosts) and molecularly identified (host and parasites). Amphipod diversity and abundance, microsporidian diversity, host phylogenetic specificity and continental-wide beta-specificity were investigated and related to each other and/or environmental variables. Fourteen microsporidian molecular operational taxonomic units (MOTUs), mainly generalist parasites, infecting 6 amphipod MOTUs were detected, expanding the current knowledge on the host range by 17 interactions. There was no difference in microsporidian diversity and host specificity among restored and near-natural streams (Boye) or between those located in urban and rural areas (Kinzig). Similarly, microsporidian diversity was generally not influenced by water parameters. In the Boye catchment, host densities did not influence microsporidian MOTU richness across restored and near-natural sites. High host turnover across the geographical range suggests that neither environmental conditions nor host diversity plays a significant role in the establishment into restored areas. Host diversity and environmental parameters do not indicate the persistence and dispersal of phylogenetic host generalist microsporidians in environments that experienced anthropogenic disturbance. Instead, these might depend on more complex mechanisms such as the production of resistant spores, host switching and host dispersal acting individually or conjointly.