From DNA to diagnostics: A case study using macroinvertebrate metabarcoding to assess the effectiveness of restoration measures in a Dutch stream.

Stream ecosystems are under pressure due to multiple stressors. Restoration measures can halt further degradation and improve their ecological status. However, assessment of the effectiveness of the implemented measures is often insufficient because of logistic and financial constraints. DNA-metabarcoding has been proposed to scale up sample processing, although its application as a diagnostic tool has received less attention. The aim of our study was to evaluate if DNA-metabarcoding of stream macroinvertebrates can be used to compute a stressor-specific index to assess the effectiveness of a stream restoration project. For this purpose, we sampled the upstream, restored, and downstream section of a recently restored lowland stream in the Netherlands. At each site, we applied three different methods of macroinvertebrate identification: morphological identification of bulk samples (morphology), DNA-metabarcoding of the same bulk samples (DNA) and metabarcoding of eDNA extracted from the water (eDNA). First, we compared the community composition identified by each method. The communities identified by morphology and DNA were highly similar, whereas the communities generated by the eDNA differed. Second, we analysed whether the identification methods could be used to assess the effectiveness of the restoration project, focussing on a stressor-specific index for flow as the restoration measures aimed at improving flow conditions. Both the morphology and bulk DNA samples indicated improved flow conditions in the restored section of the stream (i.e., less stress from the reduction or absence of flow than in the unrestored sections). Contrary, the eDNA-water samples did not differentiate the amount of stress throughout the catchment, although applying recent developments in eDNA sampling could lead to more robust results. In conclusion, this study forms proof of concept that DNA from bulk samples can be utilized to assess the effectiveness of restoration measures, showing the added value of this approach for water managers.

Eutrophication induces shifts in the trophic position of invertebrates in aquatic food webs.

Changes in the ecological stoichiometry of primary producers may have considerable implications for energy and matter transfer in food webs. We hypothesized that nutrient enrichment shifts the trophic position of omnivores towards herbivory, as the nutritional quality of primary producers increases. This hypothesis was tested by analyzing the ecological stoichiometry and stable isotope signature of primary producers and a wide range of aquatic macroinvertebrates, including primary consumers (herbivores) and secondary consumers (both potential omnivores and strict carnivores), along a eutrophication gradient in an agricultural landscape. Our results showed (1) that carbon : nutrient ratios of primary producers decreased along the eutrophication gradient, while the elemental composition of consumers remained homeostatic, and (2) that the trophic position of several omnivores and the generalist predator Notonecta decreased, while the trophic position of most other consumers remained constant. These findings suggest that shifts in the diets of aquatic invertebrates induced by increasing eutrophication may affect species interactions and food web structure in aquatic ecosystems.

Over forty years of lowland stream restoration: Lessons learned?

Stream restoration efforts have increased, but the success rate is still rather low. The underlying reasons for these unsuccessful restoration efforts remain inconclusive and need urgent clarification. Therefore, the aim of the present study was to evaluate over 40 years of stream restoration to fuel future perspectives. To this purpose we evaluated the influence of policy goals on stream restoration efforts, biophysical restoration objectives, restoration measures applied including the scale of application and monitoring efforts. Information was obtained from five stream restoration surveys that were held among the regional water authorities in the Netherlands over the last 40 years and from an analysis of the international scientific publications on stream restoration spanning the same time period. Our study showed that there was a considerable increase in stream restoration efforts, especially motivated by environmental legislation. However, proper monitoring of the effectiveness of the measures was often lacking. Furthermore, a mismatch between restoration goals and restoration measures was observed. Measures are still mainly focused on hydromorphological techniques, while biological goals remain underexposed and therefore need to be better targeted. Moreover, restoration practices occur mainly on small scales, despite the widely recognized relevance of tackling multiple stressors acting over large scales for stream ecosystem recovery. In order to increase the success rate of restoration projects, it is recommended to improve the design of the accompanying monitoring programmes, allowing to evaluate, over longer time periods, if the measures taken led to the desired results. Secondly, we advise to diagnose the dominant stressors and plan restoration measures at the appropriate scale of these stressors, generally the catchment scale.

Limitations of trait-based approaches for stressor assessment: The case of freshwater invertebrates and climate drivers.

The appeal of trait-based approaches for assessing environmental vulnerabilities arises from the potential insight they provide into the mechanisms underlying the changes in populations and community structure. Traits can provide ecologically based explanations for observed responses to environmental changes, along with predictive power gained by developing relationships between traits and environmental variables. Despite these potential benefits, questions remain regarding the utility and limitations of these approaches, which we explore focusing on the following questions: (a) How reliable are predictions of biotic responses to changing conditions based on single trait-environment relationships? (b) What factors constrain detection of single trait-environment relationships, and how can they be addressed? (c) Can we use information on meta-community processes to reveal conditions when assumptions underlying trait-based studies are not met? We address these questions by reviewing published literature on aquatic invertebrate communities from stream ecosystems. Our findings help to define factors that influence the successful application of trait-based approaches in addressing the complex, multifaceted effects of changing climate conditions on hydrologic and thermal regimes in stream ecosystems. Key conclusions are that observed relationships between traits and environmental stressors are often inconsistent with predefined hypotheses derived from current trait-based thinking, particularly related to single trait-environment relationships. Factors that can influence findings of trait-based assessments include intercorrelations of among traits and among environmental variables, spatial scale, strength of biotic interactions, intensity of habitat disturbance, degree of abiotic stress, and methods of trait characterization. Several recommendations are made for practice and further study to address these concerns, including using phylogenetic relatedness to address intercorrelation. With proper consideration of these issues, trait-based assessment of organismal vulnerability to environmental changes can become a useful tool to conserve threatened populations into the future.

Quantifying cumulative stress acting on macroinvertebrate assemblages in lowland streams.

Macroinvertebrates in lowland streams are exposed to multiple stressors from the surrounding environment. Yet, quantifying how these multiple stressors impact macroinvertebrate assemblages is challenging. The aim of this study was to develop a novel method to quantify the cumulative stress acting on macroinvertebrate assemblages in lowland streams. To this purpose, we considered 22 stressors from different stressor categories such as hydrological, morphological and chemical stressors, acting over multiple spatial scales ranging from instream to the catchment scale. Stressor intensity was categorized into classes based on impact on the macroinvertebrate assemblages. The main stream was divided into segments, after which for each stream segment, the cumulative stressor contribution from headwater catchments, from the riparian zone and from upstream was calculated. To validate the cumulative stress quantification method, the lowland stream Tungelroyse Beek in the Netherlands was used as a case study. For this stream it was shown that independently derived ecological quality scores based on macroinvertebrate samples collected at multiple sites along the stream decreased with increasing calculated cumulative stress scores, supporting the design of the cumulative stress quantification method. Based on the contribution of each specific stressor to the cumulative stress scores, the reasons for the absence and presence of macroinvertebrate species may be elucidated. Hence, the cumulative stress quantification method may help to identify and localize the most stringent stressors limiting macroinvertebrate assemblages, and can thereby provide a better focus for management resources.

Qualifying the effects of single and multiple stressors on the food web structure of Dutch drainage ditches using a literature review and conceptual models.

In September 2017, a workshop was held at Wageningen University and Research to determine the current state of knowledge of multiple stressor effects on aquatic ecosystems and to assess how to improve prediction of these effects. We developed a theoretical framework that integrates species-level responses to stressors to predict how these effects propagate through higher levels of biological organisation. Here, we present the application of the framework for drainage ditch ecosystems in the Netherlands. We used food webs to assess single and multiple stressor effects of common stressors on ditch communities. We reviewed the literature for the effects of targeted stressors (nutrients, pesticides, dredging and mowing, salinization, and siltation) on each functional group present in the food web and qualitatively assessed the relative sensitivity of groups. Using this information, we created a stressor-response matrix of positive and negative direct effects of each stressor-functional group combination. Fungicides, salinization, and sedimentation were identified as particularly detrimental to most groups, although destructive management practices, such as dredging with almost complete community removal, would take precedence depending on frequency. Using the stressor-response matrix we built, first, a series of conceptual null models of single stressor effects on food web structure and, second, a series of additive null models to illustrate potential paired-stressor effects. We compared these additive null models with published studies of the same pairs of combined single stressors to explore more complex interactions. Our approach serves as a first-step to considering multiple stressor scenarios in systems that are understudied or data-poor and as a baseline from which more complex models that include indirect effects and quantitative data may be developed. We make specific suggestions for appropriate management strategies that could be taken to support the biodiversity of these systems for individual stressors and their combined impacts.

Towards a general framework for the assessment of interactive effects of multiple stressors on aquatic ecosystems: Results from the Making Aquatic Ecosystems Great Again (MAEGA) workshop.

A workshop was held in Wageningen, The Netherlands, in September 2017 to collate data and literature on three aquatic ecosystem types (agricultural drainage ditches, urban floodplains, and urban estuaries), and develop a general framework for the assessment of multiple stressors on the structure and functioning of these systems. An assessment framework considering multiple stressors is crucial for our understanding of ecosystem responses within a multiply stressed environment, and to inform appropriate environmental management strategies. The framework consists of two components: (i) problem identification and (ii) impact assessment. Both assessments together proceed through the following steps: 1) ecosystem selection; 2) identification of stressors and quantification of their intensity; 3) identification of receptors or sensitive groups for each stressor; 4) identification of stressor-response relationships and their potential interactions; 5) construction of an ecological model that includes relevant functional groups and endpoints; 6) prediction of impacts of multiple stressors, 7) confirmation of these predictions with experimental and monitoring data, and 8) potential adjustment of the ecological model. Steps 7 and 8 allow the assessment to be adaptive and can be repeated until a satisfactory match between model predictions and experimental and monitoring data has been obtained. This paper is the preface of the MAEGA (Making Aquatic Ecosystems Great Again) special section that includes three associated papers which are also published in this volume, which present applications of the framework for each of the three aquatic systems.

Reintroduction of freshwater macroinvertebrates: challenges and opportunities.

Species reintroductions - the translocation of individuals to areas in which a species has been extirpated with the aim of re-establishing a self-sustaining population - have become a widespread practice in conservation biology. Reintroduction projects have tended to focus on terrestrial vertebrates and, to a lesser extent, fishes. Much less effort has been devoted to the reintroduction of invertebrates into restored freshwater habitats. Yet, reintroductions may improve restoration outcomes in regions where impoverished regional species pools limit the self-recolonisation of restored freshwaters. We review the available literature on macroinvertebrate reintroductions, focusing on identifying the intrinsic and extrinsic factors that determine their success or failure. Our study reveals that freshwater macroinvertebrate reintroductions remain rare, are often published in the grey literature and, of the attempts made, approximately one-third fail. We identify life-cycle complexity and remaining stressors as the two factors most likely to affect reintroduction success, illustrating the unique challenges of freshwater macroinvertebrate reintroductions. Consideration of these factors by managers during the planning process and proper documentation - even if a project fails - may increase the likelihood of successful outcomes in future reintroduction attempts of freshwater macroinvertebrates.

Biomonitoring of intermittent rivers and ephemeral streams in Europe: Current practice and priorities to enhance ecological status assessments.

Intermittent rivers and ephemeral streams (IRES) are common across Europe and dominate some Mediterranean river networks. In all climate zones, IRES support high biodiversity and provide ecosystem services. As dynamic ecosystems that transition between flowing, pool, and dry states, IRES are typically poorly represented in biomonitoring programmes implemented to characterize EU Water Framework Directive ecological status. We report the results of a survey completed by representatives from 20 European countries to identify current challenges to IRES status assessment, examples of best practice, and priorities for future research. We identify five major barriers to effective ecological status classification in IRES: 1. the exclusion of IRES from Water Framework Directive biomonitoring based on their small catchment size; 2. the lack of river typologies that distinguish between contrasting IRES; 3. difficulties in defining the 'reference conditions' that represent unimpacted dynamic ecosystems; 4. classification of IRES ecological status based on lotic communities sampled using methods developed for perennial rivers; and 5. a reliance on taxonomic characterization of local communities. Despite these challenges, we recognize examples of innovative practice that can inform modification of current biomonitoring activity to promote effective IRES status classification. Priorities for future research include reconceptualization of the reference condition approach to accommodate spatiotemporal fluctuations in community composition, and modification of indices of ecosystem health to recognize both taxon-specific sensitivities to intermittence and dispersal abilities, within a landscape context.

Oxygen drives benthic-pelagic decomposition pathways in shallow wetlands.

Oxygen availability is perceived as an important environmental factor limiting POM decomposition. In shallow wetlands, however, the impact of commonly observed anoxic conditions in the benthic layer on the relative contribution of microbes and invertebrates to POM decomposition remains largely unknown. Therefore, the aim of this study was to determine if dissolved oxygen drives benthic-pelagic decomposition pathways in shallow wetlands. Dissolved oxygen concentration, invertebrate community composition, microbial decomposition and invertebrate consumption were measured in the benthic and pelagic layer of 15 permanent drainage ditches. We showed that an increased duration of anoxic conditions in the benthic layer of the ditches was related to increased microbial decomposition in this layer, while invertebrate consumption decreased in the benthic layer and increased in the pelagic layer. The increased invertebrate consumption in the pelagic layer was related to the presence of amphipods. We concluded that anoxic conditions in the benthic layer of shallow wetlands relate to an increase in microbial decomposition and a decrease in invertebrate consumption, as detritivorous invertebrates move to the pelagic layer to consume particulate organic matter. This illustrates that environmental conditions, such as dissolved oxygen, may drive the relative importance of aquatic organisms to ecosystem functioning.

Natural and manipulated populations of the treehole mosquito, Ochlerotatus triseriatus, at its northernmost range limit in southern Ontario, Canada.

Ochlerotatus triseriatus, the eastern treehole mosquito, reaches its northernmost range limit in the extreme southeast of Canada. As a known vector of West Nile and La Crosse encephalitis viruses and a potential vector of eastern equine encephalitis, its population biology is of interest. In southern Ontario, high larval densities occur in urban woodlots within sugar maple and American beech treehole communities comprising rotifers, nematode worms, mites, other dipterans, and scirtid beetles. Treehole water was characterized by low dissolved oxygen levels and seasonally variable pH and temperature, with the latter being most influential on local populations. Densities were significantly higher (up to 503 larvae 100 ml(-1)) in tree holes close to the forest floor (<1 m) and in experimental tree holes seeded with autumn-shed maple leaves as opposed to leaves of black oak and beech. In this locality, weekly sampling showed Oc. triseriatus to be multivoltine, with mass egg hatching beginning under coldwater (<10 degrees C) conditions in March/April, and thereafter producing three successful generations with a possible, less successful fourth in late summer. Some 1st instar larvae were present in water as cold as 0.7 degree C. Compared with larval psychodids living in the same tree hole, population losses of Oc. triseriatus due to washout during major rainfall events were negligible despite high flowthrough of water derived from stemflow.