A partner-switching system controls activation of mixed-linkage ß-glucan synthesis by c-di-GMP in Sinorhizobium meliloti.

Sinorhizobium meliloti synthesizes a linear mixed-linkage (1 → 3)(1 → 4)-ß-d-glucan (ML ß-glucan, MLG) in response to high levels of cyclic diguanylate (c-di-GMP). Two proteins BgsA and BgsB are required for MLG synthesis, BgsA being the glucan synthase which is activated upon c-di-GMP binding to its C-terminal domain. Here we report that the product of bgrR (SMb20447) is a diguanylate cyclase (DGC) that provides c-di-GMP for the synthesis of MLG by BgsA. bgrR is the first gene of a hexacistronic bgrRSTUWV operon, likely encoding a partner-switching regulatory network where BgrR is the final target. Using different approaches, we have determined that the products of genes bgrU (containing a putative PP2C serine phosphatase domain) and bgrW (with predicted kinase effector domain), modulate the phosphorylation status and the activity of the STAS domain protein BgrV. We propose that unphosphorylated BgrV inhibits BgrR DGC activity, perhaps through direct protein-protein interactions as established for other partner switchers. A bgrRSTUWV operon coexists with MLG structural bgsBA genes in many rhizobial genomes but is also present in some MLG non-producers, suggesting a role of this partner-switching system in other processes besides MLG biosynthesis.

Novel mixed-linkage ß-glucan activated by c-di-GMP in Sinorhizobium meliloti.

An artificial increase of cyclic diguanylate (c-di-GMP) levels in Sinorhizobium meliloti 8530, a bacterium that does not carry known cellulose synthesis genes, leads to overproduction of a substance that binds the dyes Congo red and calcofluor. Sugar composition and methylation analyses and NMR studies identified this compound as a linear mixed-linkage (1 → 3)(1 → 4)-ß-D-glucan (ML ß-glucan), not previously described in bacteria but resembling ML ß-glucans found in plants and lichens. This unique polymer is hydrolyzed by the specific endoglucanase lichenase, but, unlike lichenan and barley glucan, it generates a disaccharidic → 4)-ß-D-Glcp-(1 → 3)-ß-D-Glcp-(1 → repeating unit. A two-gene operon bgsBA required for production of this ML ß-glucan is conserved among several genera within the order Rhizobiales, where bgsA encodes a glycosyl transferase with domain resemblance and phylogenetic relationship to curdlan synthases and to bacterial cellulose synthases. ML ß-glucan synthesis is subjected to both transcriptional and posttranslational regulation. bgsBA transcription is dependent on the exopolysaccharide/quorum sensing ExpR/SinI regulatory system, and posttranslational regulation seems to involve allosteric activation of the ML ß-glucan synthase BgsA by c-di-GMP binding to its C-terminal domain. To our knowledge, this is the first report on a linear mixed-linkage (1 → 3)(1 → 4)-ß-glucan produced by a bacterium. The S. meliloti ML ß-glucan participates in bacterial aggregation and biofilm formation and is required for efficient attachment to the roots of a host plant, resembling the biological role of cellulose in other bacteria.

The Sinorhizobium fredii HH103 Genome: A Comparative Analysis With S. fredii Strains Differing in Their Symbiotic Behavior With Soybean.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III-secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

Dead molluscan shells from multiple trophic groups as archives of nitrogen isotopic evidence of wastewater gradients in estuaries.

Increased coastal urbanization worldwide has resulted in increased nitrogen inputs to ecosystems, leading to eutrophication and other negative effects. We assessed δ15N in the dead-collected shells of three molluscan species in two estuaries in order to evaluate their ability to identify known gradients in wastewater nitrogen input, namely from private septic systems feeding directly into Waquoit Bay and from a groundwater plume reflecting wastewater injection at a municipal treatment plant in West Falmouth Harbor, Massachusetts, USA. Shells of a suspension-feeder (Geukensia demissa), a micro-algal grazer (Littorina littorea), and an omnivore (Nassarius obsoletus) were collected from lower intertidal sediments near the taxon's life habitat. We found that δ15N in dead-collected shells decreased significantly along the gradients of wastewater pollution in both estuaries, with similar results across all three trophic groups. These positive results demonstrate the potential of dead-shell assemblages for detecting spatial gradients of wastewater pollution.

Genome sequence of the soybean symbiont Sinorhizobium fredii HH103.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain that is able to nodulate legumes that develop determinate nodules, e.g., soybean, and legumes that form nodules of the indeterminate type. Here we present the genome of HH103, which consists of one chromosome and five plasmids with a total size of 7.22 Mb.

The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism and the symbiotic interaction with alfalfa.

BACKGROUND: The bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. Sinorhizobium meliloti is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in S. meliloti has remained unexplored. RESULTS: Two independent S. meliloti mutants, 2011-3.4 and 1021Deltahfq, were obtained by disruption and deletion of the hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Deltahfq revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 1021Deltahfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of nifA and fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixK is only aerobiosis dependent. Finally, we found that some of the recently identified S. meliloti sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein. CONCLUSIONS: Our results support that the S. meliloti RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified S. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.

Sinorhizobium fredii HH103 cgs mutants are unable to nodulate determinate- and indeterminate nodule-forming legumes and overproduce an altered EPS.

Sinorhizobium fredii HH103 produces cyclic beta glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgsColon, two colonslacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs::Omega) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.

Gene SMb21071 of plasmid pSymB is required for osmoadaptation of Sinorhizobium meliloti 1021 and is implicated in modifications of cell surface polysaccharides structure in response to hyperosmotic stress.

Megaplasmid pSymB of the nitrogen-fixing symbiont Sinorhizobium meliloti, implicated in adaptation to hyperosmotic stress, contains 11 gene clusters that apparently encode surface polysaccharides. However, only 2 of these clusters, containing the exo and exp genes, have been associated with the synthesis of the acidic exopolysaccharides succinoglycan and galactoglucan, respectively. The functions of the other 9 clusters remain unsolved. The involvement of one of those regions, pSymB cluster 3, on surface polysaccharide synthesis and its possible implication in osmoadaptation were investigated. In silico analysis of cluster 3 showed that it putatively encodes for the synthesis and transport of a methylated surface polysaccharide. Mutants affected in this cluster were symbiotically effective but showed defects in growth under saline and nonsaline osmotic stress. The gene SMb21071, encoding a putative initiating glycosyltransferase, is transcriptionally induced under hyperosmotic conditions. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and silver staining showed that osmotic stresses changed the profiles of surface polysaccharides of wild-type and mutants strains in different ways. The overall results suggest that cluster 3 is important for growth under saline stress and essential for growth under nonsaline hyperosmotic stress, and it appears to be implicated in maintaining and (or) modifying surface polysaccharides in response to osmotic stress.

Integrative ecotoxicological assessment of sediment in Portmán Bay (southeast Spain).

Portmán Bay, southeast Spain, contains the most seriously metal-contaminated sediments of the Mediterranean Sea. From 1958 to 1991, approximately 50 million tons of mine tailings were dumped into the bay, completely filling up the bay and dispersing over an extensive area of the continental platform and continental slope. The objective of our study was to characterize the nature and extent of metal contamination and the responses of natural communities to it and to assess the toxicity of the sediment deposits 10 years after mining had ceased. We studied the physical and chemical characteristics of the sediments and toxicity (of the porewater and sediment-water interface) using two sea urchin species (Arbacia lixula and Paracentrotus lividus). Metal bioavailability and patterns of macroinvertebrate community composition along the contamination gradient were also studied. Univariate and multivariate analyses showed positive correlation between the sediment metal concentrations associated to the all biological effects (sea urchins toxicity tests and benthic indices). The effects of sediment contamination on the benthic community structure are visible among sampling stations.

Transcriptional organization of the region encoding the synthesis of the flagellar filament in Pseudomonas fluorescens.

Pseudomonas fluorescens F113 is motile by means of type b flagella. Analysis of the region encoding the synthesis of the flagellar filament has shown a transcriptional organization different from that of type a flagella. Additionally to the promoters driving fliC, fliD, and fleQ expression, we have found promoters upstream of the flaG gene and the fliST operon. These promoters were functional in vivo. Both promoters have been mapped and appear to be dependent on the vegetative sigma factor and independent of FleQ, the master regulator of flagellum synthesis.

Carbon and nitrogen stable isotopes and metal concentration in food webs from a mining-impacted coastal lagoon.

Two food webs from the Mar Menor coastal lagoon, differing in the distance from the desert-stream through which mining wastes were discharged, were examined by reference to essential (Zn and Cu) and non-essential (Pb and Cd) metal concentrations and stable isotopes content (C and N). The partial extraction technique applied, which reflects the availability of metals to organisms after sediment ingestion, showed higher bioavailable metal concentrations in sediments from the station influenced by the mining discharges, in agreement with the higher metal concentrations observed in organisms, which in many cases exceeded the regulatory limits established in Spanish legislation concerning seafood. Spatial differences in essential metal concentrations in the fauna suggest that several organisms are exposed to metal levels above their regulation capacity. Differences in isotopic composition were found between both food webs, the wadi-influenced station showing higher delta(15)N values and lower delta(13)C levels, due to the discharge of urban waste waters and by the entrance of freshwater and allochthonous marsh plants. The linear-regressions between trophic levels (as indicated by delta(15)N) and the metal content indicated that biomagnification does not occur. In the case of invertebrates, since the "handle strategy" of the species and the physiological requirements of the organisms, among other factors, determine the final concentration of a specific element, no clear relationships between trophic level and the metal content are to be expected. For their part, fish communities did not show clear patterns in the case of any of the analyzed metals, probably because most fish species have similar metal requirements, and because biological factors also intervened. Finally, since the study deals with metals, assumptions concerning trophic transfer factors calculation may not be suitable since the metal burden originates not only from the prey but also from adsorption over the body surfaces and seawater ingestion.

Tropical land-sea couplings: Role of watershed deforestation, mangrove estuary processing, and marine inputs on N fluxes in coastal Pacific Panama.

We review data from coastal Pacific Panama and other tropical coasts with two aims. First, we defined inputs and losses of nitrogen (N) mediating connectivity of watersheds, mangrove estuaries, and coastal sea. N entering watersheds-mainly via N fixation (79-86%)-was largely intercepted; N discharges to mangrove estuaries (3-6%), small compared to N inputs to watersheds, nonetheless significantly supplied N to mangrove estuaries. Inputs to mangrove estuaries (including watershed discharges, and marine inputs during flood tides) were matched by losses (mainly denitrification and export during ebb tides). Mangrove estuary subsidies of coastal marine food webs take place by export of forms of N [DON (62.5%), PN (9.1%), and litter N (12.9%)] that provide dissimilative and assimilative subsidies. N fixation, denitrification, and tidal exchanges were major processes, and DON was major form of N involved in connecting fluxes in and out of mangrove estuaries. Second, we assessed effects of watershed forest cover on connectivity. Decreased watershed forest cover lowered N inputs, interception, and discharge into receiving mangrove estuaries. These imprints of forest cover were erased during transit of N through estuaries, owing to internal N cycle transformations, and differences in relative area of watersheds and estuaries. Largest losses of N consisted of water transport of energy-rich compounds, particularly DON. N losses were similar in magnitude to N inputs from sea, calculated without considering contribution by intermittent coastal upwelling, and hence likely under-estimated. Pacific Panama mangrove estuaries are exposed to major inputs of N from land and sea, which emphasizes the high degree of bi-directional connectivity in these coupled ecosystems. Pacific Panama is still lightly affected by human or global changes. Increased deforestation can be expected, as well as changes in ENSO, which will surely raise watershed-derived loads of N, as well as significantly change marine N inputs affecting coastal coupled ecosystems.

Stable isotopic evidence of nitrogen sources and C4 metabolism driving the world's largest macroalgal green tides in the Yellow Sea.

During recent years, rapid seasonal growth of macroalgae covered extensive areas within the Yellow Sea, developing the world's most spatially extensive "green tide". The remarkably fast accumulation of macroalgal biomass is the joint result of high nitrogen supplies in Yellow Sea waters, plus ability of the macroalgae to optionally use C4 photosynthetic pathways that facilitate rapid growth. Stable isotopic evidence shows that the high nitrogen supply is derived from anthropogenic sources, conveyed from watersheds via river discharges, and by direct atmospheric deposition. Wastewater and manures supply about half the nitrogen used by the macroalgae, fertiliser and atmospheric deposition each furnish about a quarter of the nitrogen in macroalgae. The massive green tides affecting the Yellow Sea are likely to increase, with significant current and future environmental and human consequences. Addressing these changing trajectories will demand concerted investment in new basic and applied research as the basis for developing management policies.

Long-term nutrient addition increases respiration and nitrous oxide emissions in a New England salt marsh.

Salt marshes may act either as greenhouse gas (GHG) sources or sinks depending on hydrological conditions, vegetation communities, and nutrient availability. In recent decades, eutrophication has emerged as a major driver of change in salt marsh ecosystems. An ongoing fertilization experiment at the Great Sippewissett Marsh (Cape Cod, USA) allows for observation of the results of over four decades of nutrient addition. Here, nutrient enrichment stimulated changes to vegetation communities that, over time, have resulted in increased elevation of the marsh platform. In this study, we measured fluxes of carbon dioxide (CO 2), methane (CH 4) and nitrous oxide (N2O) in dominant vegetation zones along elevation gradients of chronically fertilized (1,572 kg N ha-1 year-1) and unfertilized (12 kg N ha-1 year-1) experimental plots at Great Sippewissett Marsh. Flux measurements were performed using darkened chambers to focus on community respiration and excluded photosynthetic CO 2 uptake. We hypothesized that N-replete conditions in fertilized plots would result in larger N2O emissions relative to control plots and that higher elevations caused by nutrient enrichment would support increased CO 2 and N2O and decreased CH 4 emissions due to the potential for more oxygen diffusion into sediment. Patterns of GHG emission supported our hypotheses. Fertilized plots were substantially larger sources of N2O and had higher community respiration rates relative to control plots, due to large emissions of these GHGs at higher elevations. While CH 4 emissions displayed a negative relationship with elevation, they were generally small across elevation gradients and nutrient enrichment treatments. Our results demonstrate that at decadal scales, vegetation community shifts and associated elevation changes driven by chronic eutrophication affect GHG emission from salt marshes. Results demonstrate the necessity of long-term fertilization experiments to understand impacts of eutrophication on ecosystem function and have implications for how chronic eutrophication may impact the role that salt marshes play in sequestering C and N.

Transient coastal landscapes: Rising sea level threatens salt marshes.

Salt marshes are important coastal environments that provide key ecological services. As sea level rise has accelerated globally, concerns about the ability of salt marshes to survive submergence are increasing. Previous estimates of likely survival of salt marshes were based on ratios of sea level rise to marsh platform accretion. Here we took advantage of an unusual, long-term (1979-2015), spatially detailed comparison of changes in a representative New England salt marsh to provide an empirical estimate of habitat losses based on actual measurements. We show prominent changes in habitat mosaic within the marsh, consistent and coincident with increased submergence and coastal erosion. Model results suggest that at current rates of sea level rise, marsh platform accretion, habitat loss, and with the limitation of the widespread "coastal squeeze", the entire ecosystem might disappear by the beginning of the next century, a fate that might be likely for many salt marshes elsewhere. Meta-analysis of available data suggests that 40 to 95% of the world's salt marshes will be submerged, depending on whether sea level rise remains at current or reaches anticipated rates for the end of this century.

Eutrophication of Cape Cod estuaries: Effect of decadal changes in global-driven atmospheric and local-scale wastewater nutrient loads.

Nitrogen (N) supply by atmospheric deposition, wastewater, and fertilizers controls estuarine eutrophication. In New England, atmospheric N loads recently decreased by 50% and land-derived contributions rose about 80%, owing to national-scale emission controls and local urban development. The decrease in atmospheric deposition was large enough to balance increases in land-derived N loads, so total N loads to Waquoit Bay estuaries in Cape Cod did not change significantly between 1990 and 2014. Unchanged N regimes were corroborated by finding no differences in estuarine nutrient concentrations and macrophyte biomass between pre-2005 and in 2015. Coastal zones, subject to reasonably rapid changes in global and local driver variables, will require that assessment and management of eutrophication include adaptive strategies that capture effects of changing baselines. Management initiatives will be constrained by spatial scale of driver variables: local efforts may address wastewater and fertilizer N sources, but atmospheric sources require national or international attention.

An integrated Pan-European perspective on coastal Lagoons management through a mosaic-DPSIR approach.

A decision support framework for the management of lagoon ecosystems was tested using four European Lagoons: Ria de Aveiro (Portugal), Mar Menor (Spain), Tyligulskyi Liman (Ukraine) and Vistula Lagoon (Poland/Russia). Our aim was to formulate integrated management recommendations for European lagoons. To achieve this we followed a DPSIR (Drivers-Pressures-State Change-Impacts-Responses) approach, with focus on integrating aspects of human wellbeing, welfare and ecosystem sustainability. The most important drivers in each lagoon were identified, based on information gathered from the lagoons' stakeholders, complemented by scientific knowledge on each lagoon as seen from a land-sea perspective. The DPSIR cycles for each driver were combined into a mosaic-DPSIR conceptual model to examine the interdependency between the multiple and interacting uses of the lagoon. This framework emphasizes the common links, but also the specificities of responses to drivers and the ecosystem services provided. The information collected was used to formulate recommendations for the sustainable management of lagoons within a Pan-European context. Several common management recommendations were proposed, but specificities were also identified. The study synthesizes the present conditions for the management of lagoons, thus analysing and examining the activities that might be developed in different scenarios, scenarios which facilitate ecosystem protection without compromising future generations.

Assessment of marine ecosystem services indicators: Experiences and lessons learned from 14 European case studies.

This article shares the experiences, observations, and discussions that occurred during the completing of an ecosystem services (ES) indicator framework to be used at European Union (EU) and Member States' level. The experience base was drawn from 3 European research projects and 14 associated case study sites that include 13 transitional-water bodies (specifically 8 coastal lagoons, 4 riverine estuaries, and 1 fjord) and 1 coastal-water ecosystem. The ES pertinent to each case study site were identified along with indicators of these ES and data sources that could be used for mapping. During the process, several questions and uncertainties arose, followed by discussion, leading to these main lessons learned: 1) ES identification: Some ES that do not seem important at the European scale emerge as relevant at regional or local scales; 2) ES indicators: When direct indicators are not available, proxies for indicators (indirect indicators) might be used, including combined data on monitoring requirements imposed by EU legislation and international agreements; 3) ES mapping: Boundaries and appropriate data spatial resolution must be established because ES can be mapped at different temporal and spatial scales. We also acknowledge that mapping and assessment of ES supports the dialogue between human well-being and ecological status. From an evidence-based marine planning-process point of view, mapping and assessment of marine ES are of paramount importance to sustainable use of marine natural capital and to halt the loss of marine biodiversity. Integr Environ Assess Manag 2016;12:726-734. © 2016 SETAC.

Establishing the ecological quality status of soft-bottom mining-impacted coastal water bodies in the scope of the Water Framework Directive.

The aim of the present study was to check the usefulness of the benthic biotic indices proposed for application in the European water framework directive (WFD 2000/60/EC) for the ecological quality classification of coastal water bodies, together with some other benthic methodologies used in different countries of the world. The different approaches were applied in two marine ecosystems affected by the same heavy metal contamination source, coastal waters off Portman and the Mar Menor coastal lagoon, both in SE Spain. Two marine biotic indices proposed for application in the Directive (AMBI and BENTIX) were used, together with community descriptors (abundance, Shannon-Wiener diversity, Margalef's species richness, Pielou's evenness and Simpson's Dominance), the relative benthic index (RBI) and the abundance-biomass comparison method (ABC). Water-sediment interface toxicity bioassays using sea urchin embryos and sediment metal analysis served to check the classifications obtained. The classical community descriptors pointed to a progressive variation in benthic communities along the metal contamination gradient of Portman, but, they did not correctly characterize the environmental status of the lagoon stations. Although the RBI was the index that best classified the sites according to their degree of pollution, the selection of indicator species can bias the results. Since the AMBI, the BENTIX and the ABC method are based on the pollution resulting from organic enrichment, their application in the case of purely toxic pollution may not be successful, as was found to be the case in these two mining-polluted ecosystems. Therefore, the development of new indicator lists according to the type of pollutant may serve to improve the results obtained with organic enrichment-based indices when studying other kinds of disturbance. Finally, we found the toxicity tests to be useful tools for the environmental assessment of aquatic ecosystems, and recommend their inclusion in the WFD.