Macroinvertebrate Communities Research Articles

Assessing benthic macroinvertebrate communities’ spatial heterogeneity in Mediterranean transitional waters through eDNA metabarcoding

Transitional waters are important habitats both for biodiversity and ecological functions, providing valuable natural resources and relevant ecosystem services. However, they are highly susceptible to climate changes and anthropogenic pressures responsible for biodiversity losses and require specific biomonitoring programs. Benthic macroinvertebrates are suitable as ecological indicators of transitional waters, being affected by biological, chemical, and physical conditions of the ecosystems about their life cycles and space-use behaviour. The advent of high-throughput sequencing technologies has allowed biodiversity investigations, at the molecular level, in multiple ecosystems and for different ecological guilds. Benthic macroinvertebrate communities’ composition has been investigated, at the molecular level, mainly through DNA extracted from sediments in marine and riverine ecosystems. In this work, benthic macroinvertebrate communities are explored through eDNA metabarcoding from water samples in a Mediterranean transitional water ecosystem. This research highlighted the validity of eDNA metabarcoding as an efficient tool for the assessment of benthic macroinvertebrate community structure in transitional waters, unveiling the spatial heterogeneity of benthic macroinvertebrate communities correlated to the measured environmental gradients. The results suggest that peculiar features of transitional water ecosystems, such as shallow waters and limited currents, facilitate the assessment of benthic macroinvertebrate communities through environmental DNA analysis from surface water samples, opening for more rapid and accurate monitoring programs for these valuable ecosystems.

Context specific effects of substrate composition on the taxonomic and functional diversity of macroinvertebrate communities in temperate lowland streams.

Substrate composition has been widely recognised as a primary variable shaping lotic macroinvertebrate communities at the habitat unit level. However, fundamental understanding of how communities inhabiting mineralogical habitats (i.e., gravel, sand and silt) are structured across differing rivers is lacking. Moreover, research largely focusses on gravel beds and fine sediment in general (<2 mm) and as a result detailed field observations specifically of the sand and silt fractions are lacking. Using data from five UK streams collated from published studies, we assess taxonomic and functional biodiversity (alpha and beta diversity) at the habitat unit level (as defined by substrate composition of sand, silt and gravel). We found that the composition of taxonomic communities were clearly different in all habitat units for each individual stream (and at the landscape scale), with comparable, but less strong, distinctions between substrates for functional macroinvertebrate community composition. However, alpha diversity metrics and Local Contribution to Beta Diversity (LCBD) recorded among the different habitat units varied significantly across individual rivers, and the amount of variation explained by the habitat unit for taxonomic and functional composition demonstrated considerable differences suggesting strong context dependence. The depositional fine sediment habitats of sand and silt were found to support a discrete community composition and differing levels of alpha and beta diversity within and between rivers. We advocate that care should be taken when seeking to generalise biodiversity patterns at a landscape scale as our study highlights the high degree of context dependency when considering the role of the habitat template. Moreover, our results provide evidence that discriminating between the size fractions of fine sediment habitats (sand or silt) is important to fully elucidate the wider ecological importance of these habitats and the distinct taxonomic and functional biodiversity they support.

Benthic macroinvertebrate communities change along a karstic barrage-lake system

Barrage-lake systems are a rare natural phenomenon with specific ecological characteristics that are greatly affected by tufa formation. Our main goal was to investigate benthic macroinvertebrate communities and their longitudinal variations within the lakes arranged in cascades in the Dinaric karst hydrosystem. Each lake within the Plitvice Lakes system studied presented with a distinct community, showing the effects of the barrage-lake system on the macroinvertebrates in these lakes. We observed a downstream decrease in taxa richness, abundance and Shannon diversity index along the longitudinal profile of the studied barrage-lake system. This pattern is most probably a result of decreased organic matter and increased tufa deposition. Overall, our results suggest that the hydrosystem studied represents a sequence of sediment sinks in which lakes retain and accumulate organic matter and fauna. Therefore, each lake acts as a filtering screen and consequently appears to be a small ecosystem. Each lake has its own community characteristics, regardless of the fact that water flows from one lake to the next, connecting them and creating very similar abiotic conditions in each lake along the system.

Effects of water nutrient concentrations on stream macroinvertebrate community stoichiometry: a large-scale study

Effects of water nutrient concentrations on stream macroinvertebrate community stoichiometry: a large-scale study

Macroinvertebrate community development and resilience to channel dredging following flow restoration using effluent in an urban desert river

Macroinvertebrate community development and resilience to channel dredging following flow restoration using effluent in an urban desert river

Polychlorinated biphenyls contamination in Lagos lagoon and impacts on the benthic macroinvertebrates community structure

Polychlorinated biphenyls contamination in Lagos lagoon and impacts on the benthic macroinvertebrates community structure

Impact of wastewater treatment and drought in an Alpine region: a multidisciplinary case study

In the context of global climate change, drought occurrence in streams of alpine origin is a recent phenomenon, whose impact is still poorly investigated. In this study, we adopted a three-disciplinary approach to investigate the response of an Alpine river (NW Italy) to severe drought conditions occurred in the year 2022. We hypothesised that the considerable loss in the water flow could exacerbate wastewater treatment plant (WWTP) discharge effects, lowering dilution capacity of the stream system and then increasing chemical/microbial pollution and altering benthic community characteristics. To assess river response to drought conditions of the considered year, the concentration of the main chemical variables, faecal indicator bacteria, pathogen presence and structural/functional organisation of benthic macroinvertebrates and diatom communities were measured monthly in the reaches located upstream and downstream of a WWTP (January–December 2022). Main environmental variables, such as flow velocity, water depth, and flow regime, were also considered. A multivariate analysis approach was then applied to emphasise correlations between selected variables and flow regime.Comparing upstream and downstream sections over the considered year, a common behaviour of chemical/microbiological parameters was observed, with generally higher concentrations of nutrients and bacterial indicators downstream of the local WWTP. Moreover, a positive correlation between water scarcity and nutrients/bacterial concentrations was revealed. The macroinvertebrate communities responded accordingly, both in terms of density and biological metric shift.Interestingly, differences between the two sections were strictly associated with hydrological conditions, with higher dissimilarities found in low-flow conditions. As the magnitude and duration of drought events are projected to increase in the years to come in different parts of Europe, this work can serve as a first building block and as a hint for future studies aimed at improving our knowledge about the consequences of these events that is pivotal for planning effective management strategies.

Insights into trophic interactions: DNA metabarcoding reveals species composition in black-tailed gull fecal samples in coastal environments of South Korea.

This study explored the algae, zooplankton, macroinvertebrate, fish, and parasitic single-celled organism communities in Larus crassirostris (black-tailed gull) fecal samples from Baengnyeongdo, Hongdo, and Ulleungdo in South Korea. The fecal samples can identify key species consumed by black-tailed gull, providing insights into their, trophic interactions, and habitat dependencies. Using DNA metabarcoding, we identified algae, zooplankton, macroinvertebrate, fish, and intestinal and single-celled parasite species in the fecal samples. Parasitic single-celled organisms, such as Rhogostoma sp., Rhogostoma schuessleri, Eimeria furonis, and Aggregata eberthi, showed differing relative abundances at each sampling location, indicating variability in parasite diversity in the fecal DNA analysis of birds at each site. Intestinal parasites showed similar site-specific variability, though Clistobothrium sp. and Tetrabothrius sp. were common at all locations. Algae species, including Heterocapsa steinii, Heterocapsa niei, and Sargassum cristaefolium, also displayed habitat-specific patterns, as did zooplankton, with Calanus sp., Corycaeus speciosus, and Caprella californica being dominant on Baengnyeongdo, Hongdo, and Ulleungdo, respectively. In the macroinvertebrate communities, Octopus sinensis was prevalent at all locations but at varying abundances. Site-specific dominant fish species were also identified, with Ammodytes personatus, Decapterus maruadsi, and Arctoscopus japonicus highly predominant on Baengnyeongdo, Hongdo, and Ulleungdo, respectively. Other fish species, such as Ammodytes hexapterus, were detected in lower frequencies, suggesting a diverse diet for the seabirds. These results offer insights into the species composition and ecological dynamics in black-tailed gull populations across disparate Korean islands.

An assessment of maintenance works and their impact on macroinvertebrate communities and long-term recolonization to small lowland watercourses

Due to their small size and high anthropogenic pressure, small watercourses are particularly prone to severe siltation and are densely overgrown with macrophytes. Many of these watercourses are subject to regular maintenance works (RMW), consisting of seasonal desilting and vegetation clearance, in order to ensure unobstructed water flow. The aim of the study was to assess the impact of three types of maintenance works: dredging and mud removal (DMR), river channel vegetation removal (RCVR) and river bank vegetation removal (RBVR) on taxa species richness, macroinvertebrate density and the Shannon-Wiener diversity index, as well as their changes and long-term benthic recolonization one and two years after completion of the works. The study was carried out in 21 habitats on eight rivers in the European Central Plains Ecoregion. A total of 107 zoobenthic taxa were found at all sites, and their species composition was characteristic of highly hydrophytic waters with low hydrological and hydrochemical quality parameters. A significant decrease in macroinvertebrate taxa richness was observed one year after the works, as the average number of taxa had dropped from thirteen to eight, with a further fall to seven taxa two years after the RMW. The same was true for density, which had decreased from an average of 2496 to 786 individuals per square meter one year after the RMW, while, a gradual recolonization was recorded two years after the RMW, with an average density of 1295 individuals per square meter. The Shannon-Wiener index, which had averaged 2.528 before the RMW, also decreased, falling to 1.982 and 1.832 one and two years after. BACI statistical analyses showed that of the three types of maintenance work, desilting and bottom sediment removal had the largest negative impact, significantly reducing taxonomic composition (by an average of 53%), density (by an average of 43%), and ecological index values (by an average of 40%). Over-frequent maintenance can prevent macroinvertebrate populations from recovering, thus depleting the environment of valuable taxa, including those that provide food for fish and other vertebrates.

Principal determinants of aquatic macroinvertebrate communities in small shallow lakes and ponds

Principal determinants of aquatic macroinvertebrate communities in small shallow lakes and ponds

Insect Herbivores, Plant Sex, and Elevated Nitrogen Influence Willow Litter Decomposition and Detritivore Colonization in Early Successional Streams

Headwater streams are reliant on riparian tree leaf litterfall to fuel brown food webs. Terrestrial agents like herbivores and contaminants can alter plant growth, litter production, litter quality, and the timing of litterfall into streams, influencing aspects of the brown food web. At Mount St. Helens (USA), early successional streams are developing willow (Salix sitchensis) riparian zones. The willows are attacked by stem-boring herbivores, altering litter quality and the timing of litterfall. Within a established experimental plots, willows (male and female plants) were protected from herbivores using insecticides and provided with experimental additions of nitrogen. This enabled us to test the interacting influences of herbivores, nitrogen deposition, and willow sex on leaf litter quality, aquatic litter decomposition, and microbial and invertebrate detritivores. We found weak litter quality effects (higher N and lower C:N) for the herbivore treatment, but no effect of nitrogen deposition. Although litter decomposition rates were not strongly affected by litter treatments, detritivore communities were altered by all treatments. Nitrogen deposition resulted in decreased bacterial richness and decreased fungal diversity in-stream. Aquatic macroinvertebrate communities were influenced by the interacting effects of herbivory and nitrogen addition, with abundances highest in herbivore litter with the greatest N addition. Shredders showed the highest abundance in male, herbivore-attacked litter. The establishment of riparian willows along early successional streams and their interacting effects with herbivores and nitrogen deposition may be influencing detritivore community assembly at Mount St. Helens. More broadly, global changes like increased wet and dry N deposition and expanded ranges of key herbivores might influence tree litter decomposition in many ecosystems.

Long-Term Monitoring of Macroinvertebrate Community Assemblages and Species Composition on the Coast of Dokdo, East Sea of Korea

Dokdo Island’s diverse marine ecosystem requires long-term monitoring to understand the effects of rapid environmental changes, such as global warming, on macrobenthos species and communities. Current studies are often short-term and limited, highlighting the need for extended research to predict future changes to ecosystems. This study analyzed the environmental variables influencing macrobenthos through long-term monitoring. In total, 511 species (spp./23.4 m2) were identified with an abundance of 1709.9 individuals/m2 and a diversity of 2.9. Since 2020, there has been a decline in both the number of species and diversity, attributed to changes in sediment composition, particularly an increase in gravel and sand. The dominant species include the crustacean Abludomelita denticulata (17.6%), the polychaete Haplosyllis spongiphila (6.8%), the bivalve Glycymeris munda (5.6%), the polychaete Opisthodonta uraga (5.3%), and the bivalve Limatula japonica (3.8%). The macrobenthos community differs each year, as the dominant species G. munda has decreased in abundance since 2022 and L. japonica since 2021. Depth and gravel sediment strongly correlated with community variation. Site-specific analysis also showed significant differences, with depth, bottom temperature, and sediment composition as the influencing factors. These species thrive in cold water but their abundance declines with an increase in temperature. Continuous long-term monitoring is essential to understand and protect the Dokdo ecosystem amid rapid environmental changes. Long-term monitoring studies have revealed more species than have short-term studies, showing annual and site-specific environmental changes, including sediment erosion and bottom temperature increases. These changes have affected macrobenthos diversity, abundance, and community composition, necessitating ongoing research to determine the persistence of these trends and to protect the ecosystem.

Evaluating the response of current biotic indices and functional metrics to natural and anthropogenic predictors in disconnected pools of temporary rivers

Temporary rivers, forming the majority of river networks worldwide, are key biodiversity hotspots. Despite their great value for maintaining biodiversity and ecosystem functioning, they are often neglected in biomonitoring programs due to several challenges, such as their variable hydromorphology and the difficulty of establishing reference conditions given their dynamic nature, resulting in highly variable communities. Disconnected pools often form in temporary rivers when flow ceases, providing refuge for aquatic taxa. Given their importance for biodiversity conservation, revising and adapting biotic indices are needed. Here, we evaluate the performance of current biological indices designed for perennial rivers (macroinvertebrates, diatoms) and functional metrics (macroinvertebrates) in assessing biological quality of disconnected pools. We sampled 55 disconnected pools in Catalonia, NE Spain, covering local (e.g., physico-chemical variables, water chemistry) and regional (e.g., human influence, hydrological variables at the water body level) natural and anthropogenic gradients. Only a few macroinvertebrate biotic indices (e.g., family richness, EPT/EPT + OCH and OCH) showed strong responses to anthropogenic predictors and were unaffected by natural predictors at both local and regional scales, making them suitable for biomonitoring. Of the newly adopted functional metrics of macroinvertebrate communities tested, only two (i.e., functional redundancy of predators and response diversity based on the total community) responded strongly to anthropogenic predictors. The rest showed varying responses to the interactive effect of anthropogenic and natural predictors, requiring calibration efforts. Models assessing these metrics explained <40 % of the total variation, likely due to the interplay of colonization/extinction dynamics and density-dependent trophic interactions governing community assemblages in disconnected pools. Although some existing biological metrics could potentially be used to monitor the ecological status of disconnected pools, we call for further development of biomonitoring tools specifically designed for these habitats since they will become more widespread with global change.

Intraspecific variation in crayfish behavioral traits affects leaf litter breakdown in streams.

Although intraspecific trait variation is increasingly recognized as affecting ecosystem processes, few studies have examined the ecological significance of among-population variation in behavioral traits in natural ecosystems. In freshwater habitats, crayfish are consumers that can influence ecosystem structure (e.g., macroinvertebrate communities) and function (e.g., leaf litter breakdown). To test whether crayfish behavioral traits (activity, boldness, and foraging voracity) are major contributors of leaf litter breakdown rates in the field, we collected rusty crayfish (Faxonius rusticus) from eight streams across the midwestern USA and measured behaviors using laboratory assays. At the same streams, we measured breakdown rates of leaf packs that were accessible or inaccessible to crayfish. Our results provide evidence that among-population variation in crayfish boldness and foraging voracity was a strong predictor of leaf litter breakdown rates, even after accounting for commonly appreciated environmental drivers (water temperature and human land use). Our results suggest that less bold rusty populations (i.e., emerged from shelter more slowly) had greater direct impacts on leaf litter breakdown than bold populations (P = 0.001, r2 = 0.85), potentially because leaf packs can be both a shelter and food resource to crayfish. Additionally, we found that foraging voracity was negatively related to breakdown rates in leaf packs that were inaccessible to crayfish (P = 0.025, r2 = 0.60), potentially due to a trophic cascade from crayfish preying on other invertebrates that consume leaf litter. Overall, our results add to the growing evidence that trait variation in animals may be important for understanding freshwater ecosystem functioning.

Exploring freshwater stream bacterial communities as indicators of land use intensity

BackgroundStream ecosystems comprise complex interactions among biological communities and their physicochemical surroundings, contributing to their overall ecological health. Despite this, many monitoring programs ignore changes in the bacterial communities that are the base of food webs in streams, often focusing on stream physicochemical assessments or macroinvertebrate community diversity instead. We used 16S rRNA gene sequencing to assess bacterial community compositions within 600 New Zealand stream biofilm samples from 204 sites within a 6-week period (February–March 2010). Sites were either dominated by indigenous forests, exotic plantation forests, horticulture, or pastoral grasslands in the upstream catchment. We sought to predict each site’s catchment land use and environmental conditions based on the composition of the stream bacterial communities.ResultsRandom forest modelling allowed us to use bacterial community composition to predict upstream catchment land use with 65% accuracy; urban sites were correctly assigned 90% of the time. Despite the variation inherent when sampling across a ~ 1000-km distance, bacterial community data could correctly differentiate undisturbed sites, grouped by their dominant environmental properties, with 75% accuracy. The positive correlations between actual values and those predicted by the models built using the stream biofilm bacterial data ranged from weak (average log N concentration in the stream water, R2 = 0.02) to strong (annual mean air temperature, R2 = 0.69).ConclusionsFreshwater bacterial community data provide useful insights into land use impacts on stream ecosystems; they may be used as an additional measure to screen stream catchment attributes.

Size distribution of macroinvertebrate communities associated with live and dead coral

Size distribution of macroinvertebrate communities associated with live and dead coral

Macroinvertebrate community dynamics following wetland restoration: Insights from taxonomic structure and functional feeding groups in response to water quality

Continuous wetland restoration initiatives in China are increasing, due to the global degradation of wetland ecosystems. However, monitoring of the restoration situation remains incomplete. In this study, we investigated the effects of wetland restoration on the macroinvertebrate taxonomic structure and feeding functional groups (FFGs) in the Naolihe National Nature Reserve (NNNR). Macroinvertebrate taxonomic diversity can be used to monitor wetlands, and we hypothesized that FFGs serve the same function. We calculated the diversity index, performed a non-metric multidimensional analysis based on macroinvertebrate taxonomics and FFGs, and subsequently, performed a t-test on the results. The results showed that macroinvertebrate diversity and FFGs analyses were in general agreement with taxonomic diversity, indicating that the macroinvertebrate community in the wetland with five years of fallow land was resembled that of the natural wetland. In contrast, the macroinvertebrate community in the wetland with two years of fallow differed significantly from that in the natural wetland. Additionally, the results of the ecosystem attributes based on biomass and FFGs showed that restored wetlands exhibited lower habitat stability than natural wetlands. Nutrients (NH4+-N, NO3−-N, and total phosphorus) explained the changes in macroinvertebrate FFGs in the restored wetlands to a greater extent than in the natural wetlands. The results of this study highlight the importance of macroinvertebrate FFGs in wetland monitoring, which supports the use of macroinvertebrate FFGs in the NNNR to monitor wetland restoration.

Agricultural-Derived organochlorine pesticide residues impact on macroinvertebrate community in an Afrotropical Stream

This study evaluated the impact of pesticide application through agricultural activities in Chanchaga River, Nigeria, using macroinvertebrate data sets obtained for six months (September 2021–February 2022). Four (4) stations, characterized by various agricultural activities, were sampled along the river. Analysis of the water samples for organochlorine pesticide residues (OCP) using Gas Chromatography-Mass Spectrometry (GC/MS) at the peak of the two seasons revealed a high concentration of eleven isomers of organochlorine, which ranged from 0.01 to 0.81 μg/L, and a mean concentration that was above international drinking water standards set by the World Health Organization, the Federal Environmental Protection Agency, and the European Union. The mean concentration of detected OCP was recorded as DDT (0.72 μg/L), Dieldrin (0.59 μg/L), Paraquat (0.54 μg/L), Aldrin (0.49 μg/L), Metribuzin (0.48 μg/L), Butachlor (0.47 μg/L), Alachlor (0.28 μg/L), Atrazine (0.23 μg/L), Phenol (0.10 μg/L), Endrin (0.09 μg/L), and Benzene (0.08 μg/L). Atrazine, alachlor, metribuzin, aldrin, phenol, and endrin showed significant differences across the two seasons (p < 0.05), while dieldrin, butachlor, paraquat, benzene, and DDT showed no significant differences across the two seasons (p > 0.05). A total of 622 macroinvertebrate individuals from 19 species in 18 families from 8 orders were collected. More individuals were collected during the dry season (58.17 %) and the wet season (41.83 %). Canonical Correspondence Analysis (CCA) ordination revealed a strong relationship between species abundance and some organochlorine pesticide residues such as DDT, endrin, metribuzin, atrazine, benzene, and dieldrin. The response of macroinvertebrates to OCP indicates that Chanchaga River is a disturbed river, and the indicator organisms (Lestes sp., Coenagrion sp., Zyxomma sp., Appasus sp., Chironomus sp., Lymnaea natalensis, and Caridina nililotica) can also be used for further biomonitoring.

Declining glacier cover drives changes in aquatic macroinvertebrate biodiversity in the Cordillera Blanca, Perú.

Ongoing climate change threatens the biodiversity of glacier-fed river ecosystems worldwide through shifts in water availability and timing, temperature, chemistry, and channel stability. However, tropical glacier-fed rivers have received little attention compared to those in temperate and Arctic biomes, despite their unique biodiversity potentially responding differently due to additional stress from higher altitude locations thus lower oxygen availability, diurnal freeze-thaw cycles, and annual monsoon rainfall disturbances. However, tropical glacier-fed rivers have received little attention compared to those in temperate and Arctic biomes, despite their unique biodiversity potentially responding differently due to additional stress from higher altitude locations thus lower oxygen availability, diurnal freeze-thaw cycles, and annual monsoon rainfall disturbances. This study quantified aquatic biodiversity responses to decreasing glacier cover in the Cordillera Blanca range of the Peruvian Andes. Ten rivers were studied along a gradient of decreasing glacier cover in the Parón, Huaytapallana, and Llanganuco basins, with a specific focus on macroinvertebrates and physicochemical parameters in both the dry and wet seasons. We found higher temperatures, more stable and lower turbidity rivers as glacier cover decreased, which were related significantly to higher local diversity and lower β-diversity. Analysis of similarity revealed significant differences in the macroinvertebrate community among rivers with high, medium, or low glacier cover, illustrating turnover from specialists to generalists as glacial influence decreased. Redundancy analysis demonstrated that there were more species found to prefer stable beds and water temperatures in medium and low glacier cover in a catchment rivers. However, certain taxa in groups such as Paraheptagyia, Orthocladiinae, Anomalocosmoecus, and Limonia may be adapted to high glacial influence habitats and at risk of glacier retreat. Although species composition was different to other biomes, the Cordillera Blanca rivers showed similar benthic macroinvertebrate biodiversity responses to glacier retreat, supporting the hypothesis that climate change will have predictable effects on aquatic biodiversity in mountain ranges worldwide.

Macroinvertebrate Community as Bioindicator of Water Quality of Tambis River, Palompon, Leyte, Philippines

Macroinvertebrates have been used globally as bioindicators of water quality of rivers due to their varying tolerance to pollution. The water quality of Tambis River in Palompon, Leyte, Philippines was determined using macroinvertebrates and selected physico-chemical and ecological assessments. Employing kick-sampling method, three sampling stations were established based on the surrounding land use (forested, agricultural, and near residential). Twenty-seven macroinvertebrate families were identified. Permutational multivariate analysis of variance (PERMANOVA) revealed a significant difference in the benthic macroinvertebrate families across three stations (p=0.001). There was a decrease in the abundance of pollution-sensitive families from the forested station towards the downstream, closest to the residential area. Sensitive Ephemerellidae and tolerant Thiaridae were the most abundant in the forested and downstream area respectively. The highest diversity and evenness were observed from the agricultural area. Stream Invertebrate Grade Number - Average Level (SIGNAL 2) scores were plotted declined in all quadrants, suggesting good water quality to moderately polluted. Water temperature, pH, and velocity were the most influencing environmental variables to the macroinvertebrate assemblage as revealed by the Canonical Correspondence Analysis (CCA). Overall, the water quality of Tambis River ranged from being of good quality to moderately polluted. The data obtained in this study could serve as a baseline in providing information to formulate and implement scientific-based regulatory measures towards the conservation of Tambis River.