Freshwater ecosystems, though covering less than 1% of Earth's surface, harbor around 10% of all species. Pressures like pollution, water extraction, and habitat loss can exhibit complex interactions in their effects on freshwater biodiversity. Understanding the effects of these pressures on ecosystem services and functions of freshwater systems is crucial for effective environmental policies, and asks for availability of species data. We therefore compiled European monitoring data on macroinvertebrates from European, national, and regional biomonitoring and analysed their representativeness with respect to river size, land use as well as their suitability to derive biodiversity trends. The dataset consists of over 2.3 million macroinvertebrate abundance records from 2010 to 2015, spanning 28 European countries. Geographically, data are concentrated in central Europe and England and are not evenly representative across space or river types. They vary regarding reporting units, general sampling methods, and taxonomic levels (mainly species level). These differences hamper European-scale biodiversity comparisons. The WFD primarily focuses on assessing the "ecological status" of water bodies, which can be achieved using a menu of methods and many biodiversity research questions require improvements in harmonization of taxonomic resolutions and in method standardization to enable accurate transnational comparisons. This study emphasizes the need for improved European coordination and support in collecting, validating, exchanging, and sharing freshwater biodiversity data to enable meaningful comparisons among countries. It also delivers an open-access database that enables analyses of trends and ecological scenario development, offering further opportunities to enhance freshwater biodiversity conservation and management.

The International Barcode of Life (iBOL) initiative is building a globally accessible DNA-based system for species identification and discovery. This paper outlines the mission and strategic priorities for the iBOL community in Europe (iBOL Europe), set in a global context. The mission of iBOL Europe is to produce, curate, and provide access to a complete DNA barcode reference library of European eukaryotic biodiversity, catalyzing species discovery and enabling comprehensive, harmonized species identification and biomonitoring, and supporting the global iBOL program. Immediate objectives include completing reference libraries for priority taxa, democratizing access to sequencing technologies, and strengthening a distributed community of practice. Key actions identified span five thematic areas: community building, sample collection and taxonomic verification, sequencing infrastructure, data management, and mainstreaming DNA-based approaches to meet societal needs. The strategy emphasizes integration with European research infrastructures to ensure long-term sustainability and resilience for biodiversity genomics in Europe.

River networks are highly dynamic environments, where local conditions range from lotic to lentic, promoting the co-occurrence of highly diverse biota. These environments are threatened by various human-induced stressors, among which water scarcity affects more than half of all running waters globally. While flow intermittence occurs naturally, its spatial and temporal extension is spreading under climate change and human pressures, endangering river biota. Here, we performed a mesocosm study aiming to investigate how flow reduction during drying events affects macroinvertebrate communities from different mesoscale habitats, such as riffles and pools. The experiment was performed in a replicated flow-through mesocosm system during summer 2021. We tested the effects of both intermittent and prolonged three-month-long flow reduction on macroinvertebrate communities from riffle and pool mesohabitats in terms of community composition, and resilience and resistance functional traits (e.g., resistance form, current preference, locomotion, dispersal strategy, reproduction drift propensity, etc.). Sampling was performed before, during, and after the exposure to flow reduction to assess both the direct effect of water scarcity and the post-drought recovery of macroinvertebrate communities. We found that communities from riffle habitats were more severely affected by flow reduction during drying events, showing a more severe decline in taxonomic richness and reduced abundance of desiccation-sensitive organisms under prolonged flow reduction treatments compared to intermittent ones. During flow reduction events, we did not observe a consistent taxa turnover toward drought-tolerant taxa, with only a few resistance trait modalities (e.g., organisms with tolerance for higher water temperature or interstitial ones) significantly associated with prolonged flow reduction. Moreover, the communities from riffle mesohabitats did not fully recover even one month after normal flow conditions were re-established, showing a low post-drought resilience. In pool mesohabitats, we did not detect significant effects of intermittent or prolonged flow reduction, with a community composition dominated by generalist taxa. These findings highlight the importance of accounting for mesohabitat-specific responses to drying when evaluating the ecological consequences of increasing flow intermittence and suggest that habitat heterogeneity plays a critical role in shaping the resistance and resilience of macroinvertebrate communities under water scarcity.

Understanding the relative contributions of environmental, spatial, and temporal processes in shaping species distribution is a central objective in ecology. Bayesian species distribution models (SDMs) offer a flexible framework for this task, yet prior specification for variance components remains challenging. To address this issue, we adapt the Hierarchical Decomposition (HD) prior framework to latent Gaussian SDMs, enabling direct and transparent prior control over variance partitioning. The HD approach reparametrizes variances into a total variance and a set of interpretable proportions, structured through a decomposition tree that reflects both model architecture and ecologically meaningful groupings of effects. We discuss a principled approach for a default tree design tailored to SDMs and a practical workflow for the step-by-step implementation of the method. The framework is illustrated using presence–absence data for 39 demersal fish species from the NOAA Northeast Fisheries Science Center fall bottom trawl survey. Results demonstrate predictive performance comparable to established priors, while providing substantially improved interpretability and transparency in variance attribution and prior sensitivity analysis.

There is an urgent need for planning actions to mitigate biodiversity loss worldwide, which involves developing assessment methods to help decision-makers identifying areas most at risk and prioritizing action. This requires robust data and analyses but it also implies thinking about realistic and cost-effective measures. Fresh waters host an important part of global biodiversity but freshwater organisms are expected to be profoundly impacted by the predicted increase in water temperatures and discharge alterations associated with climate change. However, available models focus mostly on changes in air temperature, potentially failing to incorporate these impacts. Given that freshwater biodiversity is declining at an alarming and exponentially increasing rate, there is an urgent need to monitor the potential effects of climate change. Here, we modeled the distribution of freshwater macroinvertebrates across Europe for present and future conditions including recently available data on water temperature and discharge. We also included other environmental variables that might be relevant in understanding the current spatial distribution of invertebrates (e.g. geology, adjacent land use). We used 40 datasets of standardized monitoring protocols of freshwater invertebrates spanning 23 years. Then a score of the vulnerability to climate change was attributed to each taxon based on the models. Finally, the average community indicator calculated for all European rivers allowed us to identify relevant regions for monitoring climate change using a planning conservation tool.

Alpine streams are highly vulnerable to climate change and are experiencing alterations in average flow, hydrological regimes, and nutrient supply, with multiple implications for freshwater biota. This study examined aquatic macroinvertebrates from three key orders: Ephemeroptera, Plecoptera, and Trichoptera (EPT), which are typical of alpine streams and sensitive to anthropogenic pressures and environmental change. The research investigated richness, composition, and distribution of EPT taxa across glacier-fed stream classes, alongside identifying richness surrogates and indicator taxa. From 2010 to 2022, 102 sites in the Western Italian Alps were sampled and categorised into four classes based on their catchment glacier cover (CGC). Communities belonging to streams most influenced by glacier cover were distinctly well-clustered and associated with higher altitudes and lower temperatures. Results showed increasing Ephemeroptera abundance and decreasing Plecoptera richness as glacial influence declined, while EPT family richness displayed no significant variation among glacial classes. Dictyogenus Klaplek, 1904 (Plecoptera) was associated with streams with higher CGC, whereas the roles of Ephemeroptera and Trichoptera emerged in the other three classes. These findings provide a valuable starting point for better understanding EPT communities as sentinels of global change in alpine stream ecosystems.

Dung beetles (Coleoptera, Scarabaeidae) contribute to soil biogeochemical cycling via dung burial and soil mixing, yet little is known about their impact on soil biogeochemistry at the microscale. We employed planar optode imaging to simultaneously resolve oxygen and pH gradients in soil bioturbated by the tunneling species Onthophagus nuchicornis (Linnaeus, 1758). Using a "soil sandwich" setup, we monitored spatial and temporal changes in the soil microenvironments across a vertical plane over 96 h. Beetles generated a heterogeneous network of tunnels and dung balls, leading to steep oxygen and pH gradients and an 8-fold increase in the 2D oxic-anoxic interface zones. Buried dung balls exhibited persistent anoxia, confirmed via microsensor profiling, with more than 75 % of the volume remaining anoxic for over 45 h. Oxygen depletion was coupled to a rise in pH extending millimeters beyond the anoxic zone. Using fluorescent dye-labeled nanoparticles we were also able to track dung movement under waterlogged conditions, demonstrating continued, albeit reduced, beetle activity under anoxia. The combined effects on oxygen, pH, and the organic matter redistribution enhance microbial habitat heterogeneity and are expected to favor the coupling between aerobic and anaerobic processes, such as nitrification and denitrification. Finally, we obtained quantitative estimates of soil displacement and dung removal, providing direct metrics of the ecosystem services delivered by dung beetles, including enhanced soil porosity and organic matter burial. The novel methodological approach described here provides mechanistic insights into the microscale processes underlying dung beetle-mediated soil modification, sustaining their role as soil ecosystem engineers.

Despite the global ecological and societal importance of deep lakes and their associated biota and ecosystem services, the relationships between water and sediment features and the spatial patterns of macrophyte assemblages remain poorly understood in these ecosystems, especially below 4–5 m depth. We aimed to fill this gap by providing new evidence of macrophyte community assembly rules over a wide range of colonized depths (up to 20 m). The macrophyte communities of five deep volcanic lakes in Central Italy, covering a wide range of dimensions (from 1.7 to 114.5 km2), maximum depths (from 33 to 165 m), and trophic status [12.4–41.3 μg of total phosphorus (TP) L-1], were explored. We applied linear mixed effect models and multivariate Multiscale Codependence Analysis (mMCA) to investigate macrophyte depth patterns and environmental drivers at nested spatial scales ranging from micro (at the scale of single vegetation belt) to large (whole lake study site) scales. A weak or absent macrophyte spatial structure was reported for the most impacted lakes (Vico and Nemi lakes), as well as for the most pristine lakes (Bracciano and Bolsena lakes). A well-defined structure was observed exclusively in Martignano Lake, an intermediate site both in terms of trophic status (17.1 μg TP L-1) and area (2.02 km2). Overall, distinctive macrophyte patterns were found at the largest lake scale, reflecting a clear distinction between shallow (up to 3 m) and deep vegetated bands (>3 m), dominated by vascular plants and large charophytes, respectively. Conversely, no strong spatial structure was detected at the microscale (i.e., with metric resolution, comparing the different study plots with each other). The low species diversity and the constant presence of only one dominant species per vegetated band can explain this result. Beyond light availability, sediment features (TP and organic matter content) emerged as significant in determining the arrangement of macrophytes in relation to depth, offering a more informed view of macrophyte spatial processes and their functional implications in deep lakes.

Intermittent rivers represent more than half of the length of the global river network, and their extent is predicted to increase. Although flow intermittence is a natural phenomenon, it is exacerbated by the combined and interacting effects of climate change and anthropogenic pressures. This increasing intermittence represents a major threat to freshwater biodiversity and highlights the pressing need for indices that can assess the responses of freshwater biota to drying events. However, most such indices remain untested beyond their region of origin, limiting their widespread use. We addressed this gap by testing the capacity of two macroinvertebrate-based indices that respond to flow intermittence (the drought effect of habitat loss on invertebrates [DEHLI] index and the monitoring intermittent streams index [MIS-index]), developed and evaluated in United Kingdom, to distinguish between perennial and intermittent river sites in Italy. Scores for both indices and index-derived richness metrics (i.e., the taxa richness of groups of environmental association classified by the indices) were higher at perennial than at intermittent sites. Among index-derived richness metrics, only those including rheophilic taxa were lower at intermittent sites, whereas others were comparable at perennial and intermittent sites. Our results suggest that both indices have the potential for use beyond their original region of development, with a better performance of the DEHLI index due to its family-level taxonomic resolution. However, their responsiveness could be enhanced by regional adaptation to increase the pool of scoring taxa. In addition, the development of conceptual frameworks predicting assemblage composition in intermittent rivers across different biogeographical regions could guide the future development of new indices for flow intermittence.

Rice fields are one of the most important and extensive agro-ecosystems in the world. Italy is a major non-Asian rice producer, with a significant proportion of its yield originating from a vast area within the Po Valley, a region nourished by the waters of the Alps. While the biodiversity of these rice fields has been extensively documented for certain faunal groups, such as birds, there remains a paucity of research on the biodiversity of aquatic insects. A further challenge is the limited dissemination of findings, which have been primarily published in “gray” literature (local journals, newsletters and similar). Moreover, rice fields are of particular significance in the field of invasion biology, given their role in the arrival and spread of alien species. While the efficacy of rice fields as a substitute for the now-disappeared lowland natural environments is well documented, it is equally evident that traditional rice-growing techniques can require an unsustainable use of water resources, which threatens the biodiversity of the surrounding lotic systems. Here, we summarize and review multiple sources of entomological information from Italian rice fields, analyzing both publications in ISI journals and papers published in local journals (gray literature). In the near future, strategies that reduce the demand for irrigation, promote the cultivation of drought-tolerant crops, and utilize precision farming techniques will be implemented. The challenge will be balancing the need to reduce water withdrawal from rivers with the maintenance of wetlands where possible to support this pivotal component of regional biodiversity.

Drying river networks include non-perennial reaches that cease to flow or dry, and drying is becoming more prevalent with ongoing climate change. Biodiversity responses to drying have been explored mostly at local scales in a few regions, such as Europe and North America, limiting our ability to predict future global scenarios of freshwater biodiversity. Locally, drying acts as a strong environmental filter that selects for species with adaptations promoting resistance or resilience to desiccation, thus reducing aquatic α-diversity. At the river network scale, drying generates complex mosaics of dry and wet habitats, shaping metacommunities driven by both environmental and dispersal processes. By repeatedly resetting community succession, drying can enhance β-diversity in space and time. To investigate the transferability of these concepts across continents, we compiled and analyzed a unique dataset of 43 aquatic invertebrate metacommunities from drying river networks in Europe and South America. In Europe, α-diversity was consistently lower in non-perennial than perennial reaches, whereas this pattern was not evident in South America. Concomitantly, β-diversity was higher in non-perennial reaches than in perennial ones in Europe but not in South America. In general, β-diversity was predominantly driven by turnover rather than nestedness. Dispersal was the main driver of metacommunity dynamics, challenging prevailing views in river science that environmental filtering is the primary process shaping aquatic metacommunities. Lastly, α-diversity decreased as drying duration increased, but this was not consistent across Europe. Overall, drying had continent-specific effects, suggesting limited transferability of knowledge accumulated from North America and Europe to other biogeographic regions. As climate change intensifies, river drying is increasing, and our results underscore the importance of studying its effects across different regions. The importance of dispersal also suggests that management efforts should seek to enhance connectivity between reaches to effectively monitor, restore and conserve freshwater biodiversity.

Dung beetles (Coleoptera, Scarabaeoidea) support several ecological processes and services making them important ecosystem engineers. The dung beetle gut microbiota is involved in many of these ecological services. In the present study, we analyzed the microbiota of 90 individuals of three Onthophagus species feeding on different dung types. Our aim was to understand whether the species identity affected the microbiota more than the dung ingested and whether this conditioning applied equally to prokaryotes and fungi. We also compared the taxonomic and functional variability of the microorganisms to check for similarities between individuals. Using molecular analyses, we characterized the alpha and beta diversities, core and indicator taxa and taxonomic and functional composition of the gut microbiota. Alpha diversity analyses revealed diet, species and sex to influence diversity parameters but no clear differences in the diversity patterns for prokaryotes vs fungi. Conversely, all other analyses consistently showed differences in the composition patterns for prokaryotes vs fungi, with prokaryotes mostly varying according to host species identity and fungi varying according to dung type. This suggests that most prokaryotes in the dung beetle microbiota are definitive symbionts, whereas many fungi are transient symbionts. We found evidence of great similarity in the functional composition of the microbiota despite strong taxonomic dissimilarities. The results emphasize the need to consider both the prokaryotic and fungal components of the microbiota. They also suggest microbial composition analyses to be preferable to alpha diversity analyses for identifying patterns of variation that depend on phylogeny and diet.

Small streams dominate river networks and collectively support high biodiversity, but are rarely included in regulatory biomonitoring programmes. Macroinvertebrate communities are effective biomonitors of ecological condition and are routinely collected using 3-min 'kick' samples. However, this 3-min duration may not be suitable for small streams, which typically support fewer taxa at lower densities than larger rivers of equivalent condition. We evaluated the kick-sampling method at 30 sites representing a national small stream monitoring network. At each site, we collected three 5-min kick samples in 10 0.5-min component parts. We used the families collected in 15 min to represent 'total' site-scale taxonomic richness, then determined the duration needed to sample >= 65% of these taxa (a method and target comparable to those used in larger rivers). We also determined the sampling duration at which an average score per taxon (ASPT) biomonitoring index stabilized. Considering all streams, on average, 2.5-min durations captured >= 65% of taxa, but 3.5 min was required to reach this target in temporary streams, because numerous taxa occurred at low abundance. Only 54% of samples contained >= 65% of taxa after 2.5 min, compared to 70% after 3 min. In most streams, the ASPT stabilized after 2 min, whereas 3 min was required to meet this target in temporary streams. Considering the variation around any estimate of capture rates introduced by natural variability, taxonomic resolution and operator error, we suggest 3 min as the most robust sampling duration to enable condition monitoring in individual small streams and comparison with larger rivers.

Dung beetles mostly feed on mammal dung. Throughout the European Alps, the dung produced by local domestic ungulates attracts many species of dung beetles, giving rise to rich and diversified communities that play an important role in the Alpine agricultural ecosystem. There is, therefore, understandable concern about the introduction of exotic livestock, such as alpacas (Vicugna pacos (Linnaeus, 1758)), into the region. This research studied dung beetle assemblages in an Alpine valley where both cattle and alpacas are raised. We used standardized pitfall traps baited with alpaca or cow dung along altitudinal transects to assess the “attractiveness” of the two resources to dung beetles. Most species entered both trap types. The average number of species per trap did not vary significantly and the nMDS trap ordination largely overlapped, but the total number of individuals and the average number of individuals per trap were higher in traps baited with alpaca dung. This difference was largely due to the preference of the dominant species Euheptaulacus carinatus (Germar, 1824) for alpaca dung-baited traps. Moreover, both the abundance and specific richness changed with altitude, being greatest in alpaca traps during July at intermediate altitudes. IndVal analyses showed that eight out of nine species (all Aphodiinae) showed a preference for alpaca traps. Since the microclimatic conditions (i.e., temperature) of the two trap types were virtually the same, it is reasonable to consider trophic preferences as the source of the observed differences. Finally, we also sampled the beetles present in the alpaca latrines and cow dung pats deposited on pastures, providing confirmation that dung beetles do indeed visit the dung of both species. Our results emphasize the usefulness of polyphagia and the trophic flexibility of dung beetles, providing evidence that the long history of pastoralism in the Alps has not led local species to adapt to the dung of domestic ungulates. The results also suggest that alpaca breeding could be a sustainable activity since, thanks to the work of dung beetles which feed upon and move the dung into their tunnels (as occurs with cattle dung), there is less need for farmers to remove the dung from pastures.

The study of temperament and behavioral syndromes in insects is still in its early stage, and research conducted to date has mainly focused on locomotor activity and thanatosis. Dung beetles have been the subject of extensive behavioral studies; however, very few studies have addressed the expression of temperament. Those doing so only looked at subsocial and sexual horn dimorphic species, suggesting subsociality and/or sexual horn-dimorphism as possible facilitators of temperament expression. To test this assumption, we conducted a temperament study in a hornless, non-subsocial species, namely Geotrupes mutator (Marsham, 1802). We set up laboratory tests to evaluate 3 behaviors (activity, thanatosis, and distress calls) through the measurement of 7 distinct behavioral traits (3 activity-, 1 thanatosis-, and 3 call-related traits). We found high levels of individual repeatability in all activity- and thanatosis-related traits. We also identified behavioral differences between individuals, which may reflect differences in temperament. Statistical analyses revealed a negative correlation between activity and thanatosis. These results show that the temperament and behavioral syndromes related to activity and thanatosis may also be expressed in dung beetle species that are neither subsocial nor sexual horn dimorphic. By contrast, we only found one of 3 sound-related traits tested (frequency) to be clearly repeatable. Males and females presented a different structure of the stridulatory apparatus, suggesting that morphology may affect the frequency of sounds emitted.These results indicate that certain sound traits might not be good descriptors of individual temperament revealing the need for future research addressing the role of bioacoustics.

In alpine environments, open habitats alternate with wood to create a habitat mosaic that shapes insect community composition and diversity. Dung beetles are an important group of insects specialized in feeding on vertebrate dung whose availability also depends on habitat type. Although the habitat preferences of dung beetles have been extensively studied, few studies have addressed the influence of habitat structure and temperature on dung beetle communities in alpine environments. We sampled dung beetles in pastures, at the edges between pasture and wood, in inner and outer woods of two alpine areas at different altitudes (two sites per area). We found that pastures had higher mean temperatures compared to other habitat types and hosted the highest number of species. However, the interaction between habitat and altitude significantly affected species richness and abundance, suggesting that habitat type and temperature modulate the response of dung beetles in the study area. Edges hosted intermediate communities between pastures and woodlands and were populated by both pasture and woodland specialists. Our results suggest that maintaining pastures is crucial to preserving dung beetle communities.

The gut microbiota of dung beetles comprises bacteria, archaea, and fungi. Most studies have only considered the bacterial element and focused on differences between species reared in the lab. This study considered microorganisms from the gut of Trypocopris pyrenaeus and concentrated on differences among wild individuals of an alpine Italian population. We revealed remarkable interindividual variation in the taxonomic composition of microbiota. Core taxa were few, while individuals harboured many exclusive taxa. Therefore, considering only a few individuals to describe the microbiota of a species is not sufficient. The study also revealed that the nMDS ordination of individuals based on prokaryotes was different from that based on fungi, meaning that both groups should be considered in microbiota analyses and that one microbic group cannot be considered a surrogate of the other. We identified many functions potentially provided by the microbiota and found the taxonomic richness of prokaryotes to be positively correlated with their functional richness. The analysis of the functions potentially provided by these microorganisms confirmed that the gut microbiota, in addition to being essential for the health of their host, may also contribute to the effective functioning of the ecosystems of which dung beetles are part.

Catchment urbanisation results in urban streams being exposed to a multitude of stressors. Notably, stressors originating from diffuse sources have received less attention than stressors originating from point sources. Here, advances related to diffuse urban stressors and their consequences for stream benthic communities are summarised by reviewing 92 articles. Based on the search criteria, the number of articles dealing with diffuse urban stressors in streams has been increasing, and most of them focused on North America, Europe, and China. Land use was the most common measure used to characterize diffuse stressor sources in urban streams (70.7 % of the articles characterised land use), and chemical stressors (inorganic nutrients, xenobiotics, metals, and water properties, including pH and conductivity) were more frequently reported than physical or biological stressors. A total of 53.3 % of the articles addressed the impact of urban stressors on macroinvertebrates, while 35.9 % focused on bacteria, 9.8 % on fungi, and 8.7 % on algae. Regarding ecosystem functions, almost half of the articles (43.5 %) addressed changes in community dynamics, 40.3 % addressed organic matter decomposition, and 33.9 % addressed nutrient cycling. When comparing urban and non-urban streams, the reviewed studies suggest that urbanisation negatively impacts the diversity of benthic organisms, leading to shifts in community composition. These changes imply functional degradation of streams. The results of the present review summarise the knowledge gained to date and identify its main gaps to help improve our understanding of urban streams.

More than half of the world's rivers dry up periodically, but our understanding of the biological communities in dry riverbeds remains limited. Specifically, the roles of dispersal, environmental filtering and biotic interactions in driving biodiversity in dry rivers are poorly understood. Here, we conduct a large-scale coordinated survey of patterns and drivers of biodiversity in dry riverbeds. We focus on eight major taxa, including microorganisms, invertebrates and plants: Algae, Archaea, Bacteria, Fungi, Protozoa, Arthropods, Nematodes and Streptophyta. We use environmental DNA metabarcoding to assess biodiversity in dry sediments collected over a 1-year period from 84 non-perennial rivers across 19 countries on four continents. Both direct factors, such as nutrient and carbon availability, and indirect factors such as climate influence the local biodiversity of most taxa. Limited resource availability and prolonged dry phases favor oligotrophic microbial taxa. Co-variation among taxa, particularly Bacteria, Fungi, Algae and Protozoa, explain more spatial variation in community composition than dispersal or environmental gradients. This finding suggests that biotic interactions or unmeasured ecological and evolutionary factors may strongly influence communities during dry phases, altering biodiversity responses to global changes.

Habitat models rarely consider macroinvertebrate communities as ecological targets in rivers. Available approaches mainly focus on single macroinvertebrate species, not addressing the ecological needs and functionality of the whole community.This research aimed at providing an approach to model the habitat of the macroinvertebrate communities. The study was carried out in three rivers, located in Italy and characterized by a braiding morphology, gravel riverbeds, and low flows during the summer period. The approach is based on the recently developed Flow-T index, together with a Random Forest (RF) regression, which is employed to apply the Flow-T index at the mesohabitat scale. Using different datasets gathered from field data collection and 2D hydrodynamic simulations, the model was calibrated in the Trebbia River (2019 field campaign) and validated in the Trebbia, Taro, and Enza rivers (2020 field campaign).The RF model selected 12 mesohabitat descriptors as important for the macroinvertebrate community. These descriptors belong to different frequency classes of water depth, flow velocity, substrate grain size, and connectivity to the main river channel. The cross-validation R2 coefficient (R2cv) of the training dataset was 0.71, whereas the R2 coefficient (R2test) for the validation dataset was 0.63. The agreement between the simulated results and the experimental data shows sufficient accuracy and reliability. The outcomes of the study reveal that the model can identify the ecological response of the macroinvertebrate community to possible flow regime alterations and river morphological modifications.Lastly, the proposed approach allowed to extend the MesoHABSIM methodology, widely used for the fish habitat assessment, to a different ecological target community. Further applications of the approach can be related to ecological flows design in both perennial and non-perennial rivers, including river reaches in which fish fauna is absent.