Gammarus is a very diverse genus of amphipod crustaceans found throughout freshwater habitats in Europe. It comprises ca. 280 species and most of its diversity worldwide was described relying on morphological studies that have been ongoing for many decades. However, recent advances in molecular techniques have revealed a surprising amount of cryptic diversity within this group. In our study, we use an integrative approach to describe three new species from Romania, which represent divergent lineages within the Gammarus balcanicus complex. The examined material for these three lineages was collected at three distinct locations, Varciorog, Piatra Craiului Mountains and Dobrogea plateau, and is deposited at the Department of Invertebrate Zoology and Hydrobiology of the University of Lodz. For molecular species delineation and phylogeographic analyses, we used mitochondrial (COI, 16S) and nuclear (28S, EF1a, H3) markers. For the morphological study, appendages of selected individuals were dissected, mounted in glycerine gel slides, photographed, and digitally drawn using CorelDRAW 2018. Our results show that the three new species are divergent both in the case of mitochondrial and nuclear markers. These lineages originated probably in the Southern Carpathians, already in the Miocene. While two lineages are limited to singular locations, the third one colonised the whole Dobrogea plateau and went through a period of demographic expansion at ca. 100 kya. Morphological analysis shows that the three new species can be distinguished from the neotype of G. balcanicus and other closely related species by a set of characters encompassing: the amount of posteromarginal setal groups and the length of the gland cone on the antenna 2; presence/absence of calceoli on antenna 2; setal placement on maxilla 1; shape and armature on the palms of gnathopods 1–2; setal arrangement on pereopods 3–4; the shape of the posteroventral corner of epimeron 3; dorsal armature on pleonites and urosomites 1–3; and armature on telson.

The release of treated effluents from wastewater-treatment plants (WWTPs) affects not only chemical composition of water but also the benthic invertebrate community. We examined the impact of WWTPs and other smaller effluent on water quality and benthic invertebrate community composition. For 26 selected effluents, there were available benthic invertebrate data from monitoring sites upstream and downstream the WWTPs. Concentrations of selected water quality parameters were studied on 18 pairs of monitoring sites. The distance of the monitoring sites from the effluent was usually up to 5 km. An important selection criterion was the absence of a significant tributary with possible other pollution sources between the monitoring sites. Benthic invertebrate sampling was carried out in the period from 2007 to 2019; chemical data were available from 2013 to 2022 and included both dry and wet years. WWTPs discharge into the recipients accounted for 1 to 88% of the recipient flow during drought conditions (flow Q355d was considered as the limit of hydrological drought).

To illustrate the water quality situation upstream and downstream the WWTPs, the total phosphorus parameter was chosen as the average of the values measured in the summer months. The results showed a worse situation in the summer months and in dry years, which means a very possible acceleration of the deterioration in relation to the increasing trend of air temperatures. Only data from operational monitoring was used, rapid rainfall-runoff events were likely not captured which means that the situation may actually be even worse. Implemented measures are not sufficient even if effective measures exist. A change in legislation, which would bring the limits specified in Government Regulation No. 401/2015 into line with the objectives of good status of water bodies, is absolutely necessary as well as stricter limits for best available technologies in wastewater disposal. In the case of phosphorus, only respecting the goals of good ecological status will lead to an effective improvement of the eutrophication in our rivers and reservoirs.

Benthic invertebrates were studied in terms of community composition and representation of functional groups in monitoring sites upstream and downstream the WWTPs. When comparing monitoring sites where the share of discharged wastewater was higher than 5%, the ecological status of benthic invertebrates evaluated according to the WFD was strongly affected, especially in terms of the representation of sensitive species of taxa. The change in the representation of food strategies of benthic invertebrates was also significant.

This research has been supported by the PERUN Competence Centre (TAČR project SS02030040 Prediction, Evaluation and Research for Understanding National sensitivity and impacts of drought and climate change for Czechia). Water quality and benthic invertebrate data comes from the operational monitoring of the state enterprises Povodí.

The impacts of climate change are increasingly evident worldwide, including Hungary, where observable shifts in both the average precipitation and their distribution have occurred in recent decades. Consequently, numerous once-permanent small streams have transitioned to intermittent flow patterns, necessitating adaptation among resident organisms. Among these, the Chironomidae family (non-biting midges) stands out as a prevalent group found across various aquatic and wetland habitats, owing to their abundance and short life cycle, rendering them suitable for assessing the ecological status of the watercourses. Our aim was to determine whether Chironomidae assemblages can indicate the difference between permanent and intermittent streams in temperate zones. Quantitative sampling was conducted seasonally over a one-year period in the Bükkösdi-víz catchment area in Hungary. Stream sections were categorised into five types based on their flow history: 1) permanent and stable; 2) vulnerable (permanent but with recognised low flow); 3) dried more than five years before sampling; 4) dried more than one year but less than five years before sampling; and 5) dried within the year preceding sampling. We evaluated both the structural (i.e., taxonomic) and functional (i.e., traits associated with drying) composition of chironomid assemblages. Multivariate analyses revealed significant differences in assemblages between permanent streams and other types. Taxonomic composition displayed a gradient from streams that dried up within a year prior to sampling to vulnerable ones. Conversely, functional composition showed significant differentiation between vulnerable streams and those that experienced drying within the year preceding sampling. Our findings demonstrate that drying events appearing in a previously permanent river network significantly alter the taxonomic and functional composition of chironomid assemblages. Furthermore, taxonomic composition may provide insights into the timing of preceding drying events.

The Chironomidae communities in the Arctic predominantly developed during the Holocene, later than in central Europe where the midge fauna was established during the Late Weichselian. The mean July air temperature, a key factor influencing midge development, maintained a similar level during the Holocene in northern Scandinavia as it did during the Late Glacial in central Europe. The comparison between the Late Glacial midge fauna in central Europe and the Interglacial midge fauna in Scandinavia is thus justified. This study presents the Holocene stratigraphy of Chironomidae communities at Lake Kuutsjärvi in northeastern Finland. The cold climatic conditions and oligo/mesotrophic status of the lake contribute to the low abundance of chironomids. Additionally, the specific bathymetry, with a broad deep-water zone and a very narrow littoral zone, exacerbates the impact of climate. Nevertheless, as many as 91 Chironomidae morphotypes were identified in the Holocene lake sequence. The dominant taxa are adapted to low temperatures, thriving in the profundal zone during Holocene Thermal Maximum. Some species, which are typically restricted to flowing waters in central Europe, appear in stagnant waters in the Arctic due to the better oxygen saturation in oligotrophic, cold waters. The seepages, small brooks, and moss blankets along the lake shore host taxa that in temperate latitudes are typical of mountain springs. While it might be expected that North Scandinavian waters would resemble lakes in the periglacial landscape of central Europe during the ice age, midge assemblages in Lithuania, Poland, and the Carpathians during the MIS2 reveal a different taxonomic composition. In middle Europe there is a much higher domination of common, warm-adapted taxa and a lack of very cold stenotherms like Paraphaenocladius pseudirritus, Abiskomyia, and Parochlus.

Drying up has a major role in shaping communities and significantly influences the composition of the aquatic macroinvertebrate community and their diversity metrics (e.g., taxa number, number of individuals, Shannon diversity). Furthermore, the macroinvertebrate taxa groups (e.g., Odonata, Heteroptera, Coleoptera, etc.) may have different functional trait-complex, which determine how they respond to stress at individual level. However, the trait-complex appears coupled in the individual, so it is assumed that phylogenetically and taxonomically closely related species (that belong to the same taxa group) have more similar trait-complexes than more distantly related species. Based on this assumption, different taxa groups within aquatic macroinvertebrate communities may respond differently to changes in the intermittency of water flow. In our research, we performed a stressor-impact study along an intermittency gradient in the context of the intermediate disturbance hypothesis (IDH) framework, which posits that moderate level of disturbances can have beneficial effects on ecosystem structure and function. Sampling was carried out bimonthly at a total of 15 sampling sites in four intermittent streams from May 2021 to March 2022 in the Hungarian lowland region. Hydrological condition was measured by in-stream data loggers, and intermittency gradient was characterised by the number of drying days during the sampling period. Our results suggest that the moderate intensity of drought is beneficial for less competitive communities, but detrimental for communities with high dispersal ability. Revealing changes in diversity metrics and determining the unique response by taxa groups of aquatic macroinvertebrate communities along the intermittency gradient are crucial in index development, bioassessment and water management.

Lake Skadar basin, located on the border between Montenegro and Albania, is one of the key hotspots of biodiversity and endemism on a European scale. The area is characterised by high habitat heterogeneity and temporary isolation limiting species migration. It covers an area of diverse geomorphological, tectonic, and hydrogeological structures and is an important point in the context of analysing the evolutionary history of the Balkan Peninsula’s fauna. It includes an extensive and ancient system of karst springs and a relatively young lake. However, the area is at risk of biodiversity loss due to climate change and human interference associated, for example, with increasing tourism development. The aim of our study was to assess the genetic diversity and geographic distribution patterns of freshwater gammarid amphipods in the Lake Skadar basin, Balkan Peninsula. Based on morphological observations, four morphospecies were identified: Gammarus balcanicus Schäferna, 1922, G. roeselii Gervais, 1835, Pectenogammarus thoni (Schäferna, 1923) and Pectenogammarus scutarensis (Schäferna, 1923). The morphological identification was confronted with molecular data. The study was based on DNA barcoding with third-generation sequencing based on Oxford Nanopore Technologies (ONT). The obtained sequences were used to create a reference DNA barcode library in The Barcode of Life Data System (BOLD). Currently, 326 sequences have been obtained from the 32 sites studied. The analyses conducted indicate that the cryptic diversity within the conventionally described species of amphipods is high. Currently, 11 BINs (Barcode Index Number) have been assigned to the deposited sequences. Seven of the assigned BINs are unique and new for BOLD. However, preliminary species delimitation using the ASAP method indicated that 10 MOTUs (molecular operational taxonomic units) could be distinguished among the sequences obtained. Sequence analysis of G. balcanicus also indicates high genetic variability of the indicated species relative to the data present in the BOLD database. Among the analysed individuals of P. scutarensis, we also observed introgression of mtDNA from P. thoni, confirming previous studies (Jabłońska et al. 2019). Our results underscore the need to intensify research on the Balkan Peninsula to assess the diversity of local hydrobionts.

Urbanisation is one of the most serious threats to stream ecosystems worldwide. Our aim was to investigate the general effects of urbanisation in a moderately urbanised landscape and to assess the relationship between local environmental variables and biotic attributes of macroinvertebrate assemblages. We found that local habitat properties presented degraded conditions at urban sites. Urbanisation had a negative effect on the richness and Shannon diversity of macroinvertebrate assemblages and altered assemblage composition. Individual streams showed considerable variability under natural conditions, as well as in their response to urban effects. Subsequently, the mitigation of the effects of urbanisation might also need considerable variability in the type of actions required.

Benthic invertebrates belong to the biological elements utilised in the assessment of the ecological status/ecological potential of waters in accordance with the European Water Framework Directive (WDF) due to their response to various stressors, including alteration in hydro-morphological conditions, physicochemical parameters of the water, including oxygen, trophic conditions, and salinity. They are widely acknowledged as excellent indicators for biological assessment. Therefore, the aim of the research initiated in the autumn of 2022 was to assess the diversity of bottom fauna communities in the Oder River after the ecological disaster. The research was carried out at 5 sites located between 430 km and 670 km of the river course. Additionally, sampling was also done in the Gliwice Canal, which is connected to the upper Oder, and in Kłodnica River (a right-side tributary in the upper course of the Oder River).

The results of the research indicate high variability of the bottom fauna. In the Oder River, Oligochaeta, Chironomidae and Potamopyrgus antipodarum constituted the constant elements of the macrobenthos at most sites. In the Gliwice Canal, also Coenagrionidae and Physa acuta were constantly present. However, in Kłodnica, Amphipoda (Apocorophium lacustre, Gammarus tigrinus, Dikerogammarus villosus) was also a permanent element of the zoobenthos. During the research period, the percentage of particular taxa in the structure of benthic assemblages changed at all sections, both in the Oder River, the Gliwice Canal, and Kłodnica. At most sites, a high proportion of alien species, including invasive species, was found. In total, 9 alien invertebrate species were indicated – 2 species of polychaetes (Hypania invalida, Laonome xeprovala), 3 species of amphipod crustaceans (Chelicorophium curvispinum, Gammarus tigrinus, Dikerogammarus villosus), 2 species of snails (Potamopyrgus antipodarum, Physa acuta) and 2 species of bivalves (Dreissena polymorpha, Corbicula sp.). The research undertaken in 2022 is being continued, which will allow for a more complete analysis of the bottom fauna alteration over a long period of time after the ecological disaster on the Oder.

The freshwater mussel Unio crassus (thick-shelled river mussel) has not been found in the Netherlands for decades, due to its sensitivity to poor water quality (pollution, low oxygen, salinisation, and high nutrient concentrations). The mussel is an ‘ecosystem engineer’ as it alters its environment by filtering water, fixating sediment, and providing hard substrate for other organisms. The mussel extinction has therefore presumably been a drawback for other riverine species in the Netherlands as well. In recent years the abiotic conditions in Dutch rivers have significantly improved, offering opportunity for recovery of the mussel. The goal of this study was to find out if habitat conditions are suitable enough for population recovery of U. crassus in the Netherlands. The hypothesis was that the physical and chemical water quality is sufficient, but that other habitat requirements and lack of a source population pose a bottleneck for recovery. 

A feasibility study was carried out, based on literature research and available data on fish populations (host for mussel larvae). The conditions in Dutch rivers seem to fit for population recovery of U. crassus: chemical water quality (oxygen, ammonium, pH, nitrite, nitrate and, in most waters, phosphate) is sufficient, the physical habitat (substrate and flow velocity) is mostly in order and there are 21 fish species that can function as a host for the larvae of U. crassus, six of which are considered good hosts (carrying high densities of mussel larvae in their gills). Based on the analysis of the chemical, physical and biological (host) conditions of Dutch rivers, a map was created, indicating promising locations for population recovery. Permanently flowing floodplain channels that are two-sided connected to the main river, containing high densities of hosts, seem the most suitable. Because the mussel is extinct in the Netherlands, natural population recovery will have to be catalysed by colonisation from upstream waters. However, the species is rare in the entire catchment area, making it unlikely the species will reach the Netherlands on its own. In the footsteps of neighbouring countries, a reintroduction program might be considered, including detailed habitat suitability measurements on the intended locations.

The Ponto-Caspian radiation of gammaroid amphipods is one of the world’s most diverse lacustrine crustacean radiations as well as the only lacustrine amphipod radiation with a fossil record (dating back 9 Ma). Up to 40% of these species have now spread outside their native range due to human intervention. However, our evolutionary understanding of this diverse clade is fragmentary and incomplete because the relationship between fossils and extant species remains unknown. Here, we present the first morphology-based phylogeny of Ponto-Caspian gammaroids that includes 99% of extant taxa, all previously known fossils, as well as newly discovered fossil specimens from the Late Miocene (10.5 Ma) of eastern Romania. We find that all fossil species belong to a single lineage that is an evolutionary dead-end, having no living descendants despite being widespread throughout the Paratethys. This clade has an ambiguous phylogenetic relationship with extant taxa, though it is nested within the radiation’s crown group. Our analyses also reveal that the new Romanian fossils belong to a new genus with two new species. Overall, our study reveals that the complexities of Ponto-Caspian amphipod evolution cannot be fully understood only by looking at living species, highlighting the importance of including fossils in evolutionary studies.

One of the primary applications of macrozoobenthos research in freshwater habitats is water quality assessment. Macroinvertebrate community changes its structure as a response to changes in the environment, including negative anthropological influence, e.g., pollution and hydromorphological degradation. In the middle reaches of Slaná river, in February 2022 there was a huge (lasting) discharge of mine-waters, containing high amounts of iron, manganese, and sulphates, what was evident as long stretches of river coloured orange-red, and iron crust on stones and aquatic animals. To identify the extent of this negative impact on aquatic communities, macrozoobenthos was sampled in the autumn that year, and again in spring and autumn 2023. Samples were collected using benthos hand net (500 µm mash) according to AQEM on four sites, in the length of cca 20 rkm. Gočovo site (GČ) was located above the point source of mine-water, Henckovce site (HEN) just below, representing most impacted site, and further below were Rožňava-Nadabula (RVN) and Rožňava-Brzotín (RVP). Twelve invertebrate taxa groups were found in the monitored section, of which 5 belonged to permanent fauna, and 7 orders of Insecta represented temporary fauna, including in total 181 taxa. Diversity did not substantially differ among sites/years, but total abundance did. Highest total abundance in 2022 was recorded at GČ, followed by RVP with more than 3,000 ind.1.25 m^-2, and at the directly impacted HEN site only 1,018 ind.1.25 m^-2 were recorded. However, with time, the total abundance improved and in autumn 2023 1,884 ind.1.25 m^-2 were registered. Temporal changes in the community composition at HEN were evident as a very high abundance of Hydropsyche (Trichoptera) and Chironomidae representatives in 2022, decreasing in 2023. More importantly, there was an increase of more sensitive taxa – Plecoptera in general, particularly representatives of the Leuctra genus. Furthermore, in 2023 there was an increase in numbers of Gammarus individuals, sensitive to trace metal pollution, and appearance of environmentally more sensitive Habroleptoides confusa and Rhithrogena semicolorata (Ephemeroptera), and Silo piceus (Trichoptera). SIMPER analysis revealed that the difference between reference site GČ and most impacted site HEN was decreasing each succeeding sampling date, from 54.87 to 39.13%.

Some differences in the community structure were undoubtedly the result of seasonal dynamics within macrozoobenthos that occur regularly and were observed at each site. Nevertheless, changes in the dominance between taxa more or less tolerant to degradation at the site closest to the mine outlet, indicates important influence of the polluted water on the invertebrate community, but also the ability of macrozoobenthos to recover over time. Further monitoring is vital for the protecting actions to be proposed and carried out, if necessary, to preserve this river from further degradation, but also to survey the applied measures.

Lakes and ponds in the Tatra Mts, the highest part of the Carpathian Arch (Central Europe, Slovak-Polish border), were formed as remnants of the last glaciation. These unique but highly threatened aquatic biotopes contribute significantly to the preservation of ecosystem services in this region, thanks in part to the rich local biodiversity they contain. In recent years, innovative approaches such as DNA barcoding have been added to the traditional research methods that have been used to study lake biota for decades, allowing us to learn much more about their biodiversity.

Over the last 15 years, more than 200 aquatic habitats in the Tatras – of different types, at different altitudes (up to 2,200 m a.s.l.) and often remote – have been repeatedly sampled to study the genetic diversity of benthic macroinvertebrates and to create a high-quality and detailed reference library of their DNA barcodes. All groups of benthic macroinvertebrates from these glacial lakes were sequenced, especially the dominant oligochaetes and all orders of insects. In total, we obtained sequences of a standardised COI barcode fragment from almost 5,200 individuals, which are now stored in the BOLD database under the SKALP project. The first part of them was obtained by classical Sanger sequencing, but a significant part of the data (almost 4,000 barcodes, mainly from the most abundant groups Oligochaeta and Chironomidae) was produced thanks to third-generation sequencing. Massive DNA barcoding using MinION sequencer (Oxford Nanopore Technologies) has rapidly accelerated and enhanced our efforts to build the most comprehensive picture of aquatic life in these mountain lakes. The DNA barcodes represent more than 400 BINs, of which nearly 60 are unique to BOLD, highlighting the uniqueness of this area, which requires proper protection, as well as our still insufficient knowledge of its diversity. The dominant groups in terms of genetic diversity are oligochaetes (with 26 unique BINs), beetles, and dipterans, especially chironomids (19 unique BINs).

The newly created reference library of DNA barcodes is not only an exhaustive list of taxa living in Tatra lakes and ponds, but also an essential basis for correct analysis and interpretation of metabarcoding data from environmental DNA (eDNA) research, which is currently being carried out in the Tatra lakes and has a great potential for proper monitoring of their status. This study was supported by the project VEGA 2/0084/21.

European lowland rivers have been exposed to extensive anthropogenic stress for almost a century. Despite substantial improvement in water quality, novel stressors, mainly climate change, continuously endanger them. Their long-term development has often been studied, yet the impact of hydromorphological degradation on these changes was rarely considered, even though its effect on riverine biota is known to be substantial. To elucidate how such a degradation modifies macroinvertebrate response in multiple-stressor environment, we evaluated water quality, hydroclimatic and biological changes in nine Czech lowland rivers over some 20 years, regarding their hydromorphological degradation degree. We divided our sites into highly degraded and less degraded sites and found that despite comparable development in all studied environmental variables, the two site groups exhibit distinct biotic changes. Highly degraded sites experienced decreased evenness, increased proportion of non-native taxa and functional reorganisation, changes congruent with continuous deterioration. In contrast, less degraded sites displayed several favourable changes, such as increased total and potamal indicator richness, as well as assemblage stabilisation with few functional changes. Increased abundance and upsurge in non-native taxa abundance and richness was found in both site groups. Despite these changes, no significant trends in well-established biotic indices were found. Our study shows that hydromorphological degradation modifies macroinvertebrate response to changing environmental conditions, such as reduced pollution, but also rising thermal and hydrological consequences of climate change, that are expected to amplify in the near future.

Wetlands in lowland river floodplains in Central Europe have been significantly reduced and degraded. This process did not avoid even the floodplains of the Morava and the Danube Rivers. Remnants of wetlands with an area of more than 5,000 ha in the Slovak part of the Morava floodplain are protected as a Ramsar site and the NATURA 2000 area. Some of them are located directly on the territory of the capital of Slovakia Bratislava, many other protected wetlands are located in the Danube floodplain. These wetlands are not only the home of rare species and a space for recreation, but also provide breeding sites for mosquitoes, which from spring to autumn reduce the quality of life of hundreds of thousands of inhabitants of the adjacent territories. Flooding during spring and summer creates conditions for the development of an extreme number of individuals of mosquitoes of the genus Aedes. In addition to reduced comfort for residents, they also pose health risks, as mosquitoes are vectors of various diseases. In addition, these species are very mobile, which is why they have an impact on a wide area. The public is therefore demanding a solution to the “mosquito problem”, while rightly considering the protection of wetlands as one of the reasons why the mosquito calamity cannot be reduced. Is it possible to somehow combine the protection of wetlands with the reduction of mosquito plagues? We looked for an answer to this question in the recently completed Mosquito Bioregulation project, which we implemented together with Austrian partners within the Interreg SK–AT program, in the Slovakian-Austrian border area in the Moravian floodplain and in the Danube floodplain in the territory of Bratislava. In the poster, we will present the procedures for the biological control of mosquitoes, the results of mosquito monitoring in the project area, the results of the screening of mosquito-borne pathogens, the pyrethroid resistance among mosquitoes obtained during project implementation.

Simuliidae (black flies) are very efficient and opportunistic colonisers of running waters, where they can be quite numerous and diverse. The study was conducted to increase the knowledge about the Simuliidae fauna in the small and medium size rivers of Serbia. These types of rivers in Serbia are very heterogeneous. They belong to 4 different hydroecoregions (ER5-Dinaric Western Balkans, ER7-Eastern Balkans, ER10-Carpathians, and ER11-Hungarian Plain), flows into three different drainage basins (Black Sea, Adriatic, and Aegean Basin) and are located in high altitude range. All of these provide different habitats that harbour a great diversity of black flies. In the period from 2013 to 2021, larvae and pupae of Simuliidae were collected from 118 locations throughout Serbia, covering all hydroecoregions, drainage basins and altitudes from 75 to 1,437 m. A total of 36 black fly taxa were identified. Simulium ornatum was proved to be the dominant and most abundant species, with 3,033 individuals detected at 57 sites. Other common species in small and medium size rivers in Serbia were S. reptantoides, S. variegatum, S. equinum, S. argyreatum, S. reptans and S. balcanicum. Species Prosimulium tomosvaryi, S. ornatum and S. reptans were present in all hydroecoregions of Serbia. Most species were found in high altitude range and could be considered euryvalent. The species Prosimulium hirtipes was common in wadable rivers and streams, while S. brevidens was found only at high altitudes. The highest Simuliidae diversity was found at altitudes from 500 to 800 m. All black flies communities were characterised by high beta diversity, which was reflected in high values of species turnover and low values of nestedness.

With the fast development of urban areas, wastewater discharges are increasing and have a major impact on urban rivers and canals. These urban waterbodies become polluted, and the health of the ecosystem is seriously threatened. Macroinvertebrates are one of the biological quality elements used to assess the ecological status and health of rivers. The aim of this study was to investigate the macroinvertebrate communities and the present functional groups of the 16 rivers and canals in the Belgrade urban area: Danube, Sava, Kolubara, Beljanica, Pešten, Turija, Barajevska and Topčiderska rivers, Galovica, Progarska jarčina, Sibnica, Kalovita, Vizelj, Karaš, PKB, and Obrenovački canals. The samples were collected during high water levels using a multihabitat sampling approach in 2023. The ASTERIX software was used to calculate the ecological functional groups. All together 126 macroinvertebrate taxa were identified. The most abundant taxa were Insecta, Oligochaeta and Crustacea. The highest number of taxa (31) was found in the Beljanica canal while the lowest (5) was found in the Sibnica canal. All sites were characterised by the dominance of alpha- and beta-mesosaprobic organisms (tolerant of high and moderate organic matter loading), except for Turija where polysaprobic organisms dominated (species adapted to high organic load), which was also confirmed by the highest value of Zelinka & Marvan’s saprobic index (3.42). In terms of zonation preferences, most of the species recorded were typical to potamal (lower stretches of a river). When analysing the communities according to flow preference, rheophilic species dominated in the streams, preferring zones with moderate to strong flow. The medium and large rivers were dominated by rheo-limnophilic taxa that prefer slow-flowing streams and lentic zones, while the canals were dominated by indifferent taxa that have no preference for a particular flow velocity.

Subterranean freshwater ecosystems remain poorly studied, despite their significance. They harbour a diverse array of invertebrates, occupying various groundwater habitats. Notably, crustaceans constitute a significant component of these ecosystems. In addition to groundwater, they may emerge in surface waters, such as springs, during episodes of heightened groundwater levels following heavy rainfall events.

Our study focused on spring habitats within the Western Carpathians of Poland and Slovakia during the years 2022–2023. We sampled 118 individuals across 23 sites spanning four Carpathian Mountain ranges: 1. the Orava Beskid and the Żywiec Beskid; 2. Bukovec Mountains and Bieszczady; 3. Veporskie Mountains; 4. The Island Beskids. These sites encompassed springs in various environments, including fields, meadows, forests, and roadsides, representing both natural and human-transformed landscapes.

The focus of this investigation was the genus Niphargus (Malacostraca, Amphipoda), stygobiontic crustacean characterised by lack of pigmentation and absence of eyes. This taxon comprises over 400 species, with Niphargus tatrensis being one of the most typical representatives of subterranean amphipods in the Western Carpathians. Described solely based on morphology, its range has been estimated from southern Poland to Austria and Slovenia. Initial morphological observation suggested that all specimens collected in the field belong to N. tatrensis. Considering the potentially limited dispersal capabilities, our objective was to explore cryptic and pseudocryptic diversity among the collected specimens.

Genetic analyses were conducted using the mitochondrial COI gene. A total of 118 barcodes were obtained, revealing 25 haplotypes. These sequences were deposited in the BOLD database (Barcode of Life Data System), where they were assigned to eight BINs (Barcode Index Numbers), roughly equivalent to species. Additionally, application of the ASAP (Automated Sequence Analysis Pipeline) method identified five MOTUs (Molecular Operational Taxonomic Units). Finally, we confronted the observed molecular diversity patterns and morphological traits. Among species identified through genetic analysis based on BINs, we measured several morphological traits to identify characteristics of a particular BIN.

Balkan Goldenring (Cordulegaster heros) is listed in Annexes II and IV of the European Habitats Directive. This dragonfly species has a 3–4-year-long larval development, making the conservation of its larval habitats and surrounding areas crucial for its survival. It is well-known that C. heros prefers headwater habitats in shady valleys, typically with a high percentage of forest cover. In vitro laboratory experiments have also shown that the larvae of the species can survive short-term dry periods. Our aim was to investigate the importance of dry periods and land cover on the presence of the species in vivo, using a multivariate machine learning model. We focused on Bükkösdi river network (SW Hungary), where both intermittent and perennial water bodies with the occurrence of C. heros can be found. Species presence and absence data were collected through a two-year-long seasonal macroinvertebrate sampling. Hydrological logger data were gathered from 42 streams daily, and land use data were obtained from a high-resolution national dataset (NÖSZTÉP). The percentages of different land use types were calculated for the catchment area above the sampling points. As a first step, we performed a binomial GLM to select the most important hydrological and land use variables. In the second step, we used a cross-validated XGBoost model for the selected five variables, with an 80/20% train-test split, and defined the importance of these variables. From our results, we were able to predict the occurrence of the species C. heros with high accuracy (0.9 AUC). Our results demonstrate, through a two-year in vivo experiment, that areas suitable for C. heros can be characterised by a high proportion of forest cover and a low proportion of agricultural areas in the catchment area. The species can survive and potentially recolonise areas that have been periodically dry. It can be concluded that the protection of forests in the catchment area is highly important for the species’ occurrence, and while the species can survive increasing frequency of drying events up to a level, long-term drought periods and water abstraction can lead to its extinction.

Lake Ohrid is recognised as the oldest preserved lake in Europe and is characterised by high biodiversity – both in the lake and surrounding springs. Among the fauna found in the lake, Amphipoda (Crustacea) play a key role in its ecosystem as detritus-eaters and food for predators. One of the amphipod species, not only found in the lake, but also widely distributed in Europe, is Gammarus roeselii Gervais, 1835. In Central Europe the species inhabits rivers and is characterised by low molecular diversity. In contrast, in the Balkans, the species can be found in lakes or springs and its molecular variability is high, suggesting the presence of cryptic species.

The main objective of the study is to analyse the diet of G. roeselii from Lake Ohrid and adjacent springs in the context of the molecular diversity of populations found in the two habitats. In the first stage of the project, we employed DNA barcoding, based on the sequence of the mitochondrial gene encoding the COI. This will enable the determination of the G. roeselii molecular variability in the study area. The next stage of the project will be the analysis of diet, based on DNA metabarcoding, using second-generation sequencing. Various markers will be employed (COI, 18S rRNA, ITS2, 16S rRNA) to recognise possibly wide range taxonomic spectrum found in the food content. As a result of the project, we can expect differences in diet as well as variation in patterns of molecular variability of populations occurring in contrasting environments.

Functional diversity is regarded as a key component in understanding the link between ecosystem function and biodiversity and is therefore widely investigated in relation to human-induced impacts. However, information on how the intersection of roads and streams (hereafter road crossings, representing a widespread habitat transformation in relation to human development), influences the functional diversity of stream-dwelling macroinvertebrates is still missing. The general aim of our study was to provide a comprehensible picture on the impacts of road crossing structures on multiple facets of the functional diversity of stream-dwelling macroinvertebrates. In addition, we also investigated changes in trait structure. Our research showed that road crossing structures had negative impacts on functional richness and dispersion, i.e., functional diversification. However, we found no significant impact on functional divergence and evenness components. We found a decrease in functional redundancy at road crossing structures. This indicates a reduced ability of the community to recover from disturbances. Finally, we found that road crossings drive stream habitat and hydrological changes in parallel with modification of the trait composition of stream-dwelling macroinvertebrate assemblages. All these results suggest that road crossings cause notable changes in the functional diversity of stream-dwelling macroinvertebrate assemblages.

Dredging is a commonly used practice in water management to remove the accumulated sediment and vegetation from the waterbed. It has direct and indirect effects on aquatic macroinvertebrate communities, additionally it creates a modified habitat in the streambed that can be newly recolonised by macroinvertebrates. Previous studies suggests that communities can recover one year after dredging, but less is known about the potential long-lasting effects. The Space-Time Substitution Method (STSM) was used to reveal the potential long-term effects of dredging on macroinvertebrate community structure (abundance, richness and Shannon diversity and Hungarian Macroinvertebrate Multimetric Index – EQR). In 2021, nine streams were sampled during both spring and autumn following the Hungarian Water Framework Directive (WFD) protocol. Three categories were established based on the time since dredging (1, 2, and 3 years), with three streams sampled from each category, including both control and dredged sections. We found no significant differences on the richness, abundance, Shannon diversity and EQR between the control and the dredged sections. However, there were slight differences in taxonomic composition between control and dredged sites. The annual fluctuation in the proportional species richness (relative to the total number of captured species) of Odonata, Crustaceans, Gastropods, and Dipterans after dredging was observed. For Odonata, the proportion of species richness was significantly higher in all dredging categories compared to the control with no detectible trends in relation to the elapsed time. This suggests that dragonflies may be more successful in recolonising habitats following dredging. Crustaceans, Gastropods and Dipterans did not show significant differences across all categories. Plecopterans were the most sensitive to dredging, as they were absent from the community in all categories following dredging. Further studies are needed to elucidate the functional aspects of the effects of dredging on the aquatic macroinvertebrate community.

The order Mysida comprises malacostracan crustaceans that inhabit benthic and pelagic zones in various environments, including marine, brackish, freshwater, and underground waters. These small creatures are most found swimming near the bottom, sometimes forming large shoals. They can also burrow into sediments. We studied mysid populations inhabiting flowing and standing inland and estuarine waters of the peri-Adriatic region, which is a recognised biodiversity hotspot in the Mediterranean. During an extensive study across the Apennine and Balkan Peninsulas, we collected over 350 specimens from 56 sampling sites located in Italy, Croatia, Montenegro, and Albania. Based on morphological examination, these specimens were assigned to seven morphotypes within the genera Diamysis, Limnomysis, Paramysis, and Siriella of the family Mysidae. However, a genetic study of 152 individuals using the COI mitochondrial gene revealed high diversity, with the identification of over 100 haplotypes. The sequences were deposited in BOLD and assigned to 11 Barcode Index Numbers, nine of which were unique. Analyses incorporating additional mitochondrial and nuclear markers (16S, 18S, 28S, and Histone H3) were employed to elucidate species relationships within the investigated group of crustaceans. Furthermore, the newly generated sequences significantly contribute to the DNA barcode reference library for European Mysida.

Wolbachia infection prevalence among aquatic insect species is approximately 52%, albeit within infected species, the presence of endosymbiotic bacteria is detected in fewer than 50% of individuals tested. Molecular analyses utilising single or multiple genetic markers have delineated numerous distinct lineages of Wolbachia, commonly denoted as ‘strains’. These strains are distributed in several large clades, termed ‘supergroups’, and have likely diverged for over a hundred million years. Currently, Wolbachia strains have been classified into supergroups designated from A to U. Supergroups A and B, along with E and F to a lesser extent, are widely distributed in arthropods. Conversely, Wolbachia strains from supergroups C and D are prevalent in filarial nematodes. The remaining supergroups are distributed among other arthropod clades.

The present study selected Aphelocheirus aestivalis (Insecta: Heteroptera) as a model species. This riverine water bug is the only reported species of the monogeneric family Aphelocheiridae occurring in most parts of Europe. Preliminary studies have previously revealed the presence of Wolbachia in A. aestivalis populations.

Here, we used the wsp molecular marker to screen 30 samples of A. aestivalis collected from various European locations. Wolbachia was detected in all but five samples. Notably, the prevalence of selected endosymbiont varied widely, ranging from 3% to 100% among the tested samples. Subsequent analyses based on nucleotide sequences of the wsp molecular marker identified two highly divergent Wolbachia strains infecting A. aestivalis. These strains were classified within supergroup A and were not previously reported in other host species. Additional analyses uncovered intricate spatial distribution patterns for these strains. In general, strain Aaesti1 predominantly occurred in A. aestivalis samples collected in Central Europe whereas strain Aaesti2 was more prevalent in Western European samples. Interestingly, the co-occurrence of both Wolbachia strains was also observed in samples collected from Central Europe.

The study was supported by the National Science Centre, Poland, under research project no. UMO-2019/35/D/NZ8/00251.

With its rich hydrological network, Bosnia and Herzegovina is widely known for its high freshwater aquatic biodiversity and endemism, with aquatic insects being a prominent biotic component. The latest national assessment lists 220 species of Trichoptera, 74 Plecoptera, and 58 species of Ephemeroptera, with 25, 11, and 5 endemic species, respectively. Due to their sensitivity to pollution, the presence and relative abundance of caddisflies (Trichoptera) are often used for biological assessment and monitoring of water quality. Notwithstanding a long history of their research since the late 19th century, modern scientific techniques still allow for the discovery and description of new species and subspecies nowadays. Regardless of its dependence on the available reference genetic sequence libraries, DNA barcoding is an essential auxiliary tool in biodiversity research. During 2019 and 2020, a study on the diversity of aquatic insects in five protected areas in the urban area of Canton Sarajevo was conducted. Collected samples were DNA barcoded using degenerated primers LCO1490-JJ and HCO2198-JJ. Although only larval samples were available, based on the generated DNA barcodes, a new subspecies of caddisfly, Rhyacophila fasciata delici, was discovered in the investigated areas of Dariva (river Miljacka) and Vrelo Bosne (river Bosna). Furthermore, haplotype diversity was observed. Out of nine sequenced samples, five different haplotypes were discovered: three for the specimens from the Dariva location and two for the Vrelo Bosne locality. This finding is a strong indication that the diversity of Trichopteran fauna in Bosnia and Herzegovina is far from being fully explored. It is also an important argument that additional efforts should be employed to register, describe, and protect diversity within explored areas and design additional measures of conservation and management given the high anthropogenic pressures present. We emphasise that DNA barcoding, together with morphological species identification, should be part of the biomonitoring methodology in the protected areas in Canton Sarajevo. This study once again showcases the importance of continuous research and the existence and supplementation of publicly accessible genetic databases to improve species identification and bioassessment reliability.

With today’s update, the number of species at risk of extinction on the IUCN Red List has exceeded 44,000 and that is still 28% of all assessed species. The IUCN Red List now includes 157,190 species of which 44,000 are threatened with extinction. The Mediterranean region is recognised as the second largest global Biodiversity Hotspot where at least 20% of all species are threatened with extinction. In the Red Book of the Threatened Animals of Greece (2009) aquatic macroinvertebrates, including caddisflies and stoneflies, have been vastly neglected due to lack of comprehensive occurrence data. However, for the first time, Greek caddisfly and stonefly species were included in the IUCN Red List assessed for both regional and global level. Forty-eight endemic Trichoptera and fifteen Plecoptera species were assessed under the IUCN Red List criteria. Most of the endemics are located mainly in the Aegean islands, where aquatic habitats are decreasing at alarming rates due to climate change, water deficiency and increased tourism development. Nine Trichoptera species are critically endangered (CR), twenty-two are endangered (EN) and the remaining 17 are vulnerable (VU) or near threatened (NT). Regarding Plecoptera, five species are CR and ten are EN. This is the first major step towards the protection and conservation, or at least the recognition, of threatened caddisfly and stonefly species in Greece. Robust conservation measures must be implemented as soon as possible, otherwise, a significant number of species will be lost within the next years.

Sediment is a crucial component of river ecosystems, and the balance of sediment transport and deposition significantly impacts aquatic biota. However, anthropogenic alterations of watercourses, such as dam construction or riverbed dredging, profoundly affect the grain size distribution of the bed sediment, and thereby indirectly impact benthic fauna. This study focuses on the influence of changes in bed sediment grain size due to the construction of riffle-pool sequences on the diversity of benthic invertebrates. The research was conducted on the Drawa River at four sites: Prosino, Kuźnica Drawska, Rzepowo, and Drawsko Pomorskie (NW Poland). Samples were collected before substrate deposition and 3 years after the interventions. Samples were taken at two points: one was subjected to restoration treatments, while the other served as a control. Throughout the study period, a total of 61 benthic taxa were recorded at all sites. Morphological changes in the river channel affected not only invertebrates collected from the directly impacted area but likely also individuals from the control sections. Restoration activities may have indirectly contributed to improving the benthic invertebrate biodiversity only at heavily urbanised sites (presumably through improvements in physicochemical water parameters), while no observable trend or even adverse effects were noted at other sites. This is attributed to sediment homogenisation and the loss of microhabitats at the intervention sites.

Financed by the Minister of Science under the “Regional Excellence Initiative” Program. Agreement No. RID/SP/0045/2024/01.

Water beetles play crucial roles in wetland ecosystems, including peatlands. This study investigated water beetle communities in a protected peatland area and adjacent peat mining sites. The protected area encompasses the Slowinski National Park. Sample collection was conducted using water traps and a hydrological net during the spring, summer, and autumn of 2023. The study aimed to assess water beetle diversity, identify potential bioindicators, and examine seasonal migration patterns between protected and mining areas. Results showed significant differences in beetle richness, abundance, and community structure, underscoring the importance of conservation efforts in preserving these ecosystems. Furthermore, our observations revealed that peat extraction sites harbour a higher prevalence of ubiquitous species, suggesting notable environmental alterations. Surprisingly, even with the cessation of peat extraction and improved hydrological stability during summer droughts, peat ponds within the mining area failed to act as refuges for aquatic beetles typically found in the national park.

The study highlights the urgent need for proactive measures to mitigate habitat degradation and preserve biodiversity hotspots, underscoring the importance of considering invertebrate fauna, particularly bioindicator species like water beetles, in peatland conservation alongside the traditional focus on flora.

Financed by the Minister of Science under the “Regional Excellence Initiative” Program. Agreement No. RID/SP/0045/2024/01.

Dragonflies and damselflies (Odonata) have been widely used as bioindicators of the environment in various ecological and monitoring studies. But sometimes it could be a challenge to correctly identify dragonfly nymphs, which are commonly found in freshwater samples, using only morphological characters (especially in early instars). Therefore, the use of molecular data for their accurate and rapid determination is very topical and DNA barcoding has become an essential tool in current diversity studies also of this group of insects. 

Globally, there are more than 45,000 records of Odonata in BOLD database (www.boldsystems.org) available, covering around 3,500 species. Currently, 143 species are known from Europe, of which 65 are confirmed from Slovakia (45.5% of the European fauna). Despite the high number of records in BOLD database, data from Slovakia have been missing so far. The aim of this study was to process the material collected on Slovak territory, and to build the national reference DNA barcode library, considering also local genetic diversity.

Samples of odonate specimens were collected in 2021 and 2023 from 140 locations evenly distributed across Slovakia, in co-operation with the State Nature Conservancy of Slovak Republic within the project of Monitoring of animal species and habitats of community interest. Altogether 392 individuals of 62 species were collected, covering almost all our species. Processing the collected material led to a final dataset of COI sequences comprising 244 specimens (168 adults, 74 nymphs and 2 exuviae) of 62 morphologically identified species. Although the barcoding of exuviae was not successful through conventional PCR and subsequent Sanger sequencing, by using an Oxford Nanopore MinION technology we were able to obtain two barcodes out of ten collected exuviae. 

The obtained dataset represents the reference database of DNA barcodes for almost complete dragonfly fauna of Slovakia. The apriori morphological determination of adults and nymphs was confirmed by comparing with barcodes of European Odonata published on BOLD. The DNA determination was highly congruent with morphology except few nymphs and very closely related species. By comparing the datasets, we also tried to reveal relationships between Slovak and European specimens concerning intraspecific variability. While some Slovak samples have pooled with other European samples of the same species, others revealed distinct haplotypes suggesting specific position of our region regarding genetic diversity within species. This study was supported by the project VEGA 2/0084/21.

Nematomorpha are a poorly known type of invertebrates. They are called “horsehair worms” in English literature because of old beliefs that they come from horses’ hair. There are approximately 360 species within this phylum, and for a long time they were incorrectly classified as nematodes due to their great similarity to this group of animals. The life cycle of Nematomorpha can be simple or complex. The simple life cycle includes mainly insects like Orthoptera, Coleoptera or Mantodea as definitive hosts. During the complex life cycle, Nematomorpha develop a cyst stage and in this form, they can be found mainly in Trichoptera, Diptera or Ephemeroptera, which play a role of intermediate hosts. The final host, for example mantis, become infected after eating the mentioned above hosts of the parasite. Nematomorpha are located in the abdomen of the final host and can be found in all organs except the intestine. Larval form of this parasite feeds on body fat and gonads which makes the host infertile. When the parasite becomes the adult form, it makes a strong need for the host to be near water. Once the host enters the water, the Nematomorpha escape into the aquatic environment through the host’s anus or break through the flexible covering on the abdomen between the host’s posterior segments. After that, it can mate with other Nematomorpha and the life cycle ends. Most often the host dies in water reservoir but sometimes it can escape and then still live, but it cannot reproduce or live like other invertebrates which never had parasite inside them. There was some information that Nematomorpha can be accidentally found in humans, however these parasites do not have enough adaptations to live inside the human body. The biggest problem during study of the biology and ecology of Nematomorpha is difficulty of finding them in nature. Larval stages are situated inside hosts bodies and the host does not have any visible symptoms, while adult forms are very hard to find because they are very thin and hard visible in water.

An intensive agricultural land use has had broad impacts on the entire landscape, including small streams, with direct and indirect impacts on macroinvertebrate communities. Monitoring of the impact of pesticides on freshwater ecosystems is quite challenging due to their low concentrations and an irregular occurrence in time, which depends on their application and a consecutive rainfall. Monitoring of pesticides using passive samplers is possible, but this is relatively expensive, problematic, and ambiguous with uncertain results with respect to the challenging water concentration calculation. More effective is monitoring of the pesticides in sediments that represent a sink and hotspots of chemical contamination (especially for insecticides that are usually hydrophobic) as well as habitats for many macroinvertebrate species. As a substitute for demanding monitoring with uncertain results, the calculation of indices based on the composition of the macroinvertebrate community can be used. The Species At Risk index (SPEAR pesticides) was designed to determine pesticide effects where low values indicate pesticides-affected community. This index is determined by the log abundance proportion of macroinvertebrate taxa, which are sensitive to pesticides. The aim of our presented study was to evaluate the specificity of this index at 79 small agriculture streams (3 and 4 Strahler number with a catchment area of up to 145 km² and an elevation from 190 to 550 m a.s.l.) using data from 1997 to 2022 in the Czech Republic. The streams were selected regarding the diffused source of pollution (typical for agricultural land use with concurrent exclusion of point/municipal pollution), characterised by a high nitrate to total nitrogen ratio (greater than 0.95). The results of the study indicate that the value of the SPEAR index decreases significantly with higher proportion of arable land and increases significantly with the higher proportion of forest land. With higher proportion of arable land, the abundance of Ephemeroptera, Plecoptera and Trichoptera species (EPT), and value of index of food types (RETI index) also significantly decreases and simultaneously the abundance of Diptera species and Saprobic index significantly increases. Similarly, our results showed that streams with smaller catchment area and higher slope at higher elevation are more sensitive to the effect of pesticides compared to lowland streams with higher catchment area. Also, over time, the percentage ratio of individuals of sensitive groups of taxa in the sample (EPT) decreases, especially in catchments with a higher proportion of arable land. In the same way, the SPEAR index decreases (deteriorates) over time, most significantly in catchments with a lower representation of arable land. The study has been supported by the project of the Technology Agency of the Czech Republic SS02030027.

There are gaps in the study of species that live above the waterline (supralittoral) in terms of their identification, taxonomic status, and ecological and evolutionary studies. Many of these species are poorly studied and have issues with their naming. In the past, it was thought that the “Mediterranean” fauna in the Azov-Black Sea basin was made up only of Holocene immigrants and did not form an endemic component. However, modern molecular studies have shown that some species, highly adaptable to specific salinity conditions in Pontic region, represent an older complex and form genetic lineages that are isolated from the Mediterranean basin, sometimes even at the species level.

This study investigated some isopods from the supralittoral zone in the Black Sea basin. Morphological method and molecular DNA barcoding of COI gene region were used for the analysis. The obtained molecular data were registered in the public repository BOLD, where they were assigned a BIN (Barcode Index Number) based on genetic distance to other similar sequences in the database. The standard genetic distance for invertebrates is more than 2%, meaning that animals have different BINs.

The findings revealed the isopods Armadilloniscus ellipticus (BIN: BAAK0054), Tylos ponticus (novel unique for science BIN: AFC8363), and Ligia cf. italica (novel unique for science BIN: AFD2226). Tylos ponticus and Ligia cf. italica are unique Pontic BINs, showing the marine ecosystem biota of Ukraine are the sources of endemism of the biota of the Azov-Black Sea basin. The conservation status of Tylos ponticus and Ligia cf. italica, considering they appeared to be unique Pontic lineages, needs to be reassigned in Europe. Preliminary, areal of T. ponticus may be shortened now to only Pontic region, as originally it was described from there, and its local conservation status may be reassigned to the protected species. The conservation status of Ligia cf. italica in Europe is not expected to change despite a slight decrease in range without the Black Sea. However, the new Black Sea Ligia cf. italica now may be considered localised and vulnerable.

Microsporidia are a phylogenetically diverse group of ubiquitous endoparasites related to fungi, that infect hosts from all major metazoan taxa and in all environments. They are particularly frequent in aquatic ecosystems and commonly found in freshwater gammaroid amphipods. These parasites influence the host behaviour and physiology, including predation, thermal preference, and tolerance to pollutants as well as alter biological processes, such as the distortion of the sex ratio. Moreover, concomitant dispersion with hosts, and further spill over to local species, has been reported. The aim of our study was to detect, assess the prevalence and identify putative microsporidians infecting Gammarus species flock from ancient Lake Ohrid (North Macedonia). The small subunit rRNA gene (i.e., 16S) of microsporidians from gammarids collected in the lake and from surrounding watercourses were analysed to detect the parasites and investigate their taxonomy and diversity. This is the first survey of microsporidians infecting the endemic species flock in Lake Ohrid. The results show that more than 50% of studied gammarids are infected with the parasites. Microsporidians were found in all studied species (both coming from the endemic flock and local G. roeselii and G. balcanicus populations) and in the full depth range of the lake (down to 280 metres) as well as in surrounding springs. The species of microsporidians were identified through blast searches as Dictyocoela muelleri, D. roeselum, D. duebenum, Nosema sp., and Microsporidium sp. Both private haplotypes and putative endemic lineages were recovered. Their effect on the endemic communities has yet to be assessed.

Intermittent stream sections are globally more prevalent than permanent ones, and in the future, with ongoing climate change and increasing demand for water, it is expected that dryings will get more frequent and severe. In the framework of the DRYvER project (H2020 – 869226) we processed a series of quantitative aquatic macroinvertebrate samples taken from drying river networks (DRNs). Samples were taken on six occasions in 2021–2022 from six European DRNs (Croatia, Czechia, Finland, France, Hungary, Spain). On each DRN we had between 20 to 25 sampling sites which were distributed on stream sections to represent the proportion of permanent and intermittent sites across each river network. Multihabitat sampling was conducted at each sampling site from a selected reach of 50–150 m, depending on a maximum mean wetted width of the riverbed. Quantitative samples were taken with either Surber or Hess samplers but only one type of sampling device was used in each DRN. Sampled area varied according to stream width between 0.5–1.5 m² in case of flowing conditions or between 0.1–0.5 m² if only pools were present at a site. Macroinvertebrates were processed to the genus level, except for taxonomically difficult groups which were identified at the family or higher levels. From the 639 processed samples we identified nearly 1.4 million individuals which belonged to 321 genera of 141 families of 17 higher-level taxonomic groups. We provide an overview of the basic characteristics of the macroinvertebrate fauna of each DRN and the selected methodological problems that arose during the work, such as the problem of higher-than-genus taxonomic level identification, for which our solution could serve as an example for future ecological studies.

Polish Pomerania hosts a great abundance of spring areas, and the diversity of underground water outflows is huge. The springs can be found in the forests on the moraine slopes, in stream and river valleys, or in the vicinity of lakes. The study was conducted in the spring and autumn of 2022 and its aim was to determine the factors shaping caddisfly assemblages in the springs of Polish Pomerania. Three samples covering 500 cm² each were collected at every studied water outflow, using a small net. The total number of water outlets was 35, located in 9 different areas. All springs were assigned to one of the following categories: helocrene, heloreocrene, and reocrene. Some of them formed singular water outlets, while others formed spring-fed mires. We measured the amount of dissolved oxygen, conductivity, hardness, pH, the concentration of nitrogen and phosphorus (total, inorganic, and organic forms), and the amount of chlorophyll-a and calcium. The studied springs varied regarding water chemistry properties.

The following questions were tackled: What is the diversity of caddisflies in the forest springs of Polish Pomerania? Which environmental factors influence the composition of trichopteran communities? Are there any factors that could endanger more sensitive caddisfly species?

Fifteen species of Trichoptera in the larval stage were identified. Most of them belonged to the genus Sericostoma. Nonetheless, Chaetopteryx villosa and Crunoecia irrorata – the latter protected by the Polish law and serving as indicator of undisturbed springs – were also abundant. Additionally, certain species rarely found in Pomerania, such as Beraea pullata and Ernodes articularis were noted. For each species that occurred in sufficient numbers we created distribution models in relation to different environmental variables, to better understand the underlying factors eventually shaping caddisfly communities. Noteworthy, we found that the abundance of Crunoecia irrorata shows an inverse relationship with nitrate concentration, supporting the indicator value of this species as an early sign of anthropogenic pressure. Research on the impact of anthropogenic pressures on Trichoptera communities is crucial for environmental protection and water quality assessment, as caddisflies play a fundamental role in the functioning of aquatic ecosystems and are sensitive to anthropogenic disturbances, such as nutrient loading which often translates into a deterioration of water quality and ecological integrity.

Our study is one of the most comprehensive research on the topic in northern Poland, where data are extremely scarce. Moreover, our study is particularly timely as spring ecosystems are currently facing severe anthropogenic pressures and require urgent conservation actions. Overall, we contribute solid fundamental knowledge on the biodiversity and ecosystem functioning of the springs in the area, with implications for their conservation.

Dikerogammarus villosus (Sovinsky, 1894) is a Ponto-Caspian amphipod rapidly expanding in European fresh waters. Due to the high level of carnivory and voracity, this species poses a significant threat to biological diversity. In its invasive range, it originated from two genetically distinct sources: the Danube and the Dnieper deltas, spreading in Western (the Western Lineage) and Eastern Europe (the Eastern Lineage), respectively. These phylogenetic lineages exhibit phenotypic differences. We aimed to compare food consumption and preference (willow leaves, fish tissue and alive chironomid larvae, offered separately or together) between these lineages across five seasons. Each time, consumption was measured after 24 h of the experiment. In all seasons, amphipods preferred chironomid larvae. However, the amphipods from the Western Lineage consumed relatively less plant tissue than those from the Eastern Lineage. An especially high consumption rate was exhibited by large-sized amphipods from the Western Lineage. Thus, the amphipods from the Western Lineage consume more food and are more carnivorous, while their counterparts from the Eastern Lineage have a more flexible diet. As a consequence, the Western Lineage may cause a higher threat to macroinvertebrate communities, while the Eastern Lineage may be more successful in spreading in new habitats due to higher phenotypic plasticity. These differences can be explained by the different origins of both lineages as well as phenotypic disparities between them. Our study will help to predict the patterns and consequences of the further spread of invasive amphipods in Europe.

The production of clean energy to reduce greenhouse gas emissions, diversify energy supplies, and reduce the pressure on fossil fuels is among the 17 goals of the Sustainable Development strategy. Hydropower, being the most traditional and cost-effective source of clean energy, supplies about 70% of the world’s energy needs and is considered the market leader in clean energy. However, today, it is known that hydroelectric power plants, even the small ones (SHPs), cause river fragmentation and affect the quality of aquatic systems by altering water flow velocity and sediment transport capacity. These changes impair the state of aquatic habitats leading to negative consequences on biotic communities. Macroinvertebrate communities are pivotal in aquatic food webs, linking the trophic level of producers with higher trophic levels. By bringing together species (mainly insects) with different strategies, they also contribute to detritus breaking down and transferring matter and energy provided from several sources. The diversity of species and traits make them effective indicators of the state of aquatic systems. Since the macroinvertebrates are also easy to collect, they are commonly used in monitoring programs. While some studies focused on the response of macroinvertebrate communities’ structure to SHPs construction and operation, little is known about their functional responses. Our research focused on identifying the effects of 33 intake points of 17 SHPs from the Carpathian Mountains in Romania on both the structural and functional patterns in macroinvertebrate communities. To explain the spatial and temporal differences in macroinvertebrate communities, the values of distinct indices were compared and linked to the hydromorphological changes assessed for the upstream and downstream sections of intake points. Such results are valuable for informing managers who assist the decisions in the energy sector and fostering their dialogue and collaboration with water managers and researchers.

Almost a decade has passed since the entry of the Ponto-Caspian species monkey goby (Neogobius fluviatilis Pallas, 1814) into the Nemunas basin, but there is still very limited knowledge about the impact of this invasive species on the river ecosystem. Recently, there has been an increase in the abundance of this invasive fish, which may have a significant impact on invertebrate communities. The aim of our study was to determine the N. fluviatilis dietary spectrum and feeding preferences on benthic macroinvertebrates. This knowledge would allow us to predict which groups of benthic macroinvertebrates are most likely to be affected by this invasive predator in the newly invaded Nemunas basin.

Three sites with different biotopes were selected for the samples of gobies gut content analysis and benthic macroinvertebrate assemblages assessment – one in the Nemunas River, the other in its two major tributaries, the Neris and Nevėžis rivers. In total, 131 specimens of N. fluviatilis were taken for gut content analysis followed by benthic macroinvertebrate assemblages investigation at each sampling site.

The food spectrum of the monkey goby was broad. Analysis of the stomach contents showed that the diet of N. fluviatilis is dominated by the bivalve molluscs (Sphaeriidae), larvae of Chironomidae, mayflies (Ephemeridae, Baetidae) and net-spinning caddisflies (Hydropsychidae). Results also indicated a significant ontogenetic diet shift. Macroinvertebrate samples collected from the study sites showed that Gastropoda, Amphipoda, Chironomidae larvae, Oligochaeta and Hirudinea were dominant in the communities. According to Ivlev’s secretivity index, the monkey goby was found to have a strong preference for the Chironomidae, Sphaeriidae, Ephemeridae and caddisflies (Trichoptera) larvae (E>0.4). Meanwhile, it avoids (E<0.5) Gastropoda, Amphipoda, Isopoda, Zygoptera and Dreissena. Also, although there were quite abundant Dytiscidae, Corixidae, Nepidae, Hirudinea, Megaloptera and Oligochaeta in the river sites, they were not found at all in the stomachs of gobies. However, there was no significant abundance decrease of Chironomidae, Sphaeriidae, Ephemeridae and Trichoptera larvae in macroinvertebrates samples from these rivers in the last ten years, comparing pre- and post-invasion periods of N. fluviatilis in these rivers.

This study aimed to resolve the phylogenetic position of the crayfish leech Branchiobdella kozarovi, which has not been examined previously. We sampled B. kozarovi from crayfish obtained from 37 freshwater localities in Eastern Europe for this purpose. We also included data from previous research (41 further locations) to define the species’ distribution range, using QGIS. The external and internal morphology was studied using a stereomicroscope. For phylogenetic analysis, genomic DNA was isolated and the COI and 16S genetic markers were used to reveal evolutionary relationships between European species of branchiobdellidans. Maximum likelihood analysis clustered B. kozarovi as a sister branch to the clade of B. balcanica. This contrasts with previous research based on morphological features that has suggested that B. kozarovi is relatively close to B. pentadonta and B. italica. To visualise intraspecific evolutionary relationships and haplotype diversity we used PopART v.1.7 with the median-joining (MJ) network approach. The population parameters for possible population expansion were calculated using DNAsp. We detected two divergent mitochondrial haplogroups of B. kozarovi. The first occurs in eastern Ukraine and neighbouring territories, and the second was found in the South Bug and Dnieper Lyman in Ukraine. Our data reflect the distribution of haplogroups of the host crayfish, Pontastacus leptodactylus, rather than that of geological factors, aquaculture facilities or displacement from habitats by species more resistant to crayfish plague infection. Our study provides evidence that B. kozarovi is a long-lived ectosymbiont of P. leptodactylus and is specifically endemic to that host, being rarely reported from other crayfish hosts. Populations of P. leptodactylus are found widely in Eastern Europe, the Middle East, and number of European countries. Our research suggests that its endemic crayfish symbiont also occurs more widely than previously reported.

Our main goal was to investigate the historical molecular biogeography of the morphospecies Asellus aquaticus (L.) inhabiting the Periadriatic region, i.e., the areas surrounding the Adriatic Sea (the Apennine Peninsula, southern Alps, and their foreground, western Dinarides and Hellenides). Our working hypotheses tackled the three main stages of colonisation of inland waters by crustaceans. The early stage (since ca. 40 Ma), when the emergence of landmasses (above mentioned mountain ranges as well as islands and peninsulas, e.g., Apulia, Calabria) enabled colonisation of newly established inland habitats by the organisms of Tethyan and/or Paratethyan origin. The middle stage (ca. 6–5 Ma), when the massive desiccation of main water bodies during the Messinian Salinity Crisis (Lago Mare episode) promoted habitat shifts in organisms tolerant to oligohaline conditions, divergence by the habitat loss, as well as exchange of taxa between formerly isolated river systems. The late stage (ca. 2.5–0.016 Ma), when the fluctuations of sea level and salinity during the Pleistocene glaciations caused the regression of Adriatic Sea and, therefore, connection of previously separated hydrological networks within the one huge system of River Po. The phylogenetic analyses of the mitochondrial marker (COI) suggested that the oligohaline ancestor of A. aquaticus originated most probably in the Paratethys Sea, as the main centre of diversity within the genus is located in Asia and within the morphospecies – in the Pannonian Basin, respectively. In the Periadriatic region, the most widespread European lineage, i.e., the nominotypical subspecies A. aquaticus aquaticus, is present. Nevertheless, the area is inhabited by several distinct lineages forming putative cryptic species, e.g., the Apennine, Slovenian-karstic, Montenegrin and Ohridian one. Many of them are spatially more or less restricted, except the first mentioned, which is distributed from southern Alps to northern Apennine Peninsula, but was also found in Germany, France and in a single locality in Dalmatia. The molecular clock analysis suggested the Late Miocene divergence of the Apennine lineage from the Pannonian ancestor lineage, i.e., overlapping in time with the formation of the Apennine Peninsula. The Pleistocene lowering of sea level enabled the colonisation of Dalmatia via the Po River network. On the other side, the lineages inhabited the Ohrid and Skadar Lake systems originated from the Balkan ancestors in Pliocene/Pleistocene (ca. 3–2.5 Ma), which corresponds with the date of development of the two lake basins. Similarly, the estimated time of the beginning of local karstification in Slovenian Dinarides is in line with the divergence time of lineage inhabiting this unique habitat (4–5 Ma).

The introduction of a non-native species can cause particularly severe disruptions to community dynamics and food webs, primarily through direct predation and competition for habitat and food resources. Numerous studies have demonstrated the superiority of non-native predators over their native counterparts, potentially resulting in more significant negative impacts on local prey populations. Therefore, we aimed to estimate the predatory pressure on macrozoobenthos assemblages by investigating the diet and feeding preferences of the non-indigenous invasive round goby Neogobius melanostomus (Pallas 1814) and the native European bullhead Cottus gobio (Linnaeus, 1758) in the Ploučnice River (Elbe River tributary, North Bohemia, Czech Republic), where co-occurrence of both fish species has been observed. We investigated the diet composition and potential food niche overlap of these species to shed the light on their impact on the local macrozoobenthic communities. Moreover, by combining stomach content analysis with laboratory studies on feeding efficiency and gastric evacuation rates of Neogobius melanostomus, we aim to provide a more accurate estimate of the local predation pressure exerted by the invader on specific macrozoobenthic assemblages. The research presented is part of an ongoing investigation aimed at comprehensive monitoring of changes at the sites over the years, including the relationship between benthic macroinvertebrate communities and prey composition in areas where native and invasive species coexist.

In the 1960s, several species of peracarid crustaceans were intentionally transferred from the Dniper basin to Lithuania to improve fish-feeding conditions. Transported together with the larger-bodied Ponto-Caspian amphipods, the tiny Chaetogammarus warpachowskyi became the second most widespread alien amphipod in the country. However, its small size and specific habitats make its detection and proper sampling problematic, leading to poor knowledge of its ecology. Our aim, therefore, was to investigate the habitat of this enigmatic species. We studied the amphipod assemblage composition in the main mesohabitats of a lake and a river inhabited by abundant populations of C. warpachowskyi, and then tested its substrate choice in the laboratory. The field sampling was performed in the summer and autumn. We defined five main mesohabitats in the lake site: emergent vegetation/roots below knee-depth, emergent vegetation/roots above knee-depth, Dreissena beds with some Chara at knee-depth, Chara beds at waist-depth, and pebbles at ankle-depth. Four main mesohabitats were defined at the river site: emergent vegetation/roots on the flat bank, emergent vegetation/roots along the steep bank, submerged vegetation on the sandy flat bank, and logs and stones along the steep bank. In the lake, C. warpachowskyi was the most abundant in the Dreissena beds with some Chara at knee-depth, and in the river, most of it occurred in submerged vegetation on the sandy flat bank. In both study sites, it was the least abundant in emergent vegetation, which was occupied by the larger Ponto-Caspian amphipod Pontogammarus robustoides. The substrate choice was tested in square-shaped aquaria with a petri dish in each corner, each filled with a different substrate: submerged vegetation, plant detritus, sand, or pebbles. The distribution of 50 initial specimens was evaluated after 24 hours. Most specimens chose submerged vegetation. We repeated the experiments after adding 10 specimens of P. robustoides and slight changes could be observed – without changing its main preference for submerged vegetation, C. warpachowskyi then favoured the sand a little more at the expense of plant detritus and pebbles. Our results confirmed the clinger lifestyle of C. warpachowskyi previously inferred from morphology. As such, this alien amphipod introduced a novel amphipod functional role, making ecological consequences more likely. We also acknowledge that the presence of a larger competitor may adjust its distribution across different habitats. The study was funded by the Research Council of Lithuania, Project No. S-SV-23-192.

Mayflies (Order Ephemeroptera) are amphibious insects with aquatic larvae and short-living terrestrial adults. They are characterised by higher species diversity in lotic habitats, especially the upper reaches of fast flowing streams and rivers and highland rivers. The analysed dataset in this paper included 44 sites of hilly and mountainous small to medium sized streams with predominantly hard bottom substrate (gravel, stones and rocks) classified in the three water body types (WBT) according to the Serbian national legislation: WBT3 – altitude up to 500 m (24 sites); WBT4 – altitude above 500 m (6 sites) and WBT6 – small streams outside the area of the Pannonian Plain that do not fall under WBT3 and 4 and are not covered by the regulation on the establishment of surface and groundwater bodies (14 sites). Samples were collected in spring and fall 2019 using the kick and sweep sampling method from all microhabitat types according to European standards (EN 27828:1994) with an FBA hand net (mesh size 500 μm). The biological material was pooled and transferred to sample containers and preserved with 70% ethanol. Data analyses were performed using ASTERICS 4.04 software package and FLORA software version 2013.

Insects were the most dominant group of the community, recorded at the studied sites corresponding to the communities typical for the selected watercourses. Mayflies were recorded at all sampling sites as a significant component of the community. They were represented by 8 families and 30 taxa. The family Baetidae was presented with the largest number of species (12) and the species Baetis rhodani (Pictet, 1843) was the representative of the Ephemeroptera group with a significant abundance in all three groups of studied sites. The species Ecdyonurus (Helvetoaeticus) subalpinus Klapálek, 1907 was only found in WBT4, but it was the most abundant species of the Ephemeroptera group, along with Baetis (Baetis) alpinus (Pictet, 1843).

Alpha diversity was assessed within the Ephemeroptera community in relation to WBT using Shannon entropy, Species richness and Equitability. The highest values of Shannon entropy and Species richness showed WBT4, while communities in WBT3 and WBT6 showed lower values. Slightly higher Equitability was observed in WBT4 than in the other two groups. Total beta diversity, and its components, were analysed using the procedures described by Baselga. Beta diversity was similar in all three groups. The Ephemeroptera communities in WBT4 showed the lowest value for nestedness with the highest species turnover. Beta diversity was slightly higher in WBT3, but also the nestedness component was slightly higher. Total beta diversity was slightly lower at altitudes over 500 m. Lower values of alfa and higher values of beta diversity in WBT3 leads to the conclusion that these communities were under various pressures that cause species loss (higher nestedness) which can be explained through the position of these sites in the vicinity of human activities.

Glacial lakes of the Tatra Mountains (W Carpathians, Slovakia) are unique but very vulnerable ecosystems with specific and relatively isolated invertebrate communities that are challenging to access for sampling. DNA metabarcoding can benefit the assessments of their biodiversity and ecological status by providing high-quality data and minimising the lengthy manual sorting and morphological identification of specimens. Processing the fixation medium (usually ethanol), instead of the valuable invertebrate samples themselves, offers enhancements through simple protocols that allow subsequent focus on specific organisms detected among the preserved specimens. 

In this study, we present proof of the concept by analysing freshwater invertebrate samples collected from 15 Tatra lakes in September 2020. Subsamples of their fixation medium were passed through filters with 0.45 μm pores and further processed by salt-extraction of the captured DNA. Mitochondrial COI marker was amplified using the BF3/BR2 primers and the resulting 180 libraries were sequenced on the Illumina MiSeq platform. We obtained a total of 15,037,762 pairs of demultiplexed reads, eventually clustered into 827 invertebrate OTUs (operational taxonomic units) with 406 species identified. Promising targets for further taxonomic or population studies were detected, including unique genetic lineages without any record in the reference barcode database. 

We considered fixative subsamples from three different intervals of benthos fixation, in order to analyse the impact of fixation time on the metabarcoding outcomes. Utilising generalised linear models, we identified a two-week period of samples fixation time as more appropriate than a day or month of fixation. Additionally, we confirmed the previous suggestions about the role of body size and sclerotisation for fixative DNA, finding that large freshwater species were captured more efficiently than meiofauna or terrestrial taxa. These effects emerged from the detailed presence/absence data rather than from coarse per-sample records of taxonomic richness, which highlights that metabarcoding studies and optimisation efforts can take advantage of such robust metrics to investigate even subtle trends.

Moreover, the fixative DNA metabarcoding was able to reflect the lake-specific environmental conditions. The dissimilarities in community structure clearly distinguished the forest lakes from (sub)alpine lakes, which corresponds with the accumulation of nutrients in the lower zone. This distinction remained even after combining the metabarcoding datasets from various sampling strategies (lake water, sediment, several approaches to processing the benthic samples and their fixative). The comparison revealed an even broader range of taxa, as well as high consistency of fixative with benthic samples. Supported by the project VEGA 2/0084/21.

The faunistic and taxonomic research of mayflies in Kosovo is still in progress. The mayfly fauna of the Karadak Mountains in Kosovo was poorly investigated. To overcome this gap, we selected nine sampling sites that represent the major streams and ecological features of the Karadak Mountains running waters. Kosovo is separated into two ecoregions, Dinaric Western Balkan (ER5) and Hellenic (ER6). The majority of Karadak Mountain is in the Hellenic Ecoregion (ER6), and the freshwater habitats in this area are part of the Black Sea and Aegean Sea Basins.

These nine sites were sampled in the Karadak Mountains from June to September 2017. During our investigation, we discovered 19 species belonging to 10 genera and 6 families. The most diversified families were Heptageniidae (2 genera and 9 species) and Baetidae (1 genera and 4 species). The following families are, for the moment, only represented by a single species: Potamanthidae and Leptophlebiidae. The most widely distributed species of mayflies in Karadak mountains were Baetis rhodani (Pictet, 1843), Baetis alpinus (Pictet, 1843), Ecdyonurus vitoshensis (Jacob & Brasch, 1984), Ecdyonurus submontanus, (Landa, 1969), Ecdyonurus starmachi (Sowa, 1971), Ecdyonurus macani (Thomas & Sowa, 1970), and Rhithrogena braaschi Jacob, 1974) Rhithrogena gr. semicolorata (Curtis, 1834), Ephemera cf. parnassiana (Linneus, 1758), Serratella ignita (Poda, 1761), and Quatica ikonomovi (Puthz, 1971).

Finally, we want to define each species’ ecological preferences and level of vulnerability in the Karadak mountain region to provide an important tool for running water management and river quality assessments. This research adds to our understanding of the distribution of mayflies (Ephemeroptera) in Kosovo’s Karadak mountains.

Invasive species of macrozoobenthos may have a significant negative impact on aquatic ecosystems as competitors of the original representatives of the fauna, that in extreme cases can completely replace them. The reason may be the absence of natural enemies to keep them under control, strong food competition or the introduction of pathogens. They can also cause ecological and economic damage. During the regular monitoring carried out for the needs of the WFD, we found such species in the main tributaries of the Elbe River in the Czech region – Ohře, Bílina, and Ploučnice, which are managed by the Povodí Ohře, s.p.

According to previous findings and information found in the literature, we provided in 2021 an oriented survey of the occurrence of invasive species of macrozoobenthos in the lower reach of Ohře River, from the mouth to Elbe River in Litoměřice to Nechranice reservoir.