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The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics., Trends in Genetics, 39 (7), ISSN:0168-9525

The essential key to routine molecular species identification (DNA barcoding/metabarcoding) is the existence of an error-free DNA barcode reference library providing full coverage of all species. Published studies generally state the need to produce more barcodes, and control their quality, but unfortunately, the number of barcoded species is still low. However, to initiate real progress, we need to know where the gaps lie, how big they are and why they persist. Our aims were to draw and understand the current state of knowledge regarding species diversity, distribution, and barcode coverage, and offer solutions for improvement. In this study, we used two groups of aquatic insects, beetles and true bugs. We have compiled and critically evaluated an essentially complete and up-to-date European list, containing 1527 species. The list served as a basis for the barcode gap analyses in the Barcode-of-Life-Data-System (BOLD) conducted in three subsequent years (2020-2022). The overall barcode coverage of the pan-European fauna was around 50 % in both groups. The lowest coverage was in the Mediterranean, the Balkans and South-eastern Europe. The coverage in each country depended significantly on the local diversity, the number of rare, endemic species and the similarity of its fauna to that of the most active barcoding European countries. Gap analyses showed a very small increase in species coverage (1 % in European aquatic beetles) despite an ~25 % increase in the number of barcodes. Hence, it is clear that future barcoding campaigns must prioritise quality over quantity. To visibly improve reference libraries, we need to increase the involvement of taxonomic experts and focus on targeted studies and underexplored but biodiversity-rich areas.

Ancyronyx lianlabangorumsp. nov. (Coleoptera, Elmidae), a new spider riffle beetle from the Kelabit Highlands (Sarawak, northern Borneo), is described. Illustrations of the habitus and diagnostic characters of the new species and the similar, polymorphic A. pulcherrimus Kodada et al. are presented. Differences to closely related species, based on COI nucleotide sequences and morphological characters, are discussed. Ancyronyx pulcherrimus is here recorded from Sarawak for the first time, based on DNA barcoding.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics., Trends in Ecology & Evolution, 37 (3), ISSN:0169-5347, ISSN:1872-8383

Ancyronyx clisterisp. nov. (Coleoptera, Elmidae) a new spider riffle beetle discovered from northern Borneo (Brunei; Sabah and Sarawak, Malaysia) and the larva of Ancyronyx sarawacensis Jäch are described. Illustrations of the habitus and diagnostic characters of the new species and the similar and highly variable A. sarawacensis are presented. Differences to closely related species, based on DNA barcodes and morphological characters, are discussed. Association of the larva and the imago of A. sarawacensis, and the occurrence of Ancyronyx procerus Jäch in Peninsular Malaysia and Sabah are confirmed by using COI mtDNA sequences.

The riffle beetle genus Hexanchorus Sharp is, with 25 known species, the most speciose genus of the subfamily Larainae in the Neotropics and the second largest globally. An analysis of its phylogeny, based on two mitochondrial (COI, 16S) and two nuclear (18S, 28S) markers, including Hexanchorus-like, but morphologically distinct specimens, supported presence of an unknown genus. The new genus, Rumilaragen. nov., is described here with four new species (R. obscurasp. nov., R. paternasp. nov., R. riberaisp. nov., R. suppressasp. nov.) and their larvae. The separate position of the new genus is, beside molecular differences, well substantiated by the morphology of adults (the absence of sexual dimorphism, structure of the pronotum) and larvae (pleurites never reaching to sixth abdominal ventrite). H. sagittatusstat. nov. is elevated to species rank based on molecular data, and Rumilara leleupi (Delève) comb. nov. is transferred from the genus Hexanchorus and redescribed, based on its morphology.

European mountains are important areas regarding biodiversity of the continent, and they also harbour diverse freshwater fauna, which is critically endangered in terms of the current species extinction. However, sufficient knowledge of this valuable part of European biota is no longer possible without molecular data. This study focuses on the genetic diversity and distribution patterns of the classical representative of the mountain freshwater fauna, caddisfly Rhyacophila tristis, in the Western Carpathians. Based on the COI mitochondrial marker, two genetic lineages (separate BINs) were identified. BIN_W was found in 16 localities in the western part of the study area, BIN_E in 44 eastern localities. The data obtained indicate that BIN_W occurs in a significantly narrower altitude range, BIN_E is more closely related to the Balkan populations than to co-occurring BIN_W, and that the contact zone of the lineages passes through the Western Carpathians. The study revealed phylogeographic and demographic differences between lineages, supporting hypothesis of their evolutionary independence and specific ecological preferences. The obtained genetic data shifted our knowledge on the populations of the studied caddisfly and suggested patterns that could be common to other freshwater species. This could help us to protect unique freshwater ecosystems and preserve European biodiversity.

The Carpathians are one of the key biodiversity hotspots in Europe. The mountain chain uplifted during Alpine orogenesis and is characterised by a complex geological history. Its current biodiversity was highly influenced by Pleistocene glaciations. The goal of the current study was to examine the phylogenetic and demographic history of Gammarus balcanicus species complex in the Carpathians using multiple markers as well as to delimit, using an integrative approach, and describe new species hidden so far under the name G. balcanicus. Results shown that divergence of the studied lineages reaches back to the Miocene, which supports the hypothesis of their survival in multiple micro refugia. Moreover, the increase of their diversification rate in the Pleistocene suggests that glaciation was the driving force of their speciation. The climatic changes during and after the Pleistocene also played a major role in the demography of the local Carpathian lineages. Comparison of diversity patterns and phylogenetic relationships of both, the mitochondrial and nuclear markers, provide evidence of putative hybridisation and retention of ancient polymorphism (i.e., incomplete lineage sorting). The morphological examination supported the existence of two morphological types; one we describe as G. stasiuki sp. nov. and another we redescribe as G. tatrensis (S. Karaman, 1931).

The riffle beetle genus Hexanchorus Sharp, 1882 is distributed from Mexico to Argentina, forming an important component of the freshwater invertebrate fauna of Latin America. With 21 described species, Hexanchorus represents one of the most speciose Larainae genera, but its real diversity is likely much higher. We analysed material from a relatively small area in Ecuador, resulting in the first record of H.cordillierae for Ecuador and discovery of four new species and one subspecies: Hexanchorusvirilissp. n., Hexanchorusrostratussp. n., Hexanchorusshepardisp. n., Hexanchorusonoreisp. n. and Hexanchorusonoreisagittatusssp. n. For delimiting and characterizing species, both morphological and molecular (mtCOI DNA barcodes) data were used. A distribution map of Hexanchorus species is provided based on published records.

We describe here a new species in the genus Disersus Sharp, 1882 from the Otongachi Reserve in Ecuador. Disersus otongachi sp.nov. is externally similar to other representatives of the genus, however, this species can be clearly distinguished for significantly longer antennae and the unique shape of the male genitalia.

The genus Onychelmis Hinton, 1941 was for a long time regarded as a small taxon with only three known species distributed in the Andes. A study of new material from Ecuador, using morphological and molecular data, has resulted in the discovery of five new species: Onychelmis lenkae sp. nov., O. lobata sp. nov., O. minor sp. nov., O. onorei sp. nov. and O. splendida sp. nov. We also revised the entire genus and redescribed the three known species, O. longicollis (Sharp, 1882), O. leleupi Delève, 1968 and O. whiteheadi Spangler & Santiago, 1991. Habitus photographs of adults are provided, together with line drawings of male and female genitalia, and schematic illustrations of the distribution of femoral tomentum for each species. DNA sequences for barcoding the COI mtDNA fragment were used to support species delimitation and to suggest possible relationships among species. The revision includes a key to adults of all species of Onychelmis and notes on the biogeography of the genus, with an updated distribution map.

Essential for implementation of molecular tools in bioassessments are comprehensive databases with high-quality reference DNA sequence data. A recent survey arising from the DNAqua-Net EU COST Action WG1 has revealed significant gaps in the barcode reference data of aquatic biota both on a taxonomic and a geographical level (Weigand 2019). Although reference barcode libraries of all main freshwater groups are continuously improved through a number of national DNA-barcoding campaigns, still little data is available from Central and Eastern Europe. In Slovakia, the barcode coverage of the aquatic biota was very low when DNAqua-Net started. At the end of 2016 only 165 public records (60 OTUs) were available in BOLD (< 3 % of aquatic macroinvertebrates reported from Slovakia), and most of them were mined from GenBank. This led us to launch the campaign “Barcoding aquatic biota of Slovakia” (www.AquaBOL.SK), aiming to improve this unpleasant state. Since many common species in Slovakia are covered by barcodes from other countries, initially we focused on least explored habitat types within Europe - alpine glacial lakes in Tatra Mts and karstic springs. Both are located in Western and Slovak part of Eastern Carpathians, which are considered one of the most valuable biodiversity centers in Europe. More than 2700 sequences of COI-5P (337 BINs), originating in Slovakia, are currently in our dataset. Mostly they represent aquatic insects (86 %) and crustaceans (12 %), followed by minor taxonomic groups. From insects, the best covered orders are Coleoptera (43 % of sequences, 21% of species), followed by Trichoptera (22 % of sequences, 24 % of species) and Ephemeroptera (18 % of sequences, 33 % of species). And even if other groups (Hemiptera, Plecoptera) represent only around 5 % of all sequences each, their species coverage is already 21 % or 28 %, respectively. Relatively high number of unique BINs (35 out of 337) from our area, and separate intraspecific lineages within BINs of many widespread species, supports the premise that the Western Carpathians belong to the most important biodiversity hotspots. Our local reference database, continuously updated, is useful for the analysis and interpretation of metabarcoding data - a prerequisite for effective and nondestructive monitoring of aquatic biotopes including vulnerable, threatened, and protected habitats. It is also beneficial for a number of other purposes such as taxonomic revisions and phylogenetic analyses. This contribution was partially supported by VEGA2/0030/17, VEGA2/0084/2021 and COST CA15219.

As global biodiversity declines, there is an increasing need to create an educated and engaged society. Having people of all ages participate in measuring biodiversity where they live helps to create awareness. Recently, the use of environmental DNA (eDNA) for biodiversity surveys has gained momentum. Here, we explore whether sampling eDNA and sequencing it can be used as a means of rapidly surveying urban biodiversity for educational purposes. We sampled 2 × 1 L of water from each of 15 locations in the city of Trondheim, Norway, including a variety of freshwater, marine, and brackish habitats. DNA was extracted, amplified in triplicate targeting the barcoding fragment of COI gene, and sequenced. The obtained data were analyzed on the novel mBRAVE platform, an online open-access software and computing resource. The water samples were collected in 2 days by two people, and the laboratory analysis was completed in 5 days by one person. Overall, we detected the presence of 506 BINs identified as belonging to 435 taxa, representing at least 265 putative species. On average, only 5.4% of the taxa were shared among six replicates per site. Based on the observed diversity, three distinct clusters were detected and related to the geographic distribution of sites. There were some taxa shared between the habitats, with a substantial presence of terrestrial biota. Here we propose a new form of BioBlitz, where with noninvasive sampling effort combined with swift processing and straightforward online analyses, hundreds of species can be detected. Thus, using eDNA analysis of water is useful for rapid biodiversity surveys and valuable for educational purposes. We show that rapid eDNA surveys, combined with openly available services and software, can be used as an educational tool to raise awareness about the importance of biodiversity. Fil: Hupalo, Kamil. Universitat Essen; Alemania Fil: Majaneva, Markus. Norwegian Institute For Nature Research.; Noruega Fil: Czachur, Molly Victoria. STELLENBOSCH UNIVERSITY (SUN); Fil: Sire, Lucas. Universite de Tours; Francia Fil: Marquina, Daniel. Stockholms Universitet; Suecia Fil: Lijtmaer, Dario Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina Fil: Ivanov, Vladislav. University of Oulu; Finlandia Fil: Leidenberger, Sonja. University of Skövde; Suecia Fil: Čiampor, Fedor Jr. Slovak Academy of Sciences; Eslovaquia Fil: Čiamporová-Zaťovičová, Zuzana. Slovak Academy of Sciences; Eslovaquia Fil: Mendes, Izabela S.. Universidade Federal de Minas Gerais; Brasil Fil: Desiderato, Andrea. Universidade Federal do Paraná; Brasil Fil: Topstad, Lasse. Norwegian University of Science and Technology; Noruega Fil: Meganck, Kenny. Royal Museum for Central Africa; Bélgica Fil: Hariz Z. A., Danial. Universiti Sains Malaysia; Malasia Fil: Kjærstad, Gaute. Norwegian University of Science and Technology; Noruega Fil: Lin, Xiao Long. Nankai University; China Fil: Price, Benjamin. Natural History Museum; Reino Unido Fil: Stevens, Mark. South Australian Museum; Australia. University of South Australia; Australia Fil: Ekrem, Torbjørn. Norwegian University of Science and Technology; Noruega Fil: Deiner, Kristy. Natural History Museum; Reino Unido

State of ecosystems and biodiversity protection are becoming the key interests for modern society due to climate change and negative human impacts (Leese 2018). Environmental changes in freshwaters are indicated also by benthic communities, especially in sensitive ecosystems like alpine lakes (Fjellheim 2009). Moreover, remoteness and isolation of alpine lakes make them a source of biodiversity, which is worth conserving (Hamerlík 2014). A promising tool for efficient large-scale monitoring of aquatic communities is DNA metabarcoding (Leese 2018). In this study, we applied metabarcoding to analyse macrozoobenthos of 12 lakes in the Tatra Mountains, using benthic bulk samples and eDNA filtered from water (Fig. 1). In compliance with recent publications, eDNA amplified with BF3/BR2 primers resulted in high percentage of non-invertebrate reads (Leese 2021). Based on in silico tests with the obtained sequences, we confirm that the recently developed EPTDr2n primer enables minimizing non-target amplification even with eDNA filtered from alpine-lake water (Elbrecht and Leese 2017). This ability is facilitated by 3’ end of the primer and we observed the two important mismatches in non-target sequences from our study (Leese 2021). Thus, our future analyses of eDNA (and bulk-sample fixative) will benefit from the new primer. Concerning bulk samples, a wide range of invertebrate taxa was assigned to the OTUs and they showed good congruence with previous studies using morphological determination (e.g. Krno 2006). Certain differences with (and among) the previous records per lake were observed, which could suggest ecological changes, but at the moment the influence of sampling error cannot be excluded. In eDNA, several taxa were congruent with the previous records, but their amount and read abundance was considerably lower due to non-target amplification. Apart from that, filling gaps in barcoding databases remains one of our priorities, as identification to species or genus level was not yet possible for some invertebrate OTUs. In addition, we subjected the NGS data to denoising and abundance-filtering in order to explore haplotype-level diversity (Andújar 2021). Although more comprehensive conclusions will be possible only after obtaining data from more lakes and years, already the two metabarcoding experiments presented here enabled us to efficiently detect within-species genetic diversity and identify a large variety of taxa, including groups that would otherwise be omitted or very challenging to identify. This underlines the potential of DNA methods to provide valuable ecological and biodiversity data across the tree of life for modern biomonitoring. This study was realized with support from VEGA 2/0030/17 and VEGA 2/0084/21.

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data, (undefined)

Mountain lakes, typical signs of an alpine landscape all around the world, were formed in the western part of the Carpathian Arch about 10,000-15,000 years ago, as remnants of the last continental glaciation. In the Tatra Mountains (Slovak-Polish border), more than 110 permanent lakes of glacial origin and many other small ponds exist. These (sub)alpine lakes, with their specific conditions, are very vulnerable and any change in their environment can largely impact their biodiversity, which is, despite long-term faunistic and ecological research, still not fully recognized. This shortcoming could be strongly improved by molecular approaches, which, even within population-genetic studies, often reveal unsuspected cryptic lineages or potential new species. Our long-term research is aimed at revealing the genetic diversity of aquatic macroinvertebrate species in the alpine environment of the Western Carpathians. One of the currently studied species is the glacial relict flatworm taxon Crenobia alpina (Dana, 1766) (Tricladida: Planariidae), representing an element of the permanent aquatic fauna. Totally, 88 COI haplotypes of C. alpina forming at least five well-separated genetic lineages were identified within 348 individuals collected from 45 lakes and ponds in theTatra Mts. Based on tests of selective neutrality and population stability, C. alpina populations in the Tatra lakes did not overcome recent population expansions or contractions. It seems that great genetic differences between localities and their population stability could be a consequence of the presence of natural topographic barriers (ridges, mountain peaks) dividing the mountains into small and relatively isolated valleys. Moreover, other important factors that could play a significant role are the limited dispersal ability of the species as well as its ability to reproduce asexually. Our findings were also compared with all publicly available sequences (Barcoding of Life Datasystems - BOLD and GenBank) in order to shift our data from a local to a holistic view on C. alpina. Our resultssupport the assumption of Brändle et al. 2017 that C. alpina represents a complex of cryptic lineages or species in Europe. As part of the AquaBOL.sk initiative, the data obtained contribute to the knowledge of genetic variability and barcoding of aquatic organisms in Slovakia.

BackgroundAs global biodiversity declines, there’s an increasing need to create an educated and engaged society. Having people from all ages participate in measuring biodiversity where they live helps to create awareness. Recently, the use of environmental DNA (eDNA) for biodiversity surveys has gained momentum. Here, we test whether sampling eDNA and metabarcoding can be used for rapid urban biodiversity surveys for educational purposes.Materials & MethodsWe sampled 2×1 L of water from each of 15 locations in the city of Trondheim, Norway, including a variety of freshwater, marine and brackish habitats. DNA was extracted, amplified in triplicate for the COI gene and sequenced. The obtained data were analysed on the novel mBRAVE platform, an online open access software and computing resource.ResultsThe water samples were collected in two days by two people and the lab analysis was completed in five days by one person. Overall, we detected the presence of 501 taxa identified as belonging to 435 species, representing 90 orders and 18 phyla. On average, only 5.4% of the taxa were shared among six replicates per site. Based on the observed diversity, three distinct clusters were detected and related to geographic distribution of sites. There were some taxa shared between the habitats, with a substantial presence of terrestrial biota.DiscussionOur results match expected patterns of biodiversity in the landscape and show that with minimal sampling effort, hundreds of species can be detected. Thus, using eDNA analysis of water is promising for rapid biodiversity surveys, and it is likely that more detailed results could be obtained by optimising field and lab methods for particular groups of interest. We recommend that rapid eDNA surveys, with openly available services and softwares, can be used to raise awareness in the importance of biodiversity.

A new minute riffle beetle genus Ictelmis gen. nov. and the type species Ictelmis martae sp. nov. are described from Ecuador. The description is supported by characteristic morphological features and DNA barcoding data, drawings and habitus photographs are provided. Based on molecular data and morphology it is suggested that Ictelmis is closely related with Onychelmis Hinton, and Notelmis Hinton.

Lymphocytic choriomeningitis virus (LCMV) can establish in its host a persistent infection, without any prominent symptoms. Even during this infection, when the infectious virions are not released, the virus still disseminates effectively. A very effective and fast way of infection of neighboring cells utilized by many viruses is cell-to-cell transmission. Viruses use different ways of cell-to-cell spread through the extracellular space or by intracellular means through different protrusions. We have found that LCMV strain MX may use three different types of cell-to-cell transport. Firstly, similar to vaccinia virus, it can use actin to propel the virus towards the neighboring cell. Secondly, virus can travel through the intracellular space inside the tunneling nanotubes, that connect the cells even at longer distances and thirdly, the virus may travel on the surface of the membrane of different protrusions connecting two cells. We have also proved that the cells infected by MX strain of LCMV migrate faster than the uninfected cells or cells infected with a different LCMV strain. In accordance with faster migration, the infected cells form more lamellipodia with high expression of keratin 1. In this work, we have introduced three types of cell-to-cell transmission utilized by strain MX of LCMV and showed that even if the cells are not in tight connection, the virus forces them to migrate faster to join the nearest cell. As we show in this work, the virus may use more than one strategy to move to another cell, while each strategy can substitute another. These ways of transmission are very fast and effective and may have a serious impact on the host. Moreover, targeting the cell-to-cell spread, by inhibiting for instance GTPase dynamin, could be an effective way of virus elimination. Keywords: lymphocytic choriomeningitis virus; transmission; migration; keratin 1; nucleoprotein.

Diving beetles (Dytiscidae) are the most speciose water beetle group, occurring on all continents except Antarctica. They inhabit various lotic and lentic habitats and play an important role in ecosystem functioning. In this study, we developed functional polymorphic microsatellites for two widely distributed species from one of the most diversified dytiscid genus Agabus : Agabus bipustulatus (Linnaeus, 1767) and Agabus cf. guttatus from the Agabus guttatus group sensu Foster and Bilton (1997). For microsatellites development, pooled DNA and NGS pyrosequencing were used. Microsatellites are still very useful genetic markers for studying recent population changes, but for dytiscids, highly diversified and ecologically important group of freshwater invertebrates, only 8 microsatellite loci are available for one North American species. To test utility of the developed markers, we used several populations of alpine lakes, situated in the Tatra Mountains (Western Carpathians). From the 60 tested markers, 13 loci for Agabus bipustulatus and 8 loci for A. cf . guttatus showed polymorphism. The number of alleles per locus varied from 2 to 10 and no significant linkage disequilibrium was observed. Observed/expected heterozygosity varied from 0/0.077 to 1/0.834 within populations of A. cf. guttatus and from 0/0.056 to 1/0.837 within populations of A. bipustulatus . The significant deviation from HWE was probably caused by presence of null alleles or undetected biological processes. Bayesian cluster analysis revealed differences in the cluster proportions, confirming applicability of the developed markers for future studies of population structure of both Agabus species.

Alpine lakes and ponds are unique and vulnerable aquatic habitats inhabited by specific species assemblages. In contrast to alpine streams, the genetic diversity and population structure of the organisms found in alpine standing waters are almost completely unknown. Here we present a population genetic study of a macroinvertebrate species from alpine lakes, the diving beetle Agabus bipustulatus (Linnaeus, 1767). We used partial cytochrome b (Cyt b) mtDNA gene of 560 specimens from 43 lakes and ponds distributed across 20 mountain valleys of the Tatra Mountains (Western Carpathians) and their foothills. The aims of this study were to provide detailed genetic metapopulation analysis of A. bipustulatus within a geographically small mountain system, to explore the role of ridges in isolating lake populations, and to test for the applicability of Cyt b in insect population genetic studies. We detected high genetic variability (42 haplotypes), comparable to many large spatial scale studies. There was a ...

New taxonomic data on mud-loving beetles are provided based on morphological characters and DNA barcoding. Heterocerus heydeni Kuwert, 1890 was previously considered a junior synonym of H. flexuosus Stephens, 1828, but we support the validity of the species and restore the name. H. heydeni is redescribed, based on material from Central Asia and European part of Russia. Specimens of H. hauseri Kuwert, 1893 were also studied, suggesting it as a possible junior synonym of H. heydeni. We provide new DNA barcodes for H. flexuosus and Augyles cf. flavidus and comment on Heterocerus barcode data published in Barcode Of Life Data Systems (BOLD).

The putative female, nymph and egg of Enderleina preclara are described from specimens collected in the Kukenán-tepui foothills and Acopán-tepui foothills and plateau of Venezuela. Morphological and molecular approaches were employed to characterize these stages. Diagnostic keys for males and females of the described Enderleina species are also presented. The nymph is also morphologically compared with those of other Enderleina species. Comments on the ecology of this species and erroneous information regarding the putative type locality of E. preclara are provided.

The Danube River was originally inhabited by six native sturgeon species, but currently, the sterlet (Acipenser ruthenus L.) is the only native sturgeon species still occupying the Slovak–Hungarian stretch of the Middle Danube. All sturgeon species are facing extinction, suffering from overfishing, water pollution, illegal fishing, poaching or other negative impacts. Urgent and proper actions are needed to prevent their extinction, and evaluating its genetic diversity is one of the essential tools of conservation programs. Since the management actions are primarily local in nature, we first focused on comparing and analysing local sources of fish for population recovery and natural (wild) population in the adjacent stretch of the Danube River. We used 2 fragments of mitochondrial DNA and 12 microsatellites to analyse the genotype of the three groups of sterlets,i.e.wild, broodstock and stocked individuals from Slovak part of the Danube. Mitochondrial markers of all groups were diversified similarly to populations from other parts of the Danube. This confirmed that broodstock and stocked fish belong to the original Danube population. Microsatellites revealed very similar patterns among groups compared, but we detected possible negative trends reflected in losing polymorphism in a few loci in broodstock and stocked individuals.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring., This paper is a deliverable of the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219) Working Group 1, led by Torbjørn Ekrem and Fedor Čiampor. Thanks to the University of Minho and University of Pécs for hosting workshops and working group meetings. We also thank staff at National Environment Agencies and others that provided national checklists of taxa used in biomonitoring, and otherwise assisted with checklist proof-reading: Jarmila Makovinská and Emília Mišíková Elexová (Slovakia); Steinar Sandøy and Dag Rosland (Norway); Mišel Jelič (Croatia); Marlen Vasquez (Cyprus); Adam Petrusek (Czech Republic); Kristel Panksep (Estonia); Panagiotis Kaspiditis (Greece); Matteo Montagna (Italy); Marija Katarzyte (Lithuania); Ana Rotter (Slovenia); Rosa Trabajo (Spain); Florian Altermatt (Switzerland); Kristian Meissner (Finland), Rigers Bakiu (Albania), Valentina Stamenkovic and Jelena Hinic (Macedonia); Patricia Mergen (Belgium); Gael Denys & the French Biodiversity Agency (France); Mary Kelly-Quinn (Ireland); Piotr Panek and Andrzej Zawal (Poland); Cesare Mario Puzzi (Italy); Carole Fitzpatrick (United Kingdom); Simon Vitecek (Austria); Ana Filipa Filipe (Portugal); Peter Anton Stæhr & Anne Winding (Denmark); Michael Monaghan (Germany); Alain Dohet, Lionel L'Hoste, Nora Welschbillig & Luc Ector (Luxembourg), Lujza Keresztes, (Romania). The authors also want to thank Dirk Steinke for providing the original European ERMS list for marine taxa and Florian Malard for comments on the manuscript. The preparation of the AMBI checklist was carried out in the scope of a Short-term Scientific Mission (ECOST-STSM-CA15219-150217- 082111) granted to SD visiting AZTI, Spain. ZC was supported by grants EFOP-3.6.1.-16-2016-00004 and 20765-3/2018/FEKUTSTRAT. TE was supported by the NorBOL-grant (226134/F50) from the Research Coun cil of Norway. BR, FL and MFG contributed through support from the GBOL project, which is generously funded by the German Federal Min istry of Education and Research (FKZ 01LI1101 and 01LI1501). MG contributed through support of the Polish National Science Centre, grants N N303 5794 39 and 2014/15/B/NZ8/00266. SF was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER).

Innovative nanotechnology aims to develop particles that are small, monodisperse, smart, and do not cause unintentional side effects. Uniform magnetic Fe3O4 nanoparticles (12 nm in size) were prepared by thermal decomposition of iron(III) oleate. To make them colloidally stable and dispersible in water and cell culture medium, they were modified with phosphonic acid- (PA) and hydroxamic acid (HA)-terminated poly(ethylene glycol) yielding PA-PEG@Fe3O4 and HA-PEG@Fe3O4 nanoparticles; conventional γ-Fe2O3 particles were prepared as a control. Advanced techniques were used to evaluate the properties and safety of the particles. Completeness of the nanoparticle coating was tested by real-time polymerase chain reaction. Interaction of the particles with primary human peripheral blood cells, cellular uptake, cytotoxicity, and immunotoxicity were also investigated. Amount of internalized iron in peripheral blood mononuclear cells was 72, 38, and 25 pg Fe/cell for HA-PEG@Fe3O4, γ-Fe2O3, and PA-PEG@Fe3O4, respectively. Nanoparticles were localized within the cytoplasm and in the extracellular space. No cytotoxic effect of both PEGylated nanoparticles was observed (0.12-75 μg/cm2) after 24 and 72-h incubation. Moreover, no suppressive effect was found on the proliferative activity of T-lymphocytes and T-dependent B-cell response, phagocytic activity of monocytes and granulocytes, and respiratory burst of phagocytes. Similarly, no cytotoxic effect of γ-Fe2O3 particles was observed. However, they suppressed the proliferative activity of T-lymphocytes (75 μg/cm2, 72 h) and also decreased the phagocytic activity of monocytes (15 μg/cm2, 24 h; 3-75 μg/cm2, 72 h). We thus show that newly developed particles have great potential especially in cancer diagnostics and therapy.

We studied delayed effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether (PEG-b-PLA) on the endpoints related to pubertal development and reproductive function in female Wistar rats from postnatal day 4 (PND4) to PND 176. Female pups were injected intraperitoneally, daily, from PND4 to PND7 with PEG-b-PLA (20 or 40mg/kg b.w.). Both doses of PEG-b-PLA accelerated the onset of vaginal opening compared with the control group. In the low-dose PEG-b-PLA-treated group, a significantly reduced number of regular estrous cycles, increased pituitary weight due to hyperemia, vascular dilatation and congestion, altered course of hypothalamic gonadotropin-releasing hormone-stimulated luteinizing hormone secretion, and increased progesterone serum levels were observed. The obtained data indicate that neonatal exposure to PEG-b-PLA might affect the development and function of hypothalamic-pituitary-ovarian axis (HPO), and thereby alter functions of the reproductive system in adult female rats. Our study indicates a possible neuroendocrine disrupting effect of PEG-b-PLA nanoparticles.

Aquatic biomonitoring has become an essential task in Europe and many other regions as a consequence of strong anthropogenic pressures affecting the health of lakes, rivers, oceans and groundwater. A typical assessment of the environmental quality status, such as it is required by European but also North American and other legislation, relies on matching the composition of assemblages of organisms identified using morphological criteria present in aquatic ecosystems to those expected in the absence of anthropogenic pressures. Through decade-long and difficult intercalibration exercises among networks of regulators and scientists in European countries, a pragmatic biomonitoring approach was developed and adopted, which now produces invaluable information. Nonetheless, this approach is based on several hundred different protocols, making it susceptible to issues with comparability, scale and resolution. Furthermore, data acquisition is often slow due to a lack of taxonomic experts for many taxa and regions and time-consuming morphological identification of organisms. High-throughput genetic screening methods such as (e)DNA metabarcoding have been proposed as a possible solution to these shortcomings. Such "next-generation biomonitoring", also termed "biomonitoring 2.0", has many advantages over the traditional approach in terms of speed, comparability and costs. It also creates the potential to include new bioindicators and thereby further improves the assessment of aquatic ecosystem health. However, several major conceptual and technological challenges still hinder its implementation into legal and regulatory frameworks. Academic scientists sometimes tend to overlook legal or socioeconomic constraints, which regulators have to consider on a regular basis. Moreover, quantification of species abundance or biomass remains a significant bottleneck to releasing the full potential of these approaches. Here, we highlight the main challenges for next-generation aquatic biomonitoring and outline principles and good practic, COST - European Cooperation in Science and Technology(CA15219). COST Action DNAqua-Net (CA15219), supported by the COST (European Cooperation in Science and Technology) programme

We describe here a new species of the genus Neblinagena Spangler, 1985 from the foothills of the Kukenán tepui (Venezuela). Neblinagena mira sp. n. occurs sympatrically with Neblinagena doylei Kodada & Jäch, 1999, from which it differs mainly in the shape of thorax, shorter setae around eyes, pointed apices of elytra and shape of the male genitalia. We describe also the larva of the new species, which is the first description of a larva in the genus. Larva was assigned to the adults of N. mira sp. n. using mtDNA barcodes. Molecular data were also used to estimate relationships of the new species with congeneric larvae from Auyán tepui, N. doylei and related Larainae genera.

Heterotrissocladius marcidus (Walker, 1856) (Diptera: Chironomidae) inhabits aquatic habitats across the Holarctic and Oriental regions, including remote alpine glacial lakes. It is thus a promising species for studies of population structure and genetic diversity in these highly vulnerable habitats. Here, 21 polymorphic microsatellites for H. marcidus are described, obtained using 454 GS-FLX pyrosequencing. The number of alleles per locus varied from 2 to 9 and no significant linkage disequilibrium was observed. To test applicability, 40 individuals were used from two lakes in the Tatra Mts (Western Carpathians). Observed and expected heterozygosity varied from 0 to 0.9 and 0.102 to 0.888, respectively. In both populations, three loci deviated significantly from Hardy–Weinberg equilibrium after Bonferroni correction, probably due to presence of null alleles or undetected biological processes. Application of microsatellites was tested on six alpine lake populations. Bayesian cluster analysis assigned individuals to lakes of their origin and revealed limited gene-flow between them. Five loci were successfully cross-amplified in the related midge Macropelopia sp. (Tanypodinae) and two in Pseudodiamesa branickii (Nowicki, 1873) (Diamesinae). The microsatellites described herein proved to be useful for genetic studies of alpine populations, and can provide important data for management and conservation of these threatened habitats.

SUMMARY 1. Molecular genetic techniques have been used in freshwater biology for more than 30 years. Early work focussed on studies of population structure, systematics and taxonomy. More recently, the range of studies has broadened to include ecology and adaptation. Advances in analytical methods and in technology (e.g. next-generation sequencing) and decreasing costs of data production ensure that the field will continue to develop and broaden in scope. 2. At least three factors make the application of molecular techniques to freshwater biology exciting. First, the highly variable nature of many aquatic habitats makes them excellent models for the study of environmental change on ecological and evolutionary time scales. Second, the mature state of the field of freshwater biology provides an extensive foundation of ecological knowledge of freshwater organisms and their distinct adaptations. Third, the methodological advances allow researchers to focus more on merging molecular and ecological research and less on designing studies around technical limitations. 3. We identified eight research areas in freshwater biology in which the integration of molecular and ecological approaches provides exceptional opportunities. The list is not exhaustive, but considers a broad range of topics and spans the continuum from basic to applied research. The areas identified use a combination of natural, experimental and in silico approaches. 4. With advancing molecular techniques, freshwater biology is in an unusually strong position to link the genetic basis and ecological importance of adaptations across a wide range of taxa, ecosystems and spatiotemporal scales. Our aim was to identify opportunities for the integration of molecular and ecological approaches, to motivate greater collaboration and crossover, and to promote exploitation of the synergies of bridging ecological and evolutionary freshwater research.

Planktonic crustaceans are traditionally identified based on morphological and morphometric characters. However, such characters may be hardly distinguishable and often overlap between species. A probability of misidentification is thus relatively high. Molecular techniques may increase the accuracy of identification if appropriate markers are used. Aim of our work was to develop a simple molecular procedure enabling discrimination between four species of Simocephalus occurring in Europe. PCR-RFLP technique proved to be suitable for such discrimination. Within the 709 bp fragment of mitochondrial cytochrome c oxidase subunit 1 gene we found unique combinations of restriction sites of the BbsI and SacI enzymes for Simocephalus vetulus, S. exspinosus, S. serrulatus and S. congener. PCR products of samples from several locations in Slovakia were digested with the two enzymes and electrophoresed on an agarose gel. The restriction patterns were clearly visible and easily distinguishable. This method is applicable for identifying the four species in any life-stage. Considering its simplicity and cost-effectiveness it can be widely used as a diagnostic tool for discriminating between Simocephalus species with overlapping morphologic characters.

Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, (3)H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.