Detailed DNA barcoding of mayflies in a small European country proved how far we are from having comprehensive barcode reference libraries.

Mayflies (Ephemeroptera) are among the crucial water and habitat quality bioindicators. However, despite their intensive long-term use in various studies, more reliable mayfly DNA barcode data have been produced in a negligible number of countries, and only ~40% of European species had been barcoded with less than 50% of families covered. Despite being carried out in a small area, our study presents the second-most species-rich DNA reference library of mayflies from Europe and the first comprehensive view from an important biodiversity hotspot such as the Western Carpathians. Within 1153 sequences, 76 morphologically determined species were recorded and added to the Barcode of Life Data System (BOLD) database. All obtained sequences were assigned to 97 BINs, 11 of which were unique and three represented species never barcoded before. Sequences of 16 species with high intraspecific variability were divided into 40 BINs, confirming the presence of cryptic lineages. Due to the low interspecific divergence and the non-existing barcoding gap, sequences of six species were assigned to three shared BINs. Delimitation analyses resulted in 79 and 107 putative species respectively. Bayesian and maximum-likelihood phylogenies confirmed the monophyly of almost all species and complexes of cryptic taxa and proved that DNA barcoding distinguishes almost all studied mayfly species. We have shown that it is still sufficient to thoroughly investigate the fauna of a small but geographically important area to enrich global databases greatly. In particular, the insights gained here transcend the local context and may have broader implications for advancing barcoding efforts.

50%, not great, not terrible: Pan-European gap-analysis shows the real status of the DNA barcode reference libraries in two aquatic invertebrate groups and points the way ahead.

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.

Potamophilus acuminatus (Fabricius, 1792): distribution update in North Africa confirmed by COI barcoding sequencing (Coleoptera, Elmidae).

Potamophilus acuminatus (Fabricius, 1792) is here recorded for the first time from Morocco and the recent distribution in Slovakia is updated. The North African distribution was hitherto based only on two larvae from Tunisia. DNA-barcoding confirmed the identification of specimens from Morocco after comparing ten sequences of P. acuminatus from Germany, France and Slovakia. The COI haplotype of the Moroccan samples was quite divergent (0.033-0.035 uncorrected p-distance) compared with European specimens. These high p-distances suggest the existence of a different intraspecific lineage. A brief morphological diagnosis of adults and larvae is given.

DNA barcodes evidence the contact zone of eastern and western caddisfly lineages in the Western Carpathians.

The region of the Western Carpathians is, among other aspects, very important for survival and diversity of European freshwater fauna due to the presence of a large number of (sub)mountain springs and streams. However, these ecologically and faunistically diversified habitats are still understudied in the context of genetic diversity and population structure of their inhabitants. This study focuses on genetic diversity and distribution patterns of the caddisfly Rhyacophila tristis, common and widespread representative of mountain freshwater fauna. Analysis of the COI mitochondrial marker revealed presence of the western and eastern lineages, with samples from both lineages being grouped in BOLD (Barcode of Life Data System) into separate BINs (Barcode Index Numbers). Our data indicates that eastern lineage (BIN_E) is more closely related to the Balkan populations than to co-occurring western lineage (BIN_W), and that the contact zone of the lineages passes through the W Carpathians. The study revealed phylogeographic and demographic differences between lineages, supporting hypothesis of their evolutionary independence and specific ecological preferences. The obtained genetic data of the R. tristis population from W Carpathians improved our knowledge about population genetics of this aquatic species and can contribute to understanding the state and evolution of biodiversity of freshwater ecosystems in Europe.

Fuzzy species borders of glacial survivalists in the Carpathian biodiversity hotspot revealed using a multimarker approach.

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 showed 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 a G. stasiuki sp. nov. and another we redescribe as a G. tatrensis (S. Karaman, 1931).

Disersus otongachi/ a new species of Larainae riffle beetles from Ecuador (Coleoptera: Elmidae).

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 tale of springs and streams: how different aquatic ecosystems impacted the mtDNA population structure of two riffle beetles in the Western Carpathians.

The Western Carpathians are a particularly interesting part of the Carpathian Arc. According to recent molecular data upon aquatic and terrestrial taxa, this mountain area is an important biodiversity hotspot of Europe. Moreover, the W Carpathians include rich systems of karst springs inhabited by specific fauna, where molecular diversity and phylogeographic patterns are yet to be fully explored. Our study aims to compare population genetic structure and molecular diversity of two related and commonly co-occurring riffle beetles, Elmis aenea (PWJ Müller, 1806) and Limnius perrisi (Dufour, 1843) in the springs and streams of the W Carpathians using the mitochondrial DNA barcoding fragment of the cytochrome c oxidase subunit I gene (COI). The relatively stable thermal and chemical conditions of springs throughout unfavourable climatic settings make these highly specific lotic systems potentially ideal for a long-term survival of some aquatic biota. Populations of both elmid species were relatively homogeneous genetically, with a single dominant haplotype. However, we revealed that E. aenea significantly dominated in the springs, while L. perrisi preferred streams. Relative isolation of the springs and their stable conditions were reflected in significantly higher molecular diversity of the E. aenea population in comparison to L. perrisi. The results of Bayesian Skyline Plot analysis also indicated the exceptional position of springs regarding maintaining the population size of E. aenea. On the other hand, it seems that streams in the W Carpathians provide more effective dispersal channels for L. perrisi, whose population expanded much earlier compared to E. aenea. Present study points out that different demographic histories of these two closely related elmid species are manifested by their different habitat preference and molecular diversity.

Enderleina preclara Jewett, 1960 (Plecoptera, Perlidae), from the Venezuelan Guayana-A description of a putative female, nymph and egg.

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.

Validation of Heterocerus heydeni Kuwert, 1890 based on morphology and DNA barcoding, with notes on the problems of classification of the Heteroceridae (Coleoptera).

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).

Ictelmis, a new riffle beetle genus from Ecuador (Coleoptera: Elmidae).

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.

Four new species of Hexanchorus Sharp from Ecuador (Coleoptera, Elmidae) with DNA barcoding and notes on the distribution of the genus.

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: Hexanchorusvirilis sp. n., Hexanchorusrostratus sp. n., Hexanchorusshepardi sp. n., Hexanchorusonorei sp. n. and Hexanchorusonoreisagittatus ssp. 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.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

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.

Toxicity evaluation of monodisperse PEGylated magnetic nanoparticles for nanomedicine.

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.

Phanoceroides Hinton, 1939: description of new species, morphology of larvae, and revised taxonomic position of the genus (Coleoptera: Elmidae) based on molecular evidence.

The monotypic genus Phanoceroides was initially classified within the subfamily Elminae, based solely on the distribution and type of pubescence on the body surface. Although a close relationship with Larainae was suggested (Hinton 1939, Maier 2013), its taxonomic position and phylogeny remained unresolved. Based on newly collected material, we describe here P. fernandesi sp. n. and document morphological characters and differences of adults and larvae of P. aquaticus Hinton, 1939 and P. fernandesi sp. n. To clarify the taxonomic position of Phanoceroides within Elmidae, we used molecular data (cox1 and mtDNA). The results support position of the genus within the subfamily Larainae with Hypsilara Maier & Spangler, 2011 as a close relative.

Functionalized porous silica&maghemite core-shell nanoparticles for applications in medicine: design, synthesis, and immunotoxicity.

AIM: To determine cytotoxicity and effect of silica-coated magnetic nanoparticles (MNPs) on immune response, in particular lymphocyte proliferative activity, phagocytic activity, and leukocyte respiratory burst and in vitro production of interleukin-6 (IL-6) and 8 (IL-8), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF). METHODS: Maghemite was prepared by coprecipitation of iron salts with ammonia, oxidation with NaOCl and modified by tetramethyl orthosilicate and aminosilanes. Particles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). Cytotoxicity and lymphocyte proliferative activity were assessed using [3H]-thymidine incorporation into DNA of proliferating human peripheral blood cells. Phagocytic activity and leukocyte respiratory burst were measured by flow cytometry; cytokine levels in cell supernatants were determined by ELISA. RESULTS: γ-Fe2O3&SiO2-NH2 MNPs were 13 nm in size. According to TEM, they were localized in the cell cytoplasm and extracellular space. Neither cytotoxic effect nor significant differences in T-lymphocyte and T-dependent B-cell proliferative response were found at particle concentrations 0.12-75 µg/cm2 after 24, 48, and 72 h incubation. Significantly increased production of IL-6 and 8, and GM-CSF cytokines was observed in the cells treated with 3, 15, and 75 µg of particles/cm2 for 48 h and stimulated with pokeweed mitogen (PHA). No significant changes in TNF-α and IFN-γ production were observed. MNPs did not affect phagocytic activity of monocytes and granulocytes when added to cells for 24 and 48 h. Phagocytic respiratory burst was significantly enhanced in the cultures exposed to 75 µg MNPs/cm2 for 48 h. CONCLUSIONS: The cytotoxicity and in vitro immunotoxicity were found to be minimal in the newly developed porous core-shell γ-Fe2O3&SiO2-NH2 magnetic nanoparticles.

Delayed adverse effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether on hypothalamic-pituitary-ovarian axis development and function in Wistar rats.

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.

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles.

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.

Immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles in human blood cell model.

A human blood cell model for immunotoxicity and genotoxicity testing was used to measure the response to polylactic-co-glycolic acid (PLGA-PEO) nanoparticle (NP) (0.12, 3, 15 and 75 µg/cm(2) exposure in fresh peripheral whole blood cultures/isolated peripheral blood mononuclear cell cultures from human volunteers (n = 9-13). PLGA-PEO NPs were not toxic up to dose 3 µg/cm(2); dose of 75 µg/cm(2) displays significant decrease in [(3)H]-thymidine incorporation into DNA of proliferating cells after 4 h (70% of control) and 48 h (84%) exposure to NPs. In non-cytotoxic concentrations, in vitro assessment of the immunotoxic effects displayed moderate but significant suppression of proliferative activity of T-lymphocytes and T-dependent B-cell response in cultures stimulated with PWM > CON A, and no changes in PHA cultures. Decrease in proliferative function was the most significant in T-cells stimulated with CD3 antigen (up to 84%). Cytotoxicity of natural killer cells was suppressed moderately (92%) but significantly in middle-dosed cultures (4 h exposure). On the other hand, in low PLGA-PEO NPs dosed cultures, significant stimulation of phagocytic activity of granulocytes (119%) > monocytes (117%) and respiratory burst of phagocytes (122%) was recorded. Genotoxicity assessment revealed no increase in the number of micronucleated binucleated cells and no induction of SBs or oxidised DNA bases in PLGA-PEO-treated cells. To conclude on immuno- and genotoxicity of PLGA-PEO NPs, more experiments with various particle size, charge and composition need to be done.

Two new Larainae species from Guayana region, Venezuela (Coleoptera: Elmidae).

Two new species of the subfamily Larainae (Insecta: Coleoptera: Elmidae), Hexanchorus angeli n. sp. and Hypsilara autanai n. sp., are described from Guyana region in Venezuela. We provide habitus photographs, detail drawings of both male and female genitalia, and description of morphological features important for discrimination of the new species. Molecular differences within genera were measured using 816bp fragment of mtDNA gene for cytochrome oxidase c subunit I. Sequence divergences among species are discussed.

Ancyronyx reticulatus and A. pulcherrimus, two new riffle beetle species from Borneo, and discussion about elmid plastron structures (Coleoptera: Elmidae).

Two new species of Ancyronyx Erichson, 1847 (Coleoptera: Elmidae) are described from Borneo: A. pulcherrimus (Brunei) and A. reticulatus (Sabah). Habitus views, illustrations of important characters as well as plastron structures of Ancyronyx reticulatus are presented and discussed.