Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

The aim of this study was to assess the contribution of an interferon-γ release assay (T-SPOT.TB) to the differentiation of active tuberculosis (TB) from latent TB infection by quantifying spot-forming units (sfu).The investigation was a prospective study of contacts exposed to a case of contagious TB and cases of HIV-negative culture-proven TB referred over a 16-month period. Tuberculin skin tests (TSTs) and T-SPOT.TBwere performed in 310 contacts 8–12 weeks after exposure. In subjects with culture-proven TB, T-SPOT.TBwas performed within 2 weeks of initiation of treatment. The analysis included all contacts with a positive T-SPOT.TBresult and all subjects with TB.TB contacts (n = 127) and cases (n = 58) were included. Mean±sdT-SPOT.TBresults were 107±56 (range 1–207) sfu for TB, 54±60 (7–239) sfu for contacts with positive T-SPOT.TBresults and a TST induration diameter of >5 mm, and 19±27 (7–143) sfu for contacts with positive T-SPOT.TBresults and a TST induration diameter of ≤5 mm. By receiver operating characteristic curve analysis, a threshold value of 49.5 sfu showed a sensitivity of 83% and specificity of 74% for distinguishing latent TB infection from TB.Although T-SPOT.TBresults were significantly related to disease activity, the test cannot be recommended for the diagnosis of tuberculosis.

Haplotaxis Hoffmeister, 1843 is a poorly known genus: it is rarely collected because it usually inhabits groundwater, and many species are only known from immature specimens. Even the type species, Haplotaxis gordioides (Hartmann in Oken, 1819) remains poorly defined because of the absence of reference types. Most of the Haplotaxis species have been placed in synonymy with H. gordioides since the end of the 19th century, a situation that has remained essentially unchanged until now. As a result, the species is supposedly present on all continents, except Antarctica. This observation is all the more questionable as the aquatic subterranean environment is nowadays well known to harbour many species with restricted distribution, due to the strong hydrogeographic isolation and the low dispersal abilities of its inhabitants. In this study we assessed the hypothesis of H. gordioides as a single species with wide distribution versus a complex of cryptic species with narrow distribution. We used a DNA-barcoding approach based on the COI mitochondrial marker of 46 Haplotaxis specimens collected in Switzerland, mostly as part of a countrywide sampling campaign to study groundwater macroinvertebrates. Preliminary results suggested that H. gordioides is a complex of at least 6 cryptic species in Switzerland, which has important implications both for the knowledge of the exact identity of the type species and for the synonymy of most of the species described in the 19th century. However, as it is based on a single-locus approach, this study should be seen as the first step in an integrative taxonomic process that should include additional biological material, the study of complementary markers (especially nuclear), and the morphological study of specimens.

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.

Aquatic oligochaetes are well recognized bioindicators of quality of sediments and water in watercourses and lakes. However, the difficult taxonomic determination based on morphological features compromises their more common use in eco-diagnostic analyses. To overcome this limitation, we investigated molecular barcodes as identification tool for broad range of taxa of aquatic oligochaetes. We report 185 COI and 52 ITS2 rDNA sequences for specimens collected in Switzerland and belonging to the families Naididae, Lumbriculidae, Enchytraeidae and Lumbricidae. Phylogenetic analyses allowed distinguishing 41 lineages separated by more than 10 % divergence in COI sequences. The lineage distinction was confirmed by Automatic Barcode Gap Discovery (ABGD) method and by ITS2 data. Our results showed that morphological identification underestimates the oligochaete diversity. Only 26 of the lineages could be assigned to morphospecies, of which seven were sequenced for the first time. Several cryptic species were detected within common morphospecies. Many juvenile specimens that could not be assigned morphologically have found their home after genetic analysis. Our study showed that COI barcodes performed very well as species identifiers in aquatic oligochaetes. Their easy amplification and good taxonomic resolution might help promoting aquatic oligochaetes as bioindicators for next generation environmental DNA biomonitoring of aquatic ecosystems.

Introduction: Aquatic oligochaetes represent valuable indicators of the quality of sediments of watercourses and lakes, but their difficult identification based on morphological criteria compromises their more common use for eco-diagnostic analyses. This issue could be overcome by using DNA barcodes for species identification. A 10% threshold of cytochrome c oxidase (COI) divergence was proposed for differentiating between oligochaete species based on molecular and morphological data. A Swiss database of COI sequences of aquatic oligochaetes was initiated in 2012. The aim of this study is to complement the Swiss oligochaete database of COI sequences and to confirm the relevance of this threshold for species delimitation. Methods: We sequenced the COI sequence of 216 specimens collected in different regions of Switzerland and ITS2 region of some lineages whose delimitation with COI data was doubtful. Results: We distinguished 53 lineages, among which 34 were new for Switzerland and 17 sequenced for the first time. All the lineages were separated by more than 10% of COI variation, with the exception of some species within Nais and Uncinais. In these two genera, the threshold was lowered to 8% to be congruent with the morphological analysis. The total number of lineages reported so far for Switzerland is 75, including 59 morphospecies or unidentified species and 16 cryptic species. Discussion: Our study shows that the threshold of 10% of COI divergence is generally appropriate to distinguish aquatic oligochaete lineages, but that it must be adjusted for some species. The database reported here will be complemented in the future in parallel to the development of genetic oligochaete indices.