Toxicity of sediments in eight urban stormwater management ponds: bioassessment by oligochaete community metrics used in the sediment quality triad.

Implemented for decades as part of the 'best management practices (BMPs)' for controlling urban runoff impacts on receiving waters, stormwater management ponds (SMPs) have been increasingly viewed as potential habitats for urban wildlife. However, since SMPs are subject to a lot of environmental constraints, research toward assessing their ecological quality and their actual benefits as habitats for biota is needed. In this study, the sediment toxicity of eight SMPs located in Southern Ontario, Canada was assessed using the sediment quality triad (SQT) approach. Sediment samples were collected for chemical, ecotoxicological and biological analyses. An oligochaete-based index approach (Oligochaete Index of Lake Bioindication and percentage of pollution-sensitive species) was used as the biological endpoint and integrated into a weight-of-evidence approach to assessing the general sediment quality of the ponds. Our results showed that (i) heavy metals in the sediment and (ii) chloride concentrations in the sediment interstitial water caused detrimental effects on the ecological quality of the sediments in the ponds studied. The oligochaete indices applied in this study showed value as biological endpoints to be integrated into the SQT and used for setting up sediment ecological quality goals.

The Foreign Oligochaete Species Quistadrilus multisetosus (Smith, 1900) in Lake Geneva: Morphological and Molecular Characterization and Environmental Influences on Its Distribution.

The presence of the oligochaete species Quistadrilus multisetosus (Smith, 1900) originating from North America has been mentioned for several decades in Europe, the Middle East and Russia. Its distribution and abundance in Europe is still unknown but it can be considered as potentially invasive. This species was recently discovered in Lake Geneva (Switzerland/France) and three other Swiss lakes. The aims of the present work are to report its repartition and abundance in Lake Geneva, to study its ecology and to determine its invasive potential in this lake. We also provide an identification key for correctly differentiating Q. multisetosus from the closely related species Spirosperma ferox Eisen, 1879 and Embolocephalus velutinus (Grube, 1879), and study the phylogenetic position of Q. multisetosus within several Tubificinae lineages based on the cytochrome c oxidase (COI) marker. Twenty-eight sites have been monitored since 2009 in Lake Geneva. In several sites, the COI sequence corresponding to this species was also searched for in sediment samples using high-throughput sequencing. In addition, we examined specimens collected in this lake before 2009 likely to belong to Q. multisetosus and to have been misidentified. We found that Q. multisetosus was only present in the lake downstream of a wastewater treatment plant and a combined sewer overflow in the Vidy Bay (near Lausanne) and at a site located nearby. These results confirmed the high tolerance of this species to organic matter pollution. Q. multisetosus was already present in this location in 1974 (misidentified as Spirosperma ferox), which suggests that Q. multisetosus has a limited capacity to disseminate in this lake. However, we recommend continuing monitoring its presence in Lake Geneva in the future, especially in the context of warming of waters that could contribute to the expansion of this species.

High-throughput DNA barcoding of oligochaetes for abundance-based indices to assess the biological quality of sediments in streams and lakes.

Aquatic oligochaete communities are valuable indicators of the biological quality of sediments in streams and lakes, but identification of specimens to the species level based on morphological features requires solid expertise in taxonomy and is possible only for a fraction of specimens present in a sample. The identification of aquatic oligochaetes using DNA barcodes would facilitate their use in biomonitoring and allow a wider use of this taxonomic group for ecological diagnoses. Previous approaches based on DNA metabarcoding of samples composed of total sediments or pools of specimens have been proposed for assessing the biological quality of ecosystems, but such methods do not provide precise information on species abundance, which limits the value of resulting ecological diagnoses. Here, we tested how a DNA barcoding approach based on high-throughput sequencing of sorted and genetically tagged specimens performed to assess oligochaete species diversity and abundance and the biological quality of sediments in streams and lakes. We applied both molecular and morphological approaches at 13 sites in Swiss streams and at 7 sites in Lake Geneva. We genetically identified 33 or 66 specimens per site. For both approaches, we used the same index calculations. We found that the ecological diagnoses derived from the genetic approach matched well with those of the morphological approach and that the genetic identification of only 33 specimens per site provided enough ecological information for correctly estimating the biological quality of sediments in streams and lakes.

Simultaneous preservation of the DNA quality, the community composition and the density of freshwater oligochaetes for the development of genetically based biological indices.

INTRODUCTION: Oligochaetes are recognized as valuable bioindicators of sediment quality in streams and lakes. The development of an oligochaete index based on the identification of specimens using DNA barcodes requires a method for simultaneously preserving the DNA quality and information on the specimen density and oligochaete community composition. Absolute ethanol optimally preserves DNA but fixation of freshwater oligochaetes with this medium can cause disintegration and fragmentation of specimens. Here, we investigated the possibility to preserve oligochaete specimens in low-pH formalin and in neutral buffered formalin for up to four weeks before genetic analyses and tested if the addition of absolute ethanol to formalin-fixed oligochaetes resulted in a loss of specimens and/or species. METHODS: We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of a fragment of the cytochrome c oxidase I (COI) gene on tissue fragments preserved in low-pH formalin for up to 3 weeks and in neutral buffered formalin for up to 4 weeks. In addition, we compared the density and taxonomic composition of formalin-fixed oligochaetes of several sieved sediment samples before and after the addition of absolute ethanol. RESULTS: The COI fragment of all oligochaete specimens preserved in neutral buffered formalin for up to 28 days was successfully amplified by PCR and obtained sequences were complete and of high quality. The amplification success rate for low-pH formalin fixed specimens declined after 7 days of storage. The addition of absolute ethanol to formalin-fixed oligochaete communities did not alter density or diversity estimates. DISCUSSION: Our results indicate that sediment samples can be stored in neutral buffered formalin for up to 4 weeks and the sieved material can then be transferred to absolute ethanol, without affecting DNA quality, density and community composition of oligochaetes. Based on these results, a protocol for preserving freshwater oligochaetes, describing all the steps from collection of sediments to preservation of the biological material in absolute ethanol, is proposed. This method of fixation/preservation is of relevance for establishing DNA barcode reference databases, inventories of genetic diversity and developing genetically based biological indices.

Development and implementation of eco-genomic tools for aquatic ecosystem biomonitoring: the SYNAQUA French-Swiss program.

The effectiveness of environmental protection measures is based on the early identification and diagnosis of anthropogenic pressures. Similarly, restoration actions require precise monitoring of changes in the ecological quality of ecosystems, in order to highlight their effectiveness. Monitoring the ecological quality relies on bioindicators, which are organisms revealing the pressures exerted on the environment through the composition of their communities. Their implementation, based on the morphological identification of species, is expensive because it requires time and experts in taxonomy. Recent genomic tools should provide access to reliable and high-throughput environmental monitoring by directly inferring the composition of bioindicators' communities from their DNA (metabarcoding). The French-Swiss program SYNAQUA (INTERREG France-Switzerland 2017-2019) proposes to use and validate the tools of environmental genomic for biomonitoring and aims ultimately at their implementation in the regulatory bio-surveillance. SYNAQUA will test the metabarcoding approach focusing on two bioindicators, diatoms, and aquatic oligochaetes, which are used in freshwater biomonitoring in France and Switzerland. To go towards the renewal of current biomonitoring practices, SYNAQUA will (1) bring together different actors: scientists, environmental managers, consulting firms, and biotechnological companies, (2) apply this approach on a large scale to demonstrate its relevance, (3) propose robust and reliable tools, and (4) raise public awareness and train the various actors likely to use these new tools. Biomonitoring approaches based on such environmental genomic tools should address the European need for reliable, higher-throughput monitoring to improve the protection of aquatic environments under multiple pressures, guide their restoration, and follow their evolution.

Cytochrome c oxidase barcodes for aquatic oligochaete identification: development of a Swiss reference database.

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.

DNA barcoding of formalin-fixed aquatic oligochaetes for biomonitoring.

BACKGROUND: Oligochaetes are valuable bioindicators of the quality of watercourse and lake sediments. The morphological identification of aquatic oligochaetes is difficult, prompting the development of new molecular oligochaete indices based on DNA barcoding and Next-generation sequencing of sorted specimens. In general, the samples for DNA barcoding are fixed in absolute ethanol. However, in the case of aquatic oligochaetes, this medium is not appropriate as it can induce a modification of specimen abundances and of the composition of communities. Therefore, we investigated the possibility to amplify and sequence aquatic oligochaetes fixed in formalin for a short time. We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of the cytochrome c oxydase I (COI) gene on tissue fragments fixed in formalin for different periods of time (from 1 h to 1 week) and in ethanol. RESULTS: The large majority of aquatic oligochaete specimens fixed in formalin for up to 1 week could be successfully amplified and all obtained sequences were of high quality. The amplification and sequencing success rate of formalin-fixed samples and ethanol-fixed samples was similar. These results suggest that formalin fixation of aquatic oligochaete tissues for a short time does not cause serious damages to DNA and inhibit PCR amplification. CONCLUSION: The possibility to fix aquatic oligochaetes with formalin before genetic analyses is very promising for diversity monitoring, for construction of a comprehensive DNA barcode library and for development of an index based on Next-generation sequencing analysis of samples composed of sorted specimens.

Next-Generation Sequencing of Aquatic Oligochaetes: Comparison of Experimental Communities.

Aquatic oligochaetes are a common group of freshwater benthic invertebrates known to be very sensitive to environmental changes and currently used as bioindicators in some countries. However, more extensive application of oligochaetes for assessing the ecological quality of sediments in watercourses and lakes would require overcoming the difficulties related to morphology-based identification of oligochaetes species. This study tested the Next-Generation Sequencing (NGS) of a standard cytochrome c oxydase I (COI) barcode as a tool for the rapid assessment of oligochaete diversity in environmental samples, based on mixed specimen samples. To know the composition of each sample we Sanger sequenced every specimen present in these samples. Our study showed that a large majority of OTUs (Operational Taxonomic Unit) could be detected by NGS analyses. We also observed congruence between the NGS and specimen abundance data for several but not all OTUs. Because the differences in sequence abundance data were consistent across samples, we exploited these variations to empirically design correction factors. We showed that such factors increased the congruence between the values of oligochaetes-based indices inferred from the NGS and the Sanger-sequenced specimen data. The validation of these correction factors by further experimental studies will be needed for the adaptation and use of NGS technology in biomonitoring studies based on oligochaete communities.

Molecular barcoding of aquatic oligochaetes: implications for biomonitoring.

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.

Do results of the T-SPOT.TB interferon-gamma release assay change after treatment of tuberculosis?

BACKGROUND: Interferon-gamma (IFN-gamma) production by lymphocytes exposed to antigens specific of Mycobacterium tuberculosis has been shown to correlate with antigen load and disease activity. AIM OF STUDY: To determine whether treatment of tuberculosis (TB) led to a decrease and/or a reversion of results of a IFN-gamma release assay (T-SPOT.TB, Oxford Immunotec, UK) and thus if T-SPOT.TB could be used to monitor response to treatment. METHODS: Qualitative and quantitative analysis (SFUs: spot-forming units) of T-SPOT.TB in HIV-negative patients with TB, during initial 2 weeks of treatment (T0), at end of treatment (TE) and 6 months later (TE+6). RESULTS: Mean SFU (SD) was 75 (58; n=62) at T0, 46 (55; n=55) at TE, and 33 (46; n=41) at TE+6; positive rate was 98%, 93% and 98%, respectively. SFUs (paired samples, n=36) decreased significantly between T0 and TE; 2 reversions occurred between T0 and TE (6%), but none between TE and TE+6. Of 6 patients (17%) with an increase in SFUs between T0 and TE, 5 had a favourable outcome at TE and TE+6. CONCLUSION: Decrease in SFUs under treatment suggests a relationship with antigen load; however, persisting high SFUs were not predictive of unfavourable outcome and test reversion was rare.

Contribution of a IFN-gamma assay in contact tracing for tuberculosis in a low-incidence, high immigration area.

AIMS OF STUDY: to analyse, in contacts exposed to smear+/culture + (S+/C+) or S-/C+ TB, most of whom are foreign-born: 1) correlation between T-SPOT.TB IFN-gamma release assay (Oxford Immunotec, UK), TST and exposure scores; 2) agreement between T-SPOT.TB and TST in Bacillus of Calmette-Guérin (BCG) vaccinated and non-vaccinated subjects, and 3) impact of results of T-SPOT.TB on diagnosis and treatment for latent tuberculosis infection (LTBI). PATIENTS AND METHODS: TST and T-SPOT.TB were performed in 295 contacts (74% foreign-born) 8-12 weeks after exposure. Contacts completed five exposure scores. Data were analysed according to most recent US (ATS/CDC), British (NICE) and Swiss guidelines. RESULTS: T-SPOT.TB was positive in 115 (39%) and indeterminate in 15 subjects (5.1%). Neither TST, nor T-SPOT.TB was significantly related to exposure scores or infectiousness of the index case. In multivariate analysis, incidence of TB in country of origin was the strongest predictor of result of TST or T-SPOT.TB. Agreement between TST and T-SPOT.TB (kappa: 0.19-0.27) was low but improved in non BCG-vaccinated subjects (kappa: 0.28-0.47). According to guidelines referred to, 10-24% of subjects screened were T-SPOT.TB+/TST-: the prognosis of this group is yet undetermined. Another 10-27% were T-SPOT.TB-/TST+: present guidelines recommend withholding treatment for LTBI in these subjects although longitudinal data are still scarce. CONCLUSIONS: The lack of a relationship between T-SPOT.TB and exposure scores probably results from both the variability inherent to the design of this study (ie, multiple contact investigations, exposure in different settings) and limits in the performance of the IGRA tested. Longitudinal data are needed to clarify the risk of TB in T-SPOT.TB+/TST- individuals. Unreliability of diagnosis of LTBI in spite of the present use of IGRA in algorithms is illustrated by the wide variations in identification of LTBI according to different guidelines referred to.