CHOICE OF MACROPHYTE SUBSTRATE IN THE USE OF DIATOMS AS INDICATORS OF POND WATER QUALITY ASSESSMENT: PRELIMINARY DATA ON THE CASE OF ALALAY POND (COCHABAMBA, BOLIVIA)

Alalay Pond is a 230-hectare, shallow aquatic ecosystem within Cochabamba, the third largest city in Bolivia. With the aim to determine the suitability of epiphytic diatoms for water quality assessment in the pond and to choose a substrate that would hold a representative bioindicator epiphytic community, the macrophytes Schoenoplectus californicus subsp. tatora (Kunth) T. Koyama, Typha dominguensis Pers., Myriophyllum verticillatum L. and Azolla filiculoides Lam. were selected and differences in diatom community composition and structure were tested. Diatoms were collected during four sampling campaigns in the March-September, 2011 period, from three stations contiguous to the pelagic zone and prepared for analysis using standard, internationally used protocols. In all, 27 samples were collected from which 28 taxa characteristic of eutrophic environments were identified and 17 others could not be assigned names from the literature. Although many of the species are shared among sampling sites, the epiphytic communities developing at each station were different in structure and composition. Although, there are marked temporal variations in community features on each of the macrophytes, Shannon-Wiener and Pielou indexes, as well as canonical correspondence analysis, showed no marked differences within a single campaign and station among the 4 macrophytes. Shifts in structure and composition are denoted less commonly by species replacement and more often by changes in percent relative abundance of dominant and rare species. Very few species are restricted to a station or seem to show strong preference for a particular substrate. Taking into account growth, structural and ecological characteristics, as well as some phenological features of the macrophytes, S. californicus subsp. tatora and T. dominguensis are the most suitable substrates for water quality assessments in the pond. As demonstrated by multivariate analyses, among-site and time-scale differences in community composition and structure were attributable to alkalinity, chemical oxygen demand (COD), ammonium-nitrogen, conductivity and dissolved oxygen (DO). More frequent sampling, diversification of substrates and extending the study for a longer period are recommended to establish a monitoring program during any future restoration of the pon

Metadata standards and practical guidelines for specimen and DNA curation when building barcode reference libraries for aquatic life

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.info:eu-repo/semantics/publishedVersio

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Aquatic biofilms can act as natural environmental DNA samplers

Diatoms, macroinvertebrates and fish communities are widely used for the assessment of the ecological status of rivers and lakes. Metabarcoding studies of these communities are usually performed from “bulk” samples in the case of diatoms and macroinvertebrates; and from water samples in the case of fish.Recent studies, suggest that aquatic biofilms can physically act as environmental catchers of environmental DNA (eDNA) (e.g. Mariani et al. 2019). Thus, we propose an alternative metabarcoding approach to study macroinvertebrates and fishes directly from this matrix.The capacity of aquatic biofilms to catch macroinvertebrate eDNA was tested from a previous study in Mayotte Island were both biofilm samples and macroinvertebrate morphological inventories were available at same river sites (Rivera et al. 2021). First, macroinvertebrate specimens were identified based on their morphological characteristics. Second, DNA was extracted from biofilms, and macroinvertebrate communities were targeted using a standard COI barcode. The resulting morphological and molecular inventories were compared. Our results showed that both methods provided comparable structures and diversities for macroinvertebrate communities when using unassigned OTUs suggesting that macroinvertebrate DNA is present in biofilms and representative of the communities. However, after taxonomic assignment of OTUs, diversity and richness were no longer correlated. Indeed, many constraints were observed as the need for: a) more specific primers to avoid co-amplification of untargeted taxa inhabiting biofilms, b) primers targeting shorter barcodes to sequence more easily degraded eDNA that may be captured in biofilms, and c) a reference database well adapted to our tropical study sites. Finally, even if the results of this first study were encouraging, we wanted to test the biofilm approach on organisms that do not inhabit this environmental matrix in order to be able to distinguish between intra or extra-cellular DNA.Based on these observations, a second study looking for a fish eDNA signal in aquatic biofilms was performed. Environmental biofilm and water samples were collected in parallel at littoral sites at Lake Geneva. DNA was extracted from these samples, and fish communities were targeted using a standard 12S barcode. The molecular inventories derived from the biofilm and the water samples were compared. Both methods provide comparable floristic lists, providing a novel approach for ecological studies related to fish phenology using eDNA in biofilms.Our results open the door to the study of diatoms, macroinvertebrates and fish communities through metabarcoding from a single matrix reducing sampling efforts and costs

Diatom metabarcoding applied to large scale monitoring networks: Optimization of bioinformatics strategies using Mothur software

Benthic diatoms are routinely used as ecological indicators in rivers. A standardized methodology is based on biofilm sampling, species identification, and counting under microscope. DNA-metabarcoding is an alternative methodology that can identify species and assess their proportion based on high-throughput DNA sequencing. Sequence data is analyzed with bioinformatics tools, and several strategies can be chosen. The strategy choice can affect communities composition and structure, and therefore the resulting ecological assessment. We wanted to optimize the bioinformatics strategy to obtain the closest results to microscopy. This was done in the framework of the Mothur pipeline. Here, 447 samples from French rivers were analyzed in the monitoring context of the European Water Framework Directive. Samples were analyzed both with DNA metabarcoding and microscopy. A usual bioinformatics strategy in Mothur includes clustering DNA-sequences into Operational Taxonomic Units (OTUs). Different algorithms exist for this. From a subsample of 142 samples, we showed that some strategies (Furthest neighbor) gave closer results to microscopy than others (Opticlust) in terms of community structure and diatom index values. However, we showed that OTU clustering was not necessary for ecological monitoring: Direct taxonomic assignment of individual sequence units (ISU) gave similar results to those obtained in microscopy. Interestingly, direct assignment enabled the detection of more species 2 to 3 times faster in terms of computation time compared to the OTU strategy. However, it remained important to remove low quality and chimeric sequences; if not, biomonitoring results differed greatly from microscopy. We showed that it was preferable to have a loose taxonomical identification threshold instead of a stringent one. This allowed detecting more species, which could participate in the index calculation and increased its performance. Indeed, in diatoms, phylogenetically neighbor species often have similar ecologies, and this explains why it is preferable, in a biomonitoring framework, to identify more species with less stringency instead of identifying few species with stringency. Finally, the best strategy (direct assignment of filtered ISU with a loose taxonomical threshold of 60%) was applied to the 447 samples covering a large diversity of ecological qualities. These data were then used to produce quality index values, using a quantification correction factor taking into account species biovolumes. Compared to microscopy, the DNA-based method assigned the same quality class for 66% of the samples, and 72% of the samples had an index value (ranging from 0 to 20) with less than one point difference from microscopy

eDNA metabarcoding from aquatic biofilms allows studying spatial and temporal fluctuations of fish communities from Lake Geneva

International audienceAbstract Fish communities are now studied non‐invasively using environmental DNA (eDNA) recovered from water samples. The objective of this study is to evaluate the possibility of surveying these communities using fish eDNA passively “captured” by aquatic biofilms. To this end, biofilm samples developing on natural and artificial substrates were collected every 2 weeks for a year and a half in a large lake (Lake Geneva). DNA was extracted from biofilms and fish communities were targeted using a standard 12S barcode with a metabarcoding approach. The fish eDNA signal recovered from biofilms revealed temporal and spatial changes in fish communities. These changes were linked to fish habitat preferences and spawning season. Peaks in the eDNA signal of some taxa fitted with their spawning period reported in the literature. We evidenced that the nature of the biofilm's substrate (natural or artificial) does not affect the image obtained of the fish community composition. Furthermore, by using biofilms grown on artificial substrates, the studied temporal window of the eDNA signal can be controlled. With biofilms acting as environmental passive samplers, our results open up the possibility to accurately monitor fish communities and their temporal and spatial changes with eDNA in a faster and less expensive way than with the classical water filtration approach

Exploring the capacity of aquatic biofilms to act as environmental DNA samplers: Test on macroinvertebrate communities in rivers

International audienceAquatic biofilms are heterogeneous assemblages of microorganisms surrounded by a matrix of extracellular polymeric substances (EPS). Recent studies suggest that aquatic biofilms can physically act as sorptive sponges of DNA. We took the opportunity from already available samples of stone biofilms and macroinvertebrates specimens collected in parallel at the same sites to test the capacity of biofilms to act as DNA samplers of macroinvertebrate communities in streams. Macroinvertebrate communities are usually studied with metabarcoding using the DNA extracted from their bodies bulk samples, which remains a time-consuming approach and involves the destruction of all individual specimens from the samples. The ability of biofilms to capture DNA was explored on 19 rivers sites of a tropical island (Mayotte Island, France). First, macroinvertebrate specimens were identified based on their morphological characteristics. Second, DNA was extracted from biofilms, and macroinvertebrate communities were targeted using a standard COI barcode. The resulting morphological and molecular inventories were compared. They provided comparable structures and diversities for macroinvertebrate communities when one is working with the unassigned OTU data. After taxonomic assignment of the OTU data, diversity and richness were no longer correlated. The ecological assessment derived from morphological bulk samples was conserved by the biofilms samples. We also showed that the biofilm method allows to detect a higher diversity for some organisms (Cnidaria), that is hardly accessible with the morphological method. The results of this study exploring the DNA signal captured by natural biofilms are encouraging. However, a more detailed study integrating more replicates and comparing the biodiversity signal based on both morphological and molecular bulk macroinvertebrate samples to the one captured by biofilms will be necessary. Better understanding how the DNA signal captured by natural biofilms represents the biodiversity of a given sampling site is necessary before considering its use for bioassessment applications

Fish eDNA metabarcoding from aquatic biofilm samples: Methodological aspects

International audienceFish eDNA metabarcoding is usually performed from filtered water samples. The volume of filtered water depends on the study scope and can rapidly become time consuming according to the number of samples that have to be processed. To avoid time allocated to filtration, passive DNA samplers have been used to recover fish eDNA from marine environments faster. In freshwater ecosystems, aquatic biofilms were used to catch eDNA from macroinvertebrates. Here, we test the capacity of aquatic biofilms to entrap fish eDNA in a large lake and, therefore, the possibility to perform fish eDNA metabarcoding from this matrix compared to the traditional fish eDNA approach from filtered water samples. Methodological aspects of the use of aquatic biofilms for fish eDNA metabarcoding (e.g. PCR replicates, biological replicates, bioinformatics pipeline, reference database and taxonomic assignment) were validated against a mock community. When using biofilms from habitats sheltered from wind and waves, biofilm and water approach provided similar inventories. Richness and diversity were comparable between both approaches. Approaches differed only for rare taxa. Our results illustrate the capacity of aquatic biofilms to act as passive eDNA samplers of fish eDNA and, therefore, the possibility to use biofilms to monitor fish communities efficiently from biofilms. Furthermore, our results open up avenues of research to study a diversity of biological groups (among which bioindicators as diatoms, macroinvertebrates and fish) from eDNA isolated from a single environmental matrix reducing sampling efforts, analysis time and costs

DNA metabarcoding and microscopic analyses of sea turtles biofilms: Complementary to understand turtle behavior.

Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior

Filling reference libraries with diatom environmental sequences: strengths and weaknesses

International audienc