To denoise or to cluster? That is not the question. Optimizing pipelines for COI metabarcoding and metaphylogeography

Background: The recent blooming of metabarcoding applications to biodiversity studies comes with some relevant methodological debates. One such issue concerns the treatment of reads by denoising or by clustering methods, which have been wrongly presented as alternatives. It has also been suggested that denoised sequence variants should replace clusters as the basic unit of metabarcoding analyses, missing the fact that sequence clusters are a proxy for species-level entities, the basic unit in biodiversity studies. We argue here that methods developed and tested for ribosomal markers have been uncritically applied to highly variable markers such as cytochrome oxidase I (COI) without conceptual or operational (e.g., parameter setting) adjustment. COI has a naturally high intraspecies variability that should be assessed and reported, as it is a source of highly valuable information. We contend that denoising and clustering are not alternatives. Rather, they are complementary and both should be used together in COI metabarcoding pipelines. Results: Using a COI dataset from benthic marine communities, we compared two denoising procedures (based on the UNOISE3 and the DADA2 algorithms), set suitable parameters for denoising and clustering, and applied these steps in diferent orders. Our results indicated that the UNOISE3 algorithm preserved a higher intra-cluster variability. We introduce the program DnoisE to implement the UNOISE3 algorithm taking into account the natural variability (measured as entropy) of each codon position in protein-coding genes. This correction increased the number of sequences retained by 88%. The order of the steps (denoising and clustering) had little infuence on the fnal outcome. Conclusions: We highlight the need for combining denoising and clustering, with adequate choice of stringency parameters, in COI metabarcoding. We present a program that uses the coding properties of this marker to improve the denoising step. We recommend researchers to report their results in terms of both denoised sequences (a proxy for haplotypes) and clusters formed (a proxy for species), and to avoid collapsing the sequences of the latter into a single representative. This will allow studies at the cluster (ideally equating species-level diversity) and at the intra-cluster level, and will ease additivity and comparability between studies

DNA metabarcoding of littoral hard-bottom communities: high diversity and database gaps revealed by two molecular markers

Biodiversity assessment of marine hard-bottom communities is hindered by the high diversity and size-ranges of the organisms present. We developed a DNA metabarcoding protocol for biodiversity characterization of structurally complex natural marine hard-bottom communities. We used two molecular markers: the "Leray fragment" of mitochondrial cytochrome c oxidase (COI), for which a novel primer set was developed, and the V7 region of the nuclear small subunit ribosomal RNA (18S). Eight different shallow marine littoral communities from two National Parks in Spain (one in the Atlantic Ocean and another in the Mediterranean Sea) were studied. Samples were sieved into three size fractions from where DNA was extracted separately. Bayesian clustering was used for delimiting molecular operational taxonomic units (MOTUs) and custom reference databases were constructed for taxonomic assignment. Despite applying stringent filters, we found high values for MOTU richness (2,510 and 9,679 MOTUs with 18S and COI, respectively), suggesting that these communities host a large amount of yet undescribed eukaryotic biodiversity. Significant gaps are still found in sequence reference databases, which currently prevent the complete taxonomic assignment of the detected sequences. In our dataset, 85% of 18S MOTUs and 64% of COI MOTUs could be identified to phylum or lower taxonomic level. Nevertheless, those unassigned were mostly rare MOTUs with low numbers of reads, and assigned MOTUs comprised over 90% of the total sequence reads. The identification rate might be significantly improved in the future, as reference databases are further completed. Our results show that marine metabarcoding, currently applied mostly to plankton or sediments, can be adapted to structurally complex hard bottom samples. Thus, eukaryotic metabarcoding emerges as a robust, fast, objective and affordable method to comprehensively characterize the diversity of marine benthic communities dominated by macroscopic seaweeds and colonial or modular sessile metazoans. The 18S marker lacks species-level resolution and thus cannot be recommended to assess the detailed taxonomic composition of these communities. Our new universal primers for COI can potentially be used for biodiversity assessment with high taxonomic resolution in a wide array of marine, terrestrial or freshwater eukaryotic communities

Enjoying the warming Mediterranean: transcriptomic responses to temperature changes of a thermophilous keystone species in benthic communities

Information about the genomic processes underlying responses to temperature changes is still limited in non‐model marine invertebrates. In this sense, transcriptomic analyses can help to identify genes potentially related to thermal responses. We here investigated, via RNA‐seq, whole‐transcriptomic responses to increased and decreased temperatures in a thermophilous keystone sea urchin, Arbacia lixula, whose populations are increasing in the Mediterranean. This species is a key driver of benthic communities' structure due to its grazing activity. We found a strong response to experimentally induced cold temperature (7°C), with 1,181 differentially expressed transcripts relative to the control condition (13°C), compared to only 179 in the warm (22°C) treatment. A total of 84 (cold treatment) and three (warm treatment) gene ontology terms were linked to the differentially expressed transcripts. At 7°C the expression of genes encoding different heat shock proteins (HSPs) was upregulated, together with apoptotic suppressor genes (e.g., Bcl2), genes involved in the infection response and/or pathogen‐recognition (e.g., echinoidin) and ATP‐associated genes, while protein biosynthesis and DNA replication pathways were downregulated. At 22°C neither HSPs induction nor activation of the previously mentioned pathways were detected, with the exception of some apoptotic‐related activities that were upregulated. Our results suggest a strong transcriptional response associated with low temperatures, and support the idea of low water temperature being a major limitation for A. lixula expansion across deep Mediterranean and northern Atlantic waters

Facebook Ads Monitor: An Independent Auditing System for Political Ads on Facebook

The 2016 United States presidential election was marked by the abuse of targeted advertising on Facebook. Concerned with the risk of the same kind of abuse to happen in the 2018 Brazilian elections, we designed and deployed an independent auditing system to monitor political ads on Facebook in Brazil. To do that we first adapted a browser plugin to gather ads from the timeline of volunteers using Facebook. We managed to convince more than 2000 volunteers to help our project and install our tool. Then, we use a Convolution Neural Network (CNN) to detect political Facebook ads using word embeddings. To evaluate our approach, we manually label a data collection of 10k ads as political or non-political and then we provide an in-depth evaluation of proposed approach for identifying political ads by comparing it with classic supervised machine learning methods. Finally, we deployed a real system that shows the ads identified as related to politics. We noticed that not all political ads we detected were present in the Facebook Ad Library for political ads. Our results emphasize the importance of enforcement mechanisms for declaring political ads and the need for independent auditing platforms

Electrophoretic deposition of nanostructured-TiO2/chitosan composite coatings on stainless steel

Novel chitosan composite coatings containing titania nanoparticles (n-TiO2) for biomedical applications were developed by electrophoretic deposition (EPD) from ethanol–water suspensions. The optimal ethanol–water ratio was studied in order to avoid bubble formation during the EPD process and to ensure homogeneous coatings. Different n-TiO2 contents (0.5–10 g L−1) were studied for a fixed chitosan concentration (0.5 g L−1) and the properties of the electrophoretic coatings obtained were characterized. Coating composition was analyzed by thermogravimetric analysis (TG), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. Scanning electron microscopy (SEM) was employed to study both the surface and the cross section morphology of the coatings, and the thicknesses (2–6 μm) of the obtained coatings were correlated with the initial ceramic content. Contact angle measurements, as a preliminary study to predict hypothetic protein attachment on the coatings, were performed for different samples and the influence of a second chitosan layer on top of the coatings was also tested. Finally, the electrochemical behavior of the coatings, evaluated by polarization curves in DMEM at 37 °C, was studied in order to assess the corrosion resistance provided by the n-TiO2/chitosan coatings

East is East and West is West: Population genomics and hierarchical analyses reveal genetic structure and adaptation footprints in the keystone species Paracentrotus lividus (Echinoidea)

Aim The Atlanto‐Mediterranean edible purple sea urchin, Paracentrotus lividus, is a commercially exploited keystone species in benthic communities. Its browsing activity can deeply modify the littoral landscape, and changes in its abundance are of major conservation concern. This species is facing nowadays contrasting anthropogenic pressures linked to predator release, exploitation and sea warming. Management of this key species requires knowledge of its genetic structure, connectivity and local adaptation. Our goal was to assess the current global status of the species under a genomic perspective. Location Atlanto‐Mediterranean shores from Morocco and France to Turkey. Methods We used genotyping by sequencing (GBS) of 241 individuals belonging to 11 populations spanning the known range of distribution of the species. We obtained 3,348 loci for population genomics and outlier analyses. Results We identified significant genetic structure and a gradient matching the longitudinal position of the localities. A hierarchical analysis revealed two main clusters (Atlantic and Mediterranean) and subtler patterns of differentiation within them. Candidate markers for selection identified between and within these two main clusters were mostly different, likely indicating different selective pressures. Adaptation to maximum salinity and maximum temperature appeared as an important driver of the transition between Atlantic and Mediterranean basins. Other stressors, such as minimum temperature or range of temperature, seem to define the structuring within the Mediterranean

The two sides of the Mediterranean: Population genomics of the Black Sea urchin Arbacia lixula (Linnaeus 1758) in a Warming Sea

Global environmental changes may have a profound impact on ecosystems. In this context, it is crucial to gather biological and ecological information of the main species in marine communities to predict and mitigate potential effects of shifts in their distribution, abundance, and interactions. Using genotyping by sequencing (GBS), we assessed the genetic structure of a keystone species in the Mediterranean shallow littoral ecosystems, the black sea urchin Arbacia lixula. This bioengineer species can shape their communities due to its grazing activity and it is experiencing an ongoing expansion with increasing temperatures. The population genomic analyses on 5,241 loci sequenced in 240 individuals from 11 Mediterranean sampled populations revealed that all populations were diverse and showed significant departure from equilibrium. Albeit genetic differentiation was in general shallow, a significant break separated the western and eastern Mediterranean populations, a break not detected in previous studies with less resolutive markers. Notably, no clear effect of the Almería-Oran front, an important break in the Atlanto-Mediterranean transition, could be detected among the western basin populations, where only a slight differentiation of the two northernmost populations was found. Despite the generally low levels of genetic differentiation found, we identified candidate regions for local adaptation by combining different genomic analysis with environmental data. Salinity, rather than temperature, seemed to be an important driver of genetic structure in A. lixula. Overall, from a population genomics standpoint, there is ample scope for A. lixula to continue thriving and adapting in the warming Mediterranean

Efficiency of artificial collectors for quantitative assessment of sea urchin settlement rates

Summary: We tested the suitability of three different kinds of artificial collectors designed for quantitative assessment of echinoid settlement rates: (1) nylon nets containing plastic biofilter balls, (2) vertical scrub brushes with vegetal bristles and (3) horizontal triangular mats of coconut fibre. We measured the collecting efficiency by counting the number of post-larvae of two sea urchin species (Paracentrotus lividus and Arbacia lixula) gathered by each collector and deployed in two geographic areas: Tenerife (Canary Islands, eastern Atlantic) and Tossa de Mar (Costa Brava, northwestern Mediterranean). The plastic biofilter ball collector proved to be the most efficient design, collecting more settlers of both sea urchin species under all assayed conditions and showing a higher reproducibility than the other two designs. We therefore suggest using plastic biofilter balls in future studies aimed at quantifying echinoid settlement rates

The physical oceanography of the transport of floating marine debris

Marine plastic debris floating on the ocean surface is a major environmental problem. However, its distribution in the ocean is poorly mapped, and most of the plastic waste estimated to have entered the ocean from land is unaccounted for. Better understanding of how plastic debris is transported from coastal and marine sources is crucial to quantify and close the global inventory of marine plastics, which in turn represents critical information for mitigation or policy strategies. At the same time, plastic is a unique tracer that provides an opportunity to learn more about the physics and dynamics of our ocean across multiple scales, from the Ekman convergence in basin-scale gyres to individual waves in the surfzone. In this review, we comprehensively discuss what is known about the different processes that govern the transport of floating marine plastic debris in both the open ocean and the coastal zones, based on the published literature and referring to insights from neighbouring fields such as oil spill dispersion, marine safety recovery, plankton connectivity, and others. We discuss how measurements of marine plastics (both in situ and in the laboratory), remote sensing, and numerical simulations can elucidate these processes and their interactions across spatio-temporal scales

Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.Peer ReviewedPostprint (published version