Exploring the potential of metabarcoding to disentangle macroinvertebrate community dynamics in intermittent streams

Taxonomic sufficiency represents the level of taxonomic detail needed to detect ecological patterns to a level that match the requirement of a study. Most bioassessments apply the taxonomic sufficiency concept and assign specimens to the family or genus level given time constraints and the difficulty to correctly identify species. This holds particularly true for stream invertebrates because small and morphologically similar larvae are hard to distinguish. Low taxonomic resolution may hinder detecting true community dynamics, which thus leads to incorrect inferences about community assembly processes. DNA metabarcoding is a new, affordable and cost-effective tool for the identification of multiple species from bulk samples of organisms. As it provides high taxonomic resolution, it can be used to compare results obtained from different identification levels. Measuring the effect of taxonomic resolution on the detection of community dynamics is especially interesting in extreme ecosystems like intermittent streams to test if species at intermittent sites are subsets of those from perennial sources or if independently recruiting taxa exist. Here we aimed to compare the performance of morphological identification and metabarcoding to detect macroinvertebrate community dynamics in the Trebbia River (Italy). Macroinvertebrates were collected from four perennial and two intermittent sites two months after flow resumption and before the next dry phase. The identification level ranged from family to haplotype. Metabarcoding and morphological identifications found similar alpha diversity patterns when looking at family and mixed taxonomic levels. Increasing taxonomic resolution with metabarcoding revealed a strong partitioning of beta diversity in nestedness and turnover components. At flow resumption, beta diversity at intermittent sites was dominated by nestedness when family-level information was employed, while turnover was evidenced as the most important component when using Operational Taxonomic Units (OTUs) or haplotypes. The increased taxonomic resolution with metabarcoding allowed us to detect species adapted to deal with intermittency, like the chironomid Cricotopus bicinctus and the ephemeropteran Cloeon dipterum. Our study thus shows that family and mixed taxonomic level are not sufficient to detect all aspects of macroinvertebrate community dynamics. High taxonomic resolution is especially important for intermittent streams where accurate information about species-specific habitat preference is needed to interpret diversity patterns induced by drying and the nestedness/ turnover components of beta diversity are of interest to understand community assembly processes

The onset and solidification path of a basaltic melt by in situ differential scanning calorimetry (DSC) and ex situ investigations

The in situ differential scanning calorimetry (DSC) technique has been applied to investigate the solidification paths of a basaltic liquid. The starting glass was heated up to 1300◦C, kept at this superliquidus temperature for 2 h and cooled at rates (∆T/∆t) of 7, 60, 180, 1000, and 1800◦C/h, down to 800 and 600◦C. Glass transition temperature (Tg), crystallization temperature (Tx_HR) and melting temperature (Tm) were measured by in situ DSC spectra on heating. Tx measured along the cooling paths (Tx_CR) shows exothermic peaks that change from a single symmetric shape (7 and 60◦C/h) to multi-component patterns (180, 1000, and 1800◦C/h). The recovered products characterized by field emission gun source of the scanning electron microscopy and electron probe micro-analyzer-wavelength dispersive spectrometers show a phase assemblage of spinel (sp), clinopyroxene (cpx), melilite (mel), plagioclase (plg), and glass. Moreover, crystal size distributions (CSDs) and growth rates (Gmax and GCSD) were also determined. The crystal content slightly increases from 7 to 1800◦C/h. Faceted sp are present in all the run products with an amount always <2 area%. Cpx increases from 7 to 1800◦C/h, changing its texture from almost faceted to dendritic between 60 and 180◦C/h. The area% of mel follows an asymmetric Gaussian trend, while plg nucleates only at 7◦C/h with a content <2 area%. The coupling of DSC and SEM outcomes indicate that sp nucleate first, followed by cpx and mel (and/or plg). The increment of ∆T/∆t causes an increase of the CSD slope (m) and crystal population density per size (n0 ), as well as a decrease of the crystal size, for both cpx and sp. The log-linear CSD segments with different slopes at 7 and 60◦C/h suggest multiple nucleation events and crystal growth by coarsening. Gmax and GCSD for cpx and sp directly measured on the actual crystallization time by DSC spectra, both increase with the increasing of ∆T/∆t. The onset temperature of crystallization (Txi ) decreases as ∆T/∆t increases, following an exponential trend that defines the uppermost portion of a time-transformation-temperature-like curve. This analytical model allows us to quantitatively model the kinetic crystallization paths of dry basalts

Non‐specific amplification compromises environmental DNA metabarcoding with COI

1. Metabarcoding extra-organismal DNA from environmental samples is now a key technique in aquatic biomonitoring and ecosystem health assessment. However, choice of genetic marker and primer set is a critical consideration when designing experiments, especially so when developing community standards and legislative frameworks. Mitochondrial cytochrome c oxidase subunit I (COI), the standard DNA barcode marker for animals, with its extensive reference library, taxonomic discriminatory power, and predictable sequence variation, is the natural choice for many metabarcoding applications such as the bulk sequencing of invertebrates. However, the overall utility of COI for environmental sequencing of targeted taxonomic groups has yet to be fully scrutinised. 2. Here, by using a case study of marine and freshwater fishes from the British Isles, we quantify the in silico performance of twelve mitochondrial primer pairs from COI, cytochrome b, 12S and 16S, in terms of reference library coverage, taxonomic discriminatory power, and primer universality. We subsequently test in vitro three COI primer pairs and one 12S pair for their specificity, reproducibility, and congruence with independent datasets derived from traditional survey methods at five estuarine and coastal sites in the English Channel and North Sea coast. 3. Our results show that for aqueous extra-organismal DNA at low template concentrations, both metazoan and fish-targeted COI primers perform poorly in comparison to 12S, exhibiting low levels of reproducibility due to non-specific amplification of prokaryotic and non-target eukaryotic DNAs. 4. An ideal metabarcode would have an extensive reference library for which custom primer sets can be designed for either broad assessments of biodiversity or taxon specific surveys, but unfortunately, low primer specificity hinders the use of COI, while the paucity of reference sequences is problematic for 12S. The latter, however, can be mitigated by expanding the concept of DNA barcodes to include whole mitochondrial genomes generated by genome-skimming existing tissue collections

Troglitazone suppresses telomerase activity independently of PPARγ in estrogen-receptor negative breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Breast cancer is one the highest causes of female cancer death worldwide. Many standard chemotherapeutic agents currently used to treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In recent years, more specific targeted therapies have been introduced. It is known that telomerase is active in over 90% of breast cancer tumors but inactive in adjacent normal tissues. The prevalence of active telomerase in breast cancer patients makes telomerase an attractive therapeutic target. Recent evidence suggests that telomerase activity can be suppressed by peroxisome proliferator activated receptor gamma (PPARγ). However, its effect on telomerase regulation in breast cancer has not been investigated.</p> <p>Methods</p> <p>In this study, we investigated the effect of the PPARγ ligand, troglitazone, on telomerase activity in the MDA-MB-231 breast cancer cell line. Real time RT-PCR and telomerase activity assays were used to evaluate the effect of troglitazone. MDA-MB-231 cells had PPARγ expression silenced using shRNA interference.</p> <p>Results</p> <p>We demonstrated that troglitazone reduced the mRNA expression of hTERT and telomerase activity in the MDA-MB-231 breast cancer cell line. Troglitazone reduced telomerase activity even in the absence of PPARγ. In agreement with this result, we found no correlation between PPARγ and hTERT mRNA transcript levels in breast cancer patients. Statistical significance was determined using Pearson correlation and the paired Student's <it>t </it>test.</p> <p>Conclusions</p> <p>To our knowledge, this is the first time that the effect of troglitazone on telomerase activity in breast cancer cells has been investigated. Our data suggest that troglitazone may be used as an anti-telomerase agent; however, the mechanism underlying this inhibitory effect remains to be determined.</p

Connecting high-throughput biodiversity inventories: Opportunities for a site-based genomic framework for global integration and synthesis

High‐throughput sequencing (HTS) is increasingly being used for the characterization and monitoring of biodiversity. If applied in a structured way, across broad geographical scales, it offers the potential for a much deeper understanding of global biodiversity through the integration of massive quantities of molecular inventory data generated independently at local, regional and global scales. The universality, reliability and efficiency of HTS data can potentially facilitate the seamless linking of data among species assemblages from different sites, at different hierarchical levels of diversity, for any taxonomic group and regardless of prior taxonomic knowledge. However, collective international efforts are required to optimally exploit the potential of site‐based HTS data for global integration and synthesis, efforts that at present are limited to the microbial domain. To contribute to the development of an analogous strategy for the nonmicrobial terrestrial domain, an international symposium entitled “Next Generation Biodiversity Monitoring” was held in November 2019 in Nicosia (Cyprus). The symposium brought together evolutionary geneticists, ecologists and biodiversity scientists involved in diverse regional and global initiatives using HTS as a core tool for biodiversity assessment. In this review, we summarize the consensus that emerged from the 3‐day symposium. We converged on the opinion that an effective terrestrial Genomic Observatories network for global biodiversity integration and synthesis should be spatially led and strategically united under the umbrella of the metabarcoding approach. Subsequently, we outline an HTS‐based strategy to collectively build an integrative framework for site‐based biodiversity data generation