High‐throughput sequencing on preservative ethanol is effective at jointly examining infraspecific and taxonomic diversity,although bioinformatics pipelines do not perform equally

1. High‐throughput sequencing of amplicons (HTSA) has been proposed as an effective approach to evaluate taxonomic and genetic diversity at the same time. However, there are still uncertainties as to how the results produced by different bioinformatics treatments impact the conclusions drawn on biodiversity and population genetics indices.
2. We evaluated the ability of six bioinformatics pipelines to recover taxonomic and genetic diversity from HTSA data obtained from controlled assemblages. To that end, 20 assemblages were produced using 354 colonies of Botrylloides spp., sampled in the wild in ten marinas around Brittany (France). We used DNA extracted from preservative ethanol (ebDNA) after various time of storage (3, 6, and 12 months), and from a bulk of preserved specimens (bulkDNA). DNA was amplified with primers designed for targeting this ascidian genus. Results obtained from HTSA data were compared with Sanger sequencing on individual zooids (i.e., individual barcoding).
3. Species identification and relative abundance determined with HTSA data from either ebDNA or bulkDNA were similar to those obtained with traditional individual barcoding. However, after 12 months of storage, the correlation between HTSA and individual‐based data was lower than after shorter durations. The six bioinformatics pipelines were able to depict accurately the genetic diversity using standard population genetics indices (H_S and F_ST), despite producing false positives and missing rare haplotypes. However, they did not perform equally and dada2 was the only pipeline able to retrieve all expected haplotypes.
4. This study showed that ebDNA is a nondestructive alternative for both species identification and haplotype recovery, providing storage does not last more than 6 months before DNA extraction. Choosing the bioinformatics pipeline is a matter of compromise, aiming to retrieve all true haplotypes while avoiding false positives. We here recommend to process HTSA data using dada2, including a chimera‐removal step. Even if the possibility to use multiplexed primer sets deserves further investigation to expand the taxonomic coverage in future similar studies, we showed that primers targeting a particular genus allowed to reliably analyze this genus within a complex community.

     

Targeted-antibacterial-plasmids (TAPs) combining conjugation and CRISPR/Cas systems achieve strain-specific antibacterial activity

The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on targeted-antibacterial-plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step toward the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.     

Tobacco pollen tubes as cellular models for ion dynamics: improved spatial and temporal resolution of extracellular flux and free cytosolic concentration of calcium and protons using pHluorin and YC3.1 CaMeleon

The presence of both calcium (Ca²⁺) and proton (H⁺) apical gradients is necessary for polarized cell elongation to occur in pollen tubes. So far, most of these studies have been carried out in lily pollen tubes, using chemical probes. Yet, lily is a refractory model for molecular genetics, with no easy protocol available for the construction of stable transgenic lines. Tobacco, however, is well suited for both transformation and cell biology, with sexual organs that are accessible, easy to handle and visualize. Pollen tubes are in an ideal size range for sub-cellular imaging analyses using modern microscopy techniques. Ion homeostasis in tobacco pollen tubes has not been precisely characterized so far. Here, we characterize the H⁺ and Ca²⁺ spatial and temporal patterns in tobacco pollen tubes by the use of two fluorescent genetic probes, pHluorin and the YC3.1 yellow CaMeleon, and direct measurement of extracellular flux by ion-sensitive vibrating probes. A distinct 0.4 pH unit acidic gradient was found to stretch from the tip up to 40 μm into the tube shank. This gradient intensity displayed 1–4 min period oscillations and is reduced in the non-growing phase of an oscillatory cycle. Furthermore, sub-membrane and sub-apical alkaline domains were detected. Extracellular H⁺ fluxes oscillated between 10 and 40 pmol cm⁻² s⁻¹. Fourier and continuous wavelet analyses showed tubes with one or two major oscillatory components in both extra and intracellular H⁺ oscillations. Cytosolic Ca²⁺ was imaged by confocal microscopy, showing a V-shaped 40 μm gradient extending from the tip, from 0.2 to 1.0 μM, which oscillates with a 1–4 min period, but with only one major oscillatory component. Extracellular Ca²⁺ fluxes oscillate in most pollen tubes, between 2 and 50 pmol cm⁻² min⁻¹ and, like in H⁺, with one or two major oscillatory peaks. A combination of confocal and widefield microscopy showed that H⁺ and Ca²⁺ displayed different patterns and shapes inside the cell, sometimes suggesting a structurally complementary role for these 2 second messengers in the growth process. These data suggest that fluxes at the apex of the pollen tube are directly responsible for establishment and maintenance of the gradient. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of a New Set of Polypeptidic Sex Pheromones from Cuttlefish (Sepia officinalis)

Marine Biotechnology - The common English Channel cuttlefish (Sepia officinalis) reproduces every year on very localized coastal spawning areas after a west–east horizontal migration of...     

Within-thallus variation in polyphenolic content and antifouling activity in Sargassum vulgare

The brown seaweed Sargassum vulgare C. Agardh abounds along the southeast coast of Rio de Janeiro State, Brazil, creating unique habitats, and its polyphenols play an important ecological role as antifouling agents. In order to understand more precisely this defensive strategy of S. vulgare, we collected this macroalga at Ilha de Itacuruçá in May and October 2009. Thalli were separated according to tissue type: pneumatocysts, receptacles, leaflets, and axis. Phenolic extracts from each specific part of the plant were obtained and the associated antifouling activity was tested in order to assess whether a tissue specialisation/antifouling activity pattern exists. Preliminary separation process was carried out on a phenolic extract to separate the polyphenols from the remaining compounds. Such fractionation allowed us to study the involvement of strictly phenolic compounds in antifouling activity. Within-thallus variation in both phenolic content and antifouling activity were highlighted, and seasonal variation in both those characters was also observed. For both experiments, pneumatocyst and leaflet phenolic extracts were the most active against the attachment of Perna perna, but no correlation was observed between phenolic concentration and antifouling activity. On the contrary, the extract containing only phenolic compounds was twice less active than the extract from which 80 % of the polyphenols were removed. These results led us to hypothesise that polar compounds other than polyphenols are involved in the antifouling activity.     

Toxicity impacts in the environmental footprint method: calculation principles

The International Journal of Life Cycle Assessment - The EU environmental footprint (EF) is a life cycle assessment (LCA)-based method which aims at assessing the environmental impacts of...     

The effect of compact formulations on the environmental profile of northern european granular laundry detergents

Regular (1988) and compact granular (1992, 1998) laundry detergents were compared on the basis of two distinct, complementary approaches: Environmental Risk Assessment and Life-Cycle Assessment. The results are presented in this paper and an accompanying paper in this volume (Part II: Life-Cycle Assessment). Exposure data from The Netherlands and Sweden were used for this retrospective analysis. The time period studied (1988–1998) spans many innovations in laundry detergents, one of which was the introduction of compact detergents. The aquatic risk assessment resulted in risk quotients below 1 for all detergent ingredients in both countries over the period studied. Furthermore, it showed that risk quotients decrease two to five-fold between 1988 and 1998 in each country due to the introduction of compact detergents. Slightly lower risk quotients were observed in Sweden, when compared to The Netherlands, attributable to the lower water hardness resulting in lower detergent usage per wash cycle in that country. If water hardnesses were equal, the outcome of the product risk assessments would also be the same in the two countries.