Monitoring Strategy for Eight Amphibian Species in French Guiana,South America

Although dramatic amphibian declines have been documented worldwide, only few of such events have been quantitatively documented for the tropical forests of South America. This is due partly to the fact that tropical amphibians are patchily distributed and difficult to detect. We tested three methods often used to monitor population trends in amphibian species in a remote lowland tropical forest of French Guiana. These methods are capture-mark-recapture (CMR), estimation of the number of calling males with repeated counts data and distance sampling, and rates of occupancy inferred by presence/absence data. We monitored eight diurnal, terrestrial amphibian species including five Dendrobatidae and three Bufonidae. We found that CMR, the most precise way of estimating population size, can be used only with two species in high density patches where the recapture rate is high enough. Only for one of the species (Dendrobates tinctorius), a low coefficient of variation (CV = 0.19) can be achieved with 15 to 20 capture events. For dendrobatid species with day-calling males, audio surveys yield a better probability of detection with only 8 audio surveys needed; quantitative estimates can be achieved by computing the number of calling males inferred from audio counts or distance sampling analysis. We therefore suggest that an efficient monitoring protocol for Neotropical amphibian species should include a combination of sighting and audio techniques, and we discuss the need of implementing a large-scale monitoring in order to provide a baseline for comparison with future changes.     

Life history parameters and diet of Risso's dolphins,Grampus griseus,from southeastern South Africa

The life history of Risso's dolphins (Grampus griseus) remains poorly known and data from strandings can help provide important information. Data from 126 Risso's dolphins stranded or bycaught along the southeastern coastline of South Africa between 1958 and 2017 were analyzed in relation to their sex, age structure, and diet. Mean estimated length at birth was 146.9 cm, while maximum length was 325 cm for males and 313 cm for females; small sample sizes precluded detailed examination of sexual dimorphism. Age estimates for 33 individuals (14 males, 17 females, 2 unknown sex) indicated a maximum age of 13 years (males) and 17 years (females), respectively; the oldest animal was 19 years (unknown sex). Mean length and age at attainment of sexual maturity were estimated at 280 cm and 7.1 years in males and at 282 cm and 7.7 years in females. Stomach contents from 27 individuals showed that diets of immature and mature males and females overlapped and consisted predominantly of cephalopods. Reported strandings decreased between 2000 and 2017, possibly due to a lack of reporting associated with a ban on driving on beaches or related to the collapse of the local “chokka” squid (Loligo reynaudii) fishery in 2014–2015.     

Antibacterial and leishmanicidal activities of temporin-SHd,a 17-residue long membrane-damaging peptide

Temporins are a family of short antimicrobial peptides (8–17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF_amide), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.     

Genome Scale Evolution of Myxoma Virus Reveals Host-Pathogen Adaptation and Rapid Geographic Spread

The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.     

Role of Glycoside Phosphorylases in Mannose Foraging by Human Gut Bacteria

To metabolize both dietary fiber constituent carbohydrates and host glycans lining the intestinal epithelium, gut bacteria produce a wide range of carbohydrate-active enzymes, of which glycoside hydrolases are the main components. In this study, we describe the ability of phosphorylases to participate in the breakdown of human N-glycans, from an analysis of the substrate specificity of UhgbMP, a mannoside phosphorylase of the GH130 protein family discovered by functional metagenomics. UhgbMP is found to phosphorolyze β-d-Manp-1,4-β-d-GlcpNAc-1,4-d-GlcpNAc and is also a highly efficient enzyme to catalyze the synthesis of this precious N-glycan core oligosaccharide by reverse phosphorolysis. Analysis of sequence conservation within family GH130, mapped on a three-dimensional model of UhgbMP and supported by site-directed mutagenesis results, revealed two GH130 subfamilies and allowed the identification of key residues responsible for catalysis and substrate specificity. The analysis of the genomic context of 65 known GH130 sequences belonging to human gut bacteria indicates that the enzymes of the GH130_1 subfamily would be involved in mannan catabolism, whereas the enzymes belonging to the GH130_2 subfamily would rather work in synergy with glycoside hydrolases of the GH92 and GH18 families in the breakdown of N-glycans. The use of GH130 inhibitors as therapeutic agents or functional foods could thus be considered as an innovative strategy to inhibit N-glycan degradation, with the ultimate goal of protecting, or restoring, the epithelial barrier.     

Sequence Analysis of In Vivo Defective Interfering-Like RNA of Influenza A H1N1 Pandemic Virus

Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.     

Phenoscope: an automated large‐scale phenotyping platform offering high spatial homogeneity

Increased phenotyping accuracy and throughput are necessary to improve our understanding of quantitative variation and to be able to deconstruct complex traits such as those involved in growth responses to the environment. Still, only a few facilities are known to handle individual plants of small stature for non‐destructive, real‐time phenotype acquisition from plants grown in precisely adjusted and variable experimental conditions. Here, we describe Phenoscope, a high‐throughput phenotyping platform that has the unique feature of continuously rotating 735 individual pots over a table. It automatically adjusts watering and is equipped with a zenithal imaging system to monitor rosette size and expansion rate during the vegetative stage, with automatic image analysis allowing manual correction. When applied to Arabidopsis thaliana, we show that rotating the pots strongly reduced micro‐environmental disparity: heterogeneity in evaporation was cut by a factor of 2.5 and the number of replicates needed to detect a specific mild genotypic effect was reduced by a factor of 3. In addition, by controlling a large proportion of the micro‐environmental variance, other tangible sources of variance become noticeable. Overall, Phenoscope makes it possible to perform large‐scale experiments that would not be possible or reproducible by hand. When applied to a typical quantitative trait loci (QTL) mapping experiment, we show that mapping power is more limited by genetic complexity than phenotyping accuracy. This will help to draw a more general picture as to how genetic diversity shapes phenotypic variation.     

Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D₂O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d₁₀. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.     

Genetic diversity and phylogenetic analysis of native mountain ponies of Britain and Ireland reveals a novel rare population

The conservation of unique populations of animals is critical in order to preserve valuable genetic diversity and, where populations are free‐living, maintain their irreplaceable influence upon habitat ecology. An accurate assessment of genetic diversity and structure within and between populations is crucial in order to design and implement conservation strategies in natural and domesticated species. Moreover, where it is possible to identify relic populations that are related to a structured breed an ideal opportunity presents itself to model processes that reveal historical factors that have shaped genetic diversity. The origins of native UK mountain and moorland ponies are uncertain, but they may have directly descended from prehistoric populations and potentially harbour specific adaptations to the uplands of Britain and Ireland. To date, there have been no studies of population structure and genetic diversity present within a free‐living group of ponies in the Carneddau mountain range of North Wales. Herein, we describe the use of microsatellites and SNPs together with analysis of the mitochondrial control region to quantify the extent and magnitude of genetic diversity present in the feral Carneddau pony and relate this to several recognised British and Irish pony breeds. Our results establish that the feral Carneddau ponies represent a unique and distinctive population that merits recognition as a defined population and conservation priority. We discuss the implications for conservation of this population as a unique pool of genetic diversity adapted to the British uplands and potentially of particular value in maintaining the biodiversity of these habitats.