Noninvasive population genetics: a review of sample source, diet, fragment length and microsatellite motif effects on amplification success and genotyping error rates

International audienceNoninvasive population genetics has found many applications in ecology and conservation biology. However, the technical difficulties inherent to the analysis of low quantities of DNA generally tend to limit the efficiency of this approach. The nature of samples and loci used in noninvasive population genetics are important factors that may help increasing the potential success of case studies. Here we reviewed the effects of the source of DNA (hair vs. faeces), the diet of focal species, the length of mitochondrial DNA fragments, and the length and repeat motif of nuclear microsatellite loci on genotyping success (amplification success and rate of allelic dropout). Locus-specific effects appeared to have the greatest impact, amplification success decreasing with both mitochondrial and microsatellite fragments' length, while error rates increase with amplicons' length. Dinucleotides showed best amplification success and lower error rates compared to longer repeat units. Genotyping success did not differ between hair- versus faeces-extracted DNA, and success in faeces-based analyses was not consistently influenced by the diet of focal species. While the great remaining variability among studies implies that other unidentified parameters are acting, results show that the careful choice of genetic markers may allow optimizing the success of noninvasive approaches

Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians

Buccal swabs have recently been used as a minimally invasive sampling method in genetic studies of wild populations, including amphibian species. Yet it is not known to date what is the level of reliability for microsatellite genotypes obtained using such samples. Allelic dropout and false alleles may affect the genotyping derived from buccal samples. Here we quantified the success of microsatellite amplification and the rates of genotyping errors using buccal swabs in two amphibian species, the Alpine newt Triturus alpestris and the Green tree frog Hyla arborea, and we estimated two important parameters for downstream analyses, namely the number of repetitions required to achieve typing reliability and the probability of identity among genotypes. Amplification success was high, and only one locus tested required two to three repetitions to achieve reliable genotypes, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. This sampling method which allows avoiding the controversial toe-clipping will likely prove very useful in the context of amphibian conservation

Hypoxia Reduces the Pathogenicity of Pseudomonas Aeruginosa by Decreasing the Expression of Multiple Virulence Factors

Our understanding of how the course of opportunistic bacterial infection is influenced by the microenvironment is limited. We demonstrate that the pathogenicity of Pseudomonas aeruginosa strains derived from acute clinical infections is higher than that of strains derived from chronic infections, where tissues are hypoxic. Exposure to hypoxia attenuated the pathogenicity of strains from acute (but not chronic) infections, implicating a role for hypoxia in regulating bacterial virulence. Mass spectrometric analysis of the secretome of P. aeruginosa derived from an acute infection revealed hypoxia-induced repression of multiple virulence factors independent of altered bacterial growth. Pseudomonas aeruginosa lacking the Pseudomonas prolyl-hydroxylase domain-containing protein, which has been implicated in bacterial oxygen sensing, displays reduced virulence factor expression. Furthermore, pharmacological hydroxylase inhibition reduces virulence factor expression and pathogenicity in a murine model of pneumonia. We hypothesize that hypoxia reduces P. aeruginosa virulence at least in part through the regulation of bacterial hydroxylases

The evolution of strong reproductive isolation between sympatric intertidal snails

The evolution of strong reproductive isolation (RI) is fundamental to the origins and maintenance of biological diversity, especially in situations where geographical distributions of taxa broadly overlap. But what is the history behind strong barriers currently acting in sympatry? Using whole-genome sequencing and single nucleotide polymorphism genotyping, we inferred (i) the evolutionary relationships, (ii) the strength of RI, and (iii) the demographic history of divergence between two broadly sympatric taxa of intertidal snail. Despite being cryptic, based on external morphology, Littorina arcana and Littorina saxatilis differ in their mode of female reproduction (egg-laying versus brooding), which may generate a strong post-zygotic barrier. We show that egg-laying and brooding snails are closely related, but genetically distinct. Genotyping of 3092 snails from three locations failed to recover any recent hybrid or backcrossed individuals, confirming that RI is strong. There was, however, evidence for a very low level of asymmetrical introgression, suggesting that isolation remains incomplete. The presence of strong, asymmetrical RI was further supported by demographic analysis of these populations. Although the taxa are currently broadly sympatric, demographic modelling suggests that they initially diverged during a short period of geographical separation involving very low gene flow. Our study suggests that some geographical separation may kick-start the evolution of strong RI, facilitating subsequent coexistence of taxa in sympatry. The strength of RI needed to achieve sympatry and the subsequent effect of sympatry on RI remain open questions. This article is part of the theme issue ‘Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'

Whole-genome phylogeography of the intertidal snail Littorina saxatilis

Understanding the factors that have shaped the current distributions and diversity of species is a central and longstanding aim of evolutionary biology. The recent inclusion of genomic data into phylogeographic studies has dramatically improved our understanding in organisms where evolutionary relationships have been challenging to infer. We used whole-genome sequences to study the phylogeography of the intertidal snail Littorina saxatilis, which has successfully colonized and diversified across a broad range of coastal environments in the Northern Hemisphere amid repeated cycles of glaciation. Building on past studies based on short DNA sequences, we used genome-wide data to provide a clearer picture of the relationships among samples spanning most of the species natural range. Our results confirm the trans-Atlantic colonization of North America from Europe, and have allowed us to identify rough locations of glacial refugia and to infer likely routes of colonization within Europe. We also investigated the signals in different datasets to account for the effects of genomic architecture and non-neutral evolution, which provides new insights about diversification of four ecotypes of L. saxatilis (the crab, wave, barnacle, and brackish ecotypes) at different spatial scales. Overall, we provide a much clearer picture of the biogeography of L. saxatilis, providing a foundation for more detailed phylogenomic and demographic studies

Heat stress causes spatially-distinct membrane re-modelling in K562 leukemia cells

Cellular membranes respond rapidly to various environmental perturbations. Previously we showed that modulations in membrane fluidity achieved by heat stress (HS) resulted in pronounced membrane organization alterations which could be intimately linked to the expression and cellular distribution of heat shock proteins. Here we examine heat-induced membrane changes using several visualisation methods. With Laurdan two-photon microscopy we demonstrate that, in contrast to the enhanced formation of ordered domains in surface membranes, the molecular disorder is significantly elevated within the internal membranes of cells preexposed to mild HS. These results were compared with those obtained by anisotropy, fluorescence lifetime and electron paramagnetic resonance measurements. All probes detected membrane changes upon HS. However, the structurally different probes revealed substantially distinct alterations in membrane heterogeneity. These data call attention to the careful interpretation of results obtained with only a single label. Subtle changes in membrane microstructure in the decision-making of thermal cell killing could have potential application in cancer therapy

Cell death regulation during influenza A virus infection by matrix (M1) protein: a model of viral control over the cellular survival pathway

During early infection, viruses activate cellular stress-response proteins such as heat-shock proteins (Hsps) to counteract apoptosis, but later on, they modulate these proteins to stimulate apoptosis for efficient viral dissemination. Hsp70 has been attributed to modulate viral entry, transcription, nuclear translocation and virion formation. It also exerts its anti-apoptotic function by binding to apoptosis protease-activating factor 1 (Apaf-1) and disrupting apoptosome formation. Here, we show that influenza A virus can regulate the anti-apoptotic function of Hsp70 through viral protein M1 (matrix 1). M1 itself did not induce apoptosis, but enhanced the effects of apoptotic inducers. M1-small-interfering RNA inhibits virus-induced apoptosis in cells after either virus infection or overexpression of the M1 protein. M1 binds to Hsp70, which results in reduced interaction between Hsp70 and Apaf-1. In a cell-free system, the M1 protein mediates procaspase-9 activation induced by cytochrome c/deoxyadenosine triphosphate. A study involving deletion mutants confirmed the role of the C-terminus substrate-binding domain (EEVD) of Hsp70 and amino acids 128–165 of M1 for this association. The M1 mutants, which did not co-immunoprecipitate with Hsp70, failed to induce apoptosis. Overall, the study confirms the proapoptotic function of the M1 protein during influenza virus infection

Hsp60 Is Actively Secreted by Human Tumor Cells

Background: Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. Methodology/Principal Findings: Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. Conclusions/Significance: Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likel

Retracing the history and planning the future of the red squirrel (Sciurus vulgaris) in Ireland using non-invasive genetics

The Eurasian red squirrel’s (Sciurus vulgaris) history in Ireland is largely unknown, but the original population is thought to have been driven to extinction by humans in the 17th Century, and multiple records exist for its subsequent reintroduction in the 19th 4 Century. However, it is currently unknown how these reintroductions affect the red squirrel population today, or may do so in the future. In this study, we report on the development of a DNA toolkit for the non-invasive genetic study of the red squirrel. Non-invasively collected red squirrel samples were combined with other samples collected throughout Ireland and previously published mitochondrial DNA (mtDNA) data from Ireland, Great Britain and continental Europe to give an insight into population genetics and historical introductions of the red squirrel in Ireland. Our findings demonstrate that the Irish red squirrel population is on a national scale quite genetically diverse, but at a local level contains relatively low levels of genetic diversity and evidence of genetic structure. This is likely an artefact of the introduction of a small number of genetically similar animals to specific sites. A lack of continuous woodland cover in Ireland has prevented further mixing with animals of different origins that may have been introduced even to neighbouring sites. Consequently, some of these genetically isolated populations are or may in the future be at risk of extinction. The Irish red squirrel population contains mtDNA haplotypes of both a British and Continental European origin, the former of which are now extinct or simply not recorded in contemporary Great Britain. The Irish population is therefore important in terms of red squirrel conservation not only in Ireland, but also for Great Britain, and should be appropriately managed

The consolidated European synthesis of CO2emissions and removals for the European Union and United Kingdom : 1990-2018

Acknowledgements FAOSTAT statistics are produced and disseminated with the support of its member countries to the FAO regular budget. Philippe Ciais acknowledges the support of the European Research Council Synergy project SyG-2013-610028 IMBALANCE-P and from the ANR CLAND Convergence Institute. We acknowledge the work of the entire EDGAR group (Marilena Muntean, Diego Guizzardi, Edwin Schaaf and Jos Olivier). We acknowledge Stephen Sitch and the authors of the DGVMs TRENDY v7 ensemble models for providing us with the data. Financial support This research has been supported by the H2020 European Research Council (grant no. 776810).Peer reviewedPublisher PD