Widespread, long-term admixture between grey wolves and domestic dogs across Eurasia and its implications for the conservation status of hybrids

Hybridisation between a domesticated species and its wild ancestor is an important conservation problem, especially if it results in the introgression of domestic gene variants into wild species. Nevertheless, the legal status of hybrids remains unregulated, partially because of the limited understanding of the hybridisation process and its consequences. The occurrence of hybridisation between grey wolves and domestic dogs is well-documented from different parts of the wolf geographic range, but little is known about the frequency of hybridisation events, their causes and the genetic impact on wolf populations. We analysed 61K SNPs spanning the canid genome in wolves from across Eurasia and North America and compared that data to similar data from dogs to identify signatures of admixture. The haplotype block analysis, which included 38 autosomes and the X chromosome, indicated the presence of individuals of mixed wolf-dog ancestry in most Eurasian wolf populations, but less admixture was present in North American populations. We found evidence for male-biased introgression of dog alleles into wolf populations, but also identified a first-generation hybrid resulting from mating between a female dog and a male wolf. We found small blocks of dog ancestry in the genomes of 62% Eurasian wolves studied and melanistic individuals with no signs of recent admixed ancestry, but with a dog-derived allele at a locus linked to melanism. Consequently, these results suggest that hybridisation has been occurring in different parts of Eurasia on multiple timescales and is not solely a recent phenomenon. Nevertheless, wolf populations have maintained genetic differentiation from dogs, suggesting that hybridisation at a low frequency does not diminish distinctiveness of the wolf gene pool. However, increased hybridisation frequency may be detrimental for wolf populations, stressing the need for genetic monitoring to assess the frequency and distribution of individuals resulting from recent admixture

Monitoring of species’ genetic diversity in Europe varies greatly and overlooks potential climate change impacts

Genetic monitoring of populations currently attracts interest in the context of the Convention on Biological Diversity but needs long-term planning and investments. However, genetic diversity has been largely neglected in biodiversity monitoring, and when addressed, it is treated separately, detached from other conservation issues, such as habitat alteration due to climate change. We report an accounting of efforts to monitor population genetic diversity in Europe (genetic monitoring effort, GME), the evaluation of which can help guide future capacity building and collaboration towards areas most in need of expanded monitoring. Overlaying GME with areas where the ranges of selected species of conservation interest approach current and future climate niche limits helps identify whether GME coincides with anticipated climate change effects on biodiversity. Our analysis suggests that country area, financial resources and conservation policy influence GME, high values of which only partially match species’ joint patterns of limits to suitable climatic conditions. Populations at trailing climatic niche margins probably hold genetic diversity that is important for adaptation to changing climate. Our results illuminate the need in Europe for expanded investment in genetic monitoring across climate gradients occupied by focal species, a need arguably greatest in southeastern European countries. This need could be met in part by expanding the European Union’s Birds and Habitats Directives to fully address the conservation and monitoring of genetic diversity

Molecular and biometric characterization of natural bream × roach hybrids population in the Dobczyce Reservoir (S Poland)

Natural hybrids of leuciscine cyprinids were investigated in a medium-sized submontane reservoir (49°52'N, 20°03'E, altitude 270 m) in the Carpathian part of the Vistula basin. The material included 380 hybrid specimens (TL: 24.0–39.8 cm, SL: 19.0–31.7 cm, W: 134–714 g) collected in 2006–2013. To detect their ancestry, genotypes of the 327 putative hybrids were compared to reference genotypes of 85 roaches, 115 common breams, 18 rudds, and 99 silver breams. Individuals were typed in 15 polymorphic microsatellite loci (Dubut et al. 2009), while species specific amplification of a fragment of mitochondrial cytochrome b gene was used to detect maternal origin of hybrids (Wyatt et al. 2006). Few individuals with traces of rudd and silver bream ancestry were excluded from the study. Analysis of mitochondrial DNA suggests bias towards bream maternal origin, with only 22 individuals (6,7%) with roach mitochondrial DNA. The analyzed set of hybrids was composed mostly of first generation bream × roach hybrids. Only 7 individuals (2,1%) showed a sign of backcrossing to bream, however, this finding has to be confirmed by analysis of further loci as it might result from presence of null alleles in roach. F2 generation hybrids were not detected. Biometric investigation of collected specimens comprised of selected morphometric (body height, and lengths of head, trunk, and tail) and meristic characters (counts of scales in lateral line and soft rays in anal fin) of individuals of different maternal origin. As all the analyzed differences between these categories of specimens appeared insignificant, the investigated population of hybrids may be regarded morphologically uniform

Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages

<div><p>The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.</p></div

Non-MHC immunity genes do not affect parasite load in European invasive populations of common raccoon

Abstract Understanding the evolutionary mechanisms behind invasion success enables predicting which alien species and populations are the most predisposed to become invasive. Parasites may mediate the success of biological invasions through their effect on host fitness. The evolution of increased competitive ability (EICA) hypothesis assumes that escape from parasites during the invasion process allows introduced species to decrease investment in immunity and allocate resources to dispersal and reproduction. Consequently, the selective pressure of parasites on host species in the invasive range should be relaxed. We used the case of the raccoon Procyon lotor invasion in Europe to investigate the effect of gastrointestinal pathogen pressure on non-MHC immune genetic diversity of newly established invasive populations. Despite distinct differences in parasite prevalence between analysed populations, we detected only marginal associations between two analysed SNPs and infection intensity. We argue that the differences in parasite prevalence are better explained by detected earlier associations with specific MHC-DRB alleles. While the escape from native parasites seems to allow decreased investment in overall immunity, which relaxes selective pressure imposed on immune genes, a wide range of MHC variants maintained in the invasive range may protect from newly encountered parasites

OVA and OVA-Cl bind to the same receptors, which are inhibited by mannan (Man), DS and CS and also shared with other HOCl-modified proteins and glycoproteins.

<p>(<b>A</b>, <b>B</b>, <b>C</b>) BM-DC were pre-incubated for 20 min with 1 mg/ml of indicated unlabelled proteins (A), 6 mg/ml Man, 0.2 mg/ml DS or CS (B, C) before the same volume of double-concentrated solution of AF-OVA or AF-OVA-Cl was added to give the final concentration of 5 μg/ml and the incubation was continued for 1 h (A, B) or 2 h (C) in a cell culture incubator. Following washing, cell-associated fluorescence was quantified by flow cytometry. (<b>D</b>) BM-DC were incubated for 1 h with 5 μg/ml AF-OVA-Cl, washed and either directly assessed for antigen uptake or incubated for another 1 h in medium alone before the cell-associated fluorescence was measured. (<b>E</b>) Following pre-incubation with DS or Man, BM-DC were incubated for 1 h on ice with 20 μg/ml DQ-OVA. Unbound DQ-OVA was washed out and the cells were either directly assessed for DQ-OVA binding (“4°C”) or transfer to 37°C for 2 h before the measurement. Results shown are averages ± SEM of triplicates obtained in single experiments, each repeated at least 3 times with similar results. Statistical analysis was performed with ANOVA, followed by the Tukey-Kramer post-test (A-C, E) or with the Student’s t-test (D). *, p < 0.05; NS, non-significant.</p

OVA-Cl exhibits increased immunogenicity that may be caused by enhanced uptake by APC.

<p>(<b>A</b>) BM-DC were incubated with indicated concentrations of OVA or OVA-Cl for 2 h at 37°C. When indicated, 200 ng/ml LPS was additionally included. Following washing, 0.4 × 10⁵ BM-DC were co-cultured for 2 or 3 days with 1.5 × 10⁵ CD4⁺ OT-II lymphocytes. One μCi of ³H-thymidine was added for the last 20 h of co-culture and radioactivity incorporated by proliferating lymphocytes was measured by scintillation counting. (<b>B</b>) BM-DC were incubated for 1 h at 37°C with indicated concentrations of Alexa Fluor 647-labelled OVA (AF-OVA) or OVA-Cl (AF-OVA-Cl) and, following washing, cell-associated fluorescence was measured by flow cytometry. (<b>C</b>) Unfractionated splenocytes, prepared from spleens of OT-II mice, we pre-incubated with OVA, OVA-Cl and LPS, as described in A, washed, plated at 2.5 × 10⁵/well in 0.2 ml of fresh medium and cultured for 2 days, for assessing lymphocyte proliferation, or 3 days, for assessing IFN-γ level in culture medium by ELISA. Results shown on graphs A-C are averages ± SEM of 2 (B), 3 (A) or 4 (C) replicates, obtained in single experiments which were repeated at least 3 times with similar results. (<b>D</b>) Expression of MHC-II and co-stimulatory molecules on the surface of BM-DC as well as splenic DC and macrophages was determined by flow cytometry. When indicated, BM-DC were pre-incubated overnight with LPS. Specific binding was calculated by subtracting binding of PE-conjugated control mAb from the total binding of specific mAb. The results shown are averages ±SEM from 4–6 independent experiments.</p

LPS and SR-A ligands regulate uptake, acidification and proteolysis of endocytosed antigens.

<p>(<b>A</b>) Effects of 1-day pre-treatment with 100 ng/ml LPS on the total uptake of AF-OVA-Cl, internalisation of pHr-OVA-Cl and degradation of DQ-OVA by BM-DC and PEM. (<b>B</b>, <b>C</b>) Effects on indicated ligands on the acidification of pHr-OVA-Cl-containing endosomes (B) and proteolytic digestion of DQ-OVA (C) in BM-DC and PEM. The results shown are averages +SEM from 3 independent experiments, each performed in 4 replicates. The data were analysed by the Student’s t-test (A) or by ANOVA, with the Dunnett’s post-test applied to compare the control (“medium”) with other groups (B, C). *, p < 0.05.</p

LOX-1 is capable of binding HOCl-modified proteins, but does not contribute to OVA-Cl uptake by BM-DC.

<p>(<b>A</b>) Binding of rLOX-1 to plate-adsorbed proteins. (<b>B</b>, <b>C</b>) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rLOX-1 binding to plate-adsorbed OVA-Cl (B) or GA-BSA (C). (<b>D</b>) Binding of PE-conjugated anti-mouse LOX-1 mAb and control rat IgG2a to CBA BM-DC, determined by flow cytometry. (<b>E</b>) The effect of blocking goat anti-mouse LOX-1 polyclonal Ab, relative to normal goat IgG, on AF-OVA-Cl uptake by untreated and LPS-pre-treated CBA BM-DC. (<b>F</b>) LOX-1 expression on LPS-pre-treated CBA BM-DC. Results of single experiments are shown, repeated at least twice with similar results. The data were analysed by the Student’s t-test (A, E) or by ANOVA, followed by the Tukey-Kramer post-test (B, C). *, p < 0.05; NS, non-significant.</p