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Proteases catalyze hydrolysis of amide bonds within peptides and proteins, therefore they play crucial functions for organism functioning, but also in industry to facilitate numerous processes. Feather-degrading fungus Onygena corvina (O. corvina) is loaded with numerous proteases that can be utilized for variety of applications. The most active species of these enzymes is heat-sensitive serine protease (NHSSP), from O. corvina fungi and due to its potential applications in industry is an alternative to proteinase K. The uniqueness of NHSSP relies on the ability of this enzyme to hydrolyze peptides at neutral to acidic pH values between 5.0 and 8.5, with an optimum of 6.8 and a temperature activity ranging from 15 to 50 °C making NHSSP exceptionally universal enzyme. Thus, we have performed the in-depth characterization of NHSSP substrate specificity by using a positional scanning substrate combinatorial library (PS-SCL). Afterward, we obtained a set of fluorescent substrates hydrolyzed by NHSSP that served as a leading sequence for the first tailored covalent inhibitor of this enzyme, containing a diphenylphosphonate as a warhead and MeOSuc amine protecting group. Our first inhibitor for NHSSP binds potently with target protease and is a tool for future study of this enzyme functions.
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Immunogenetic variation in natural vertebrate populations is expected to respond to spatial and temporal fluctuations in pathogen assemblages. While spatial heterogeneity in pathogen-driven selection enhances local immunogenetic adaptations and population divergence, different immune genes may yield contrasting responses to the environment. Here, we investigated population differentiation at the key pathogen recognition genes of the innate and adaptive immune system in a colonial bird species, the black-headed gull Chroicocephalus ridibundus. We assessed genetic variation at three toll-like receptor (TLR) genes (innate immunity) and the major histocompatibility complex (MHC) class I and II genes (adaptive immunity) in gulls from seven colonies scattered across Poland. As expected, we found much greater polymorphism at the MHC than TLRs. Population differentiation at the MHC class II, but not MHC-I, was significantly stronger than at neutral microsatellite loci, suggesting local adaptation. This could reflect spatial variation in the composition of extracellular parasite communities (e.g., helminths), possibly driven by sharp differences in habitat structure between colonies. Despite contrasting patterns of population differentiation, both MHC classes showed similar regimes of diversifying selection. Some significant population differentiation was also observed at TLRs, suggesting that innate immune receptors may respond to fine-scale spatial variation in pathogen pressure, although this pattern could have been enhanced by drift. Our results suggested that local adaptation at the pathogen recognition immune genes can be maintained at relatively small or moderate spatial scales in species with high dispersal potential and they highlighted the complexity of immunogenetic responses of animals to heterogeneous environments.
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Oxidative metabolism is a key component of organismal physiology and it is primarily determined by aerobic capacity, which depends on the capacity of blood to carry oxygen. However, experimental manipulations of blood oxygen-carrying capacity are rarely implemented to test ecophysiological hypotheses in vertebrate populations. Here, we combined an experimental manipulation of blood oxygen-carrying capacity with GPS tracking to test whether suboptimal (reduced) haematological performance affects foraging behaviour in a colonial waterbird, the black-headed gull, Chroicocephalus ridibundus. First, a validation of phenylhydrazine (PHZ) treatment in gulls revealed a 9-18% reduction in haematocrit and blood haemoglobin concentration (via oxidative denaturation and haemolysis of erythrocytes). Then, GPS tracking of experimental (PHZ-treated) and control (saline-treated) gulls during the incubation period provided no support for reduced or suspended engagement in energetically costly activities (long-distance foraging trips) by experimental birds. Instead, we found evidence for fine-scale alterations in foraging behaviour of PHZ-treated individuals, which resulted in fewer foraging trips per unit time, but trips that were longer in duration and distance compared with those of control birds. This suggests reduced foraging performance of experimental birds (e.g. lower capacity to find and collect food during trips) or evasion of social competition, although no differences in the total investment in foraging may also suggest compensatory physiological responses to haemolytic anaemia. Our study contributes to a better understanding of the physio-ecological nexus in non-diving colonial avian species. Whether behavioural effects of reduced aerobic capacity have any implications for gull condition and reproductive performance should be the subject of further investigation.
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Charadriiformes , Conservación de los Recursos Naturales , Humanos , Animales , Aves/fisiología , Conducta Alimentaria/fisiologíaRESUMEN
Toll-like receptors (TLRs) form a key component of animal innate immunity, being responsible for recognition of conserved microbial structures. As such, TLRs may be subject to diversifying and balancing selection, which maintains allelic variation both within and between populations. However, most research on TLRs in non-model avian species is focused on bottlenecked populations with depleted genetic variation. Here, we assessed variation at the extracellular domains of three TLR genes (TLR1LA, TLR3, TLR4) across eleven species from two passerine families of buntings (Emberizidae) and finches (Fringillidae), all having large breeding population sizes (millions of individuals). We found extraordinary TLR polymorphism in our study taxa, with >100 alleles detected at TLR1LA and TLR4 across species and high haplotype diversity (>0.75) in several species. Despite recent species divergence, no nucleotide allelic variants were shared between species, suggesting rapid TLR evolution. Higher variation at TLR1LA and TLR4 than TLR3 was associated with a stronger signal of diversifying selection, as measured with nucleotide substitutions rates and the number of positively selected sites (PSS). Structural protein modelling of TLRs showed that some PSS detected within TLR1LA and TLR4 were previously recognized as functionally important sites or were located in their proximity, possibly affecting ligand recognition. Furthermore, we identified PSS responsible for major surface electrostatic charge clustering, which may indicate their adaptive importance. Our study provides compelling evidence for the divergent evolution of TLR genes in buntings and finches and indicates that high TLR variation may be adaptively maintained via diversifying selection acting on functional ligand binding sites.
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Pinzones , Passeriformes , Animales , Receptor Toll-Like 4/genética , Pinzones/genética , Ligandos , Receptor Toll-Like 3/genética , Receptores Toll-Like/genética , Receptores Toll-Like/química , Passeriformes/genética , Evolución MolecularRESUMEN
Cathepsin K (CatK) is a lysosomal cysteine protease whose highest expression is found in osteoclasts, which are the cells responsible for bone resorption. Investigations of the functions and physiological relevance of CatK have often relied on antibody-related techniques, which makes studying its activity patterns a challenging task. Hence, we developed a set of chemical tools for the investigation of CatK activity. We show that our probe is a valuable tool for monitoring the proteolytic activation of CatK during osteoclast formation. Moreover, we demonstrate that our inhibitor of CatK impedes osteoclastogenesis and bone resorption and that CatK is stored in its active form in osteoclasts within their lysosomal compartment and mainly in the ruffled borders of osteoclasts. Given that our probe recognizes active CatK within living cells without exhibiting any observed cytotoxicity in the several models tested, we expect that it would be well suited to theranostic applications in CatK-related diseases.
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Resorción Ósea , Osteoclastos , Humanos , Osteoclastos/metabolismo , Osteogénesis , Catepsina K/metabolismo , Resorción Ósea/metabolismoRESUMEN
Haemoparasites represent a diverse group of vector-borne parasites that infect a wide range of vertebrate hosts. In birds, haemoparasite infection rates may be associated with various ecological and life history traits, including habitat choice, colony size and migration distance. Here, we molecularly assessed the prevalence of 3 main haemoparasite genera (Plasmodium, Haemoproteus and Leucocytozoon) in 2 bird species with different habitat preferences and migratory behaviour: black-headed gulls (Chroicocephalus ridibundus) and common terns (Sterna hirundo). We found that gulls showed a much higher prevalence and diversity of Plasmodium or Haemoproteus (ca. 60% of individuals infected) than terns (zero prevalence). The prevalence of Leucocytozoon was low in both species (<3%). The differences in haemoparasite prevalences may be primarily driven by varying vector encounter rate resulting from different habitat preferences, as black-headed gulls mainly use vector-rich vegetated freshwater habitats, whereas common terns often use vector-poor coastal and brackish habitats. Since common terns migrate further than black-headed gulls, our results did not provide support for an association between haemoparasite prevalence and migratory distance. In gulls, we found a negative association between colony size and infection rates, suggestive of an ideal despotic distribution, and phylogenetic analyses of detected haemoparasite lineages provided evidence for higher host specificity in Haemoproteus than Plasmodium. Our results suggest that the preference for coastal areas and less vegetated habitats in terns may reduce haemoparasite infection rates compared to other larids, regardless of their migratory distance, emphasizing the role of ecological niches in parasite exposure.
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Enfermedades de las Aves , Haemosporida , Parásitos , Plasmodium , Animales , Enfermedades de las Aves/epidemiología , Enfermedades de las Aves/parasitología , Aves/parasitología , Ecosistema , Haemosporida/genética , Humanos , Parásitos/genética , Filogenia , Plasmodium/genética , PrevalenciaRESUMEN
Bird feeders are known to be a transfer site for many important bird pathogens, such as zoonotic Salmonella spp., known to be widespread among wild birds in Poland. The aim of the study was to investigate (1) whether feeders can be a source of Salmonella spp., (2) whether the risk is the same for feeders located in cities and rural areas and (3) whether there is a different level of contamination with Salmonella spp. between old and new feeders. Data were collected in the period 12 January-28 February 2018 in four cities in Poland and nearby rural areas. In total, 204 feeders were sampled. The samples from feeders were taken after a 2-week period of feeding birds. Material for analysis consisted of the remains of food and feces. We did not find the presence of Salmonella spp. in any of the tested samples collected from bird feeders. Therefore, the estimated value of the 95% confidence interval for the binary data was 0.000-0.018. Reasons for the isolation of Salmonella spp. from feeders not being successful lie in the low intensity of bacterial shedding by infected wild birds and low survival of bacteria in the environment in bird feces-which are still not well studied.
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Food wastes are among the factors with the greatest effects on animal populations. The white stork is among bird species that clearly profit from feeding at landfills, at least in Western Europe and North Africa. However, the rate and the consequences of this feeding are still unknown in the Central-Eastern European population, which differs from the western population not only in terms of migration routes but also in the greater availability of suitable natural breeding habitats due to less intensified agriculture. The aim of the study was to describe the use of landfills and its consequences in terms of probability of nest occupation and breeding effects in different regions of Poland. Although the most important factors influencing nest-site selection and breeding effect are still habitat quality and weather conditions, distance to landfills is important in selection of nest sites. White storks use landfills most intensively late in the breeding season, independently of the density of breeding pairs. The results suggest that the use of landfills is not currently essential in the Central-Eastern European population of the white stork, does not affect breeding effect, and may be more frequent in non-breeders. However, this phenomenon is still developing and requires continuous monitoring.
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Major histocompatibility complex (MHC) genes code for key proteins of the adaptive immune system, which present antigens from intra-cellular (MHC class I) and extra-cellular (MHC class II) pathogens. Because of their unprecedented diversity, MHC genes have long been an object of scientific interest, but due to methodological difficulties in genotyping of duplicated loci, our knowledge on the evolution of the MHC across different vertebrate lineages is still limited. Here, we compared the evolution of MHC class I and class II genes in three sister clades of common passerine birds, finches (Fringillinae and Carduelinae) and buntings (Emberizidae) using a uniform methodological (genotyping and data processing) approach and uniform sample sizes. Our analyses revealed contrasting evolutionary trajectories of the two MHC classes. We found a stronger signature of pervasive positive selection and higher allele diversity (allele numbers) at the MHC class I than class II. In contrast, MHC class II genes showed greater allele divergence (in terms of nucleotide diversity) and a much stronger recombination (gene conversion) signal. Gene copy numbers at both MHC class I and class II evolved via fluctuating selection and drift (Brownian Motion evolution), but the evolutionary rate was higher at class I. Our study constitutes one of few existing examples, where evolution of MHC class I and class II genes was directly compared using a multi-species approach. We recommend that re-focusing MHC research from single-species and single-class approaches towards multi-species analyses of both MHC classes can substantially increase our understanding MHC evolution in a broad phylogenetic context.
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Pinzones , Passeriformes , Animales , Pinzones/genética , Genes MHC Clase II , Antígenos de Histocompatibilidad Clase II/genética , FilogeniaRESUMEN
BACKGROUND: Nesting in large aggregations provides several important advantages for colonially breeding birds. However, it also imposes certain costs, associated with facilitated pathogen transmission and social stress. The cost-benefit ratio is not similar for all the birds in a colony and it might be mediated by nest density. To investigate the influence of nest density on cell-mediated immune function and on physiological condition of nestlings, we arranged a cross-fostering experiment in three breeding colonies of black-headed gulls Chroicocephalus ridibundus. First, we exchanged eggs between plots of high and low nest density. Afterwards, we performed phytohaemagglutinin (PHA) skin test and we measured blood haemoglobin concentration in nearly 350 nestlings from experimental (exchanged) and control (non-exchanged) groups. RESULTS: We found that PHA response was lowest in high nest density control group, indicating that depressed immune function of offspring, likely caused by social stress, can be considered as a cost of colonial breeding. Contrastingly, body condition of nestlings was the poorest in low density control group. CONCLUSION: Nestlings hatched and raised in high nest density plots did not have higher blood haemoglobin concentration in comparison to other study groups. Furthermore, they were affected with depressed cell mediated immune function, which is possibly driven by combined maternal (corticosteroid hormones deposited in yolk) and environmental (elevated social stress) effects. These results indicate that breeders from high nest densities do not benefit by rising offspring in better quality, in terms of immune function and body condition, although, in the light of previous studies, high nest densities are occupied by birds of higher individual quality, than low density areas. Our study provides a novel insight into the mechanisms of density-dependence that govern fitness of colonially nesting birds.
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The choice of colony size may have profound consequences for individual fitness in colonially breeding birds, but at the same time it may require certain behavioural adaptations. Here, we aimed to examine behavioural divergence of common terns Sterna hirundo nesting in colonies of different size. For this purpose, we promoted establishment of small (<35 pairs) and large (>100 pairs) tern colonies under uniform ecological and environmental conditions by providing attractive patches of nesting substrate (floating rafts) at a single site. We combined video recording and GPS-tracking to assess communal and individual defence initiation rate, intra-specific aggression rate, and foraging flight characteristics. We found that birds from larger colonies more frequently engaged in communal defence and they performed longer foraging flights, while terns from smaller colonies more frequently showed individual defence behaviours. Also, intra-specific aggression rate was higher in smaller colonies, but this effect was primarily attributed to a higher proportion of edge breeding pairs, which were more aggressive. Our results suggest that various colony sizes may be associated with different behavioural syndromes, which comprise of diverse personality traits, such as social responsiveness, social tolerance, or propensity for aggression. It remains to be tested whether these behavioural differences reflect processes of phenotypic sorting among colonies of different size or whether they are a result of behavioural plasticity under different social contexts.
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Biomasa , Charadriiformes/fisiología , Conducta Sexual Animal , Conducta Social , Animales , Conducta Alimentaria , Femenino , Vuelo Animal , Masculino , ReproducciónRESUMEN
Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.
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Colorantes Fluorescentes/química , Granzimas , Imagen Óptica , Péptidos/química , Linfocitos T/enzimología , Línea Celular Tumoral , Granzimas/química , Granzimas/metabolismo , HumanosRESUMEN
Great Egret Ardea alba is one of few Western Palearctic species that underwent a rapid range expansion in the recent decades. Originally breeding in central and eastern Europe, the species has spread in northern (up to the Baltic coast) and western (up to the western France) directions and established viable breeding populations throughout almost entire continent. We monitored one of the first Great Egrets colonies established in Poland to infer migratory patterns and survival rates directly after range expansion. For this purpose, we collected resightings from over 200 Great Egret chicks marked between 2002-2017 in central Poland. Direction of migration was non-random, as birds moved almost exclusively into the western direction. Wintering grounds were located mainly in the western Europe (Germany to France) within 800-950 km from the breeding colony. First-year birds migrated farther than adults. We found some, although relatively weak, support for age-dependent survival of Great Egrets and under the best-fitted capture-recapture model, the estimated annual survival rate of adults was nearly twice higher than for first-year birds (φ ad = 0.85 ± 0.05 vs. φ fy = 0.48 ± 0.15). Annual survival rate under the constant model (no age-related variation) was estimated at φ = 0.81 ± 0.05. Our results suggest that Great Egrets rapidly adapted to novel ecological and environmental conditions during range expansion. We suggest that high survival rate of birds from central Poland and their western direction of migration may facilitate further colonization processes in western Europe.
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Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.
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Cumarinas/farmacología , Colorantes Fluorescentes/farmacología , Granzimas/análisis , Oligopéptidos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Línea Celular Tumoral , Cumarinas/síntesis química , Cumarinas/toxicidad , Diseño de Fármacos , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Granzimas/química , Humanos , Oligopéptidos/síntesis química , Oligopéptidos/toxicidad , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/toxicidad , Especificidad por SustratoRESUMEN
Melanin- and carotenoid-based ornaments often signal different aspects of individual quality or similar components of quality under different environmental conditions and, thus, they may become evolutionarily integrated into a composite sexual trait. On the other hand, functionally and developmentally different characters (e.g. coloration characters of different developmental origin) are more likely to evolve independently from each other than more similar traits. Here, we examined evolutionary correlations between the occurrence of a conspicuous melanin-based ornament (hood) and carotenoid-based bare-part ornaments within gull family. We also aimed to identify major ecological, life-history and biogeographical predictors of hood occurrence and reconstruct evolutionary history of this ornament. We found that hood occurrence was associated with red or dark coloration of unfeathered traits (bill and legs), whereas combinations of hood with yellow carotenoid-based coloration of integument were evolutionarily avoided. Also, hood occurrence correlated negatively with the occurrence of other melanin-based plumage character (mantle). Breeding latitude and habitat were identified as major predictors of hood occurrence in gulls, as hoods were recorded more frequently in low-latitude and inland (rather than marine) species. Finally, our analysis provided support for evolutionary lability in hood occurrence, with a dominance of transitions towards hood loss in the evolutionary history of gulls. The results of our study provide one of the first evidence for a correlated evolution of melanin- and carotenoid-based ornaments in an avian lineage, which supports evolutionary modularity of developmentally and functionally different coloration traits.
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Evolución Biológica , Carotenoides/metabolismo , Charadriiformes/genética , Melaninas/metabolismo , Pigmentación/genética , Animales , Charadriiformes/metabolismo , Femenino , MasculinoRESUMEN
Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.
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Trampas Extracelulares/metabolismo , Neutrófilos/enzimología , Serina Proteasas/metabolismo , Animales , Anticuerpos/química , Anticuerpos/inmunología , Candida albicans/fisiología , ADN/metabolismo , Escherichia coli/fisiología , Trampas Extracelulares/efectos de los fármacos , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/inmunología , Elastasa de Leucocito/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Microscopía Confocal , Neutrófilos/efectos de los fármacos , Piroptosis/efectos de los fármacos , Células RAW 264.7 , Serina Proteasas/química , Serina Proteasas/inmunología , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Domestic and wild mammals, domestic birds and particularly wild birds are considered to be reservoirs of many species of Enterobacteriaceae, and also important human enteric pathogens, e.g., the bacteria of the genus Campylobacter that occur in their digestive tracts. These species may be vectors of antimicrobial resistance dissemination in the environment, because they may have contact with an environment contaminated with antibiotics. Bird feeders have been suggested as potential dispersal centres between wild wintering birds whose feeding is supported by humans. Therefore, we checked for the presence of Campylobacter bacteria among great tits Parus major, the most common bird species on bird feeders in Poland. Samples (n = 787 cloacal swabs) were collected in urban and rural areas of Poland. Bacterial species were identified using multiplex PCR, and 23 (2.9%) positive tests for Campylobacter spp. were found; in ten samples, C. jejuni was detected. The odds ratio of Campylobacter infection in rural birds was over 2.5 times higher than urban birds. Ten samples with C. jejuni were tested for antibiotic resistance, and all were sensitive to azithromycin, erythromycin and gentamycin, while six isolates were resistant to tetracycline, and five were resistant to ciprofloxacin. Four Campylobacter isolates were resistant to both these antibiotics.
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Infecciones por Campylobacter/veterinaria , Farmacorresistencia Bacteriana , Pájaros Cantores/microbiología , Animales , Antibacterianos/farmacología , Campylobacter/efectos de los fármacos , Campylobacter/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , PoloniaRESUMEN
Louse flies are blood-sucking ectoparasites of birds and mammals of veterinary and medical importance. These flies damage host skin and serve as vectors for blood-associated pathogens. Little is known about their phenology, host selection, and prevalence in wild birds. Our study provided a broad-scale assessment of louse flies parasitizing Charadrii and Scolopaci shorebirds during their migration through Central Europe. We collected louse flies from 3,129 specimens in 22 species of shorebirds at a major migratory stopover site in central Poland in 2011-17. In total, 31 Ornithomya avicularia and 15 Ornithomya chloropus specimens occurred on five wader species: Common Snipe (Gallinago gallinago), Ruff (Calidris pugnax), Dunlin (Calidris alpina), Curlew Sandpiper (Calidris ferruginea), and Wood Sandpiper (Tringa glareola). Prevalences ranged from 0.23% to 4.62%. No individual birds were infested by both species of fly, and no flies were found on an additional 17 wader species. The rarer of the two flies, O. chloropus, occurred on all infested bird species, whereas O. avicularia occurred only on Common Snipe and Curlew Sandpiper. In comparison with the other four infested wader species, Common Snipe (n=1,194) was more frequently infested with O. avicularia than with O. chloropus. Our study shows that the prevalence of louse flies on shorebirds is much lower than on other avian taxa. Ecologic and evolutionary mechanisms leading to the relative rarity of louse flies on shorebirds remain to be understood.
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Migración Animal , Charadriiformes/parasitología , Dípteros/clasificación , Dípteros/fisiología , Infestaciones Ectoparasitarias/veterinaria , Animales , Infestaciones Ectoparasitarias/epidemiología , Infestaciones Ectoparasitarias/parasitología , Interacciones Huésped-Parásitos , PoloniaRESUMEN
Lon is a widely conserved housekeeping protease found in all domains of life. Bacterial Lon is involved in recovery from various types of stress, including tolerance to fluoroquinolone antibiotics, and is linked to pathogenesis in a number of organisms. However, detailed functional studies of Lon have been limited by the lack of selective, cell-permeant inhibitors. Here, we describe the use of positional scanning libraries of hybrid peptide substrates to profile the primary sequence specificity of bacterial Lon. In addition to identifying optimal natural amino acid binding preferences, we identified several non-natural residues that were leveraged to develop optimal peptide substrates as well as a potent peptidic boronic acid inhibitor of Lon. Treatment of Escherichia coli with this inhibitor promotes UV-induced filamentation and reduces tolerance to ciprofloxacin, phenocopying established lon-deletion phenotypes. It is also nontoxic to mammalian cells due to its selectivity for Lon over the proteasome. Our results provide new insight into the primary substrate specificity of Lon and identify substrates and an inhibitor that will serve as useful tools for dissecting the diverse cellular functions of Lon.
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Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Oligopéptidos/química , Biblioteca de Péptidos , Proteasa La/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Ácidos Borónicos/química , Ciprofloxacina/metabolismo , Inhibidores Enzimáticos/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Humanos , Ratones , Mutación , Oligopéptidos/metabolismo , Proteasa La/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Células RAW 264.7 , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Elevated transmission rate of pathogens and parasites is considered one of the major costs of sociality in birds. However, greater risk of infection in colonial birds might be compensated by specific immune adaptations. Here, we predicted that nestlings raised in larger colonies should invest more in their immune function. To test this hypothesis, we manipulated colony size and conduced cross-fostering experiment in a colonial waterbird, the common tern Sterna hirundo. Establishment of different size colonies under uniform environmental conditions was induced by providing large and small patches of attractive nesting area for terns (floating rafts). Then, pairs of clutches were swapped between large and small tern colonies, and skin-swelling response to phytohaemagglutinin (PHA) was assessed for nestlings from experimental and control broods. Contrary to our expectations, we found a negative effect of foster colony size on nestling PHA response (nestlings raised in the larger colony had lower PHA response). In addition, nestling PHA response correlated negatively with heterophil/lymphocyte ratio used as a measure of physiological stress. This suggested that low PHA response of nestlings raised in the larger colony could be mediated by an elevated level of social stress. We suggest that depression of immune function via social stress may constitute a strong selective pressure against large colony size in the common tern, and possibly in other colonial species. We also recommend that this largely overlooked cost of sociality should be considered in the further studies on the evolution and ecology of avian coloniality.