Early-life factors shaping the gut microbiota of Common buzzard nestlings.

BACKGROUND: Exploring the dynamics of gut microbiome colonisation during early-life stages is important for understanding the potential impact of microbes on host development and fitness. Evidence from model organisms suggests a crucial early-life phase when shifts in gut microbiota can lead to immune dysregulation and reduced host condition. However, our understanding of gut microbiota colonisation in long-lived vertebrates, especially during early development, remains limited. We therefore used a wild population of common buzzard nestlings (Buteo buteo) to investigate connections between the early-life gut microbiota colonisation, environmental and host factors. RESULTS: We targeted both bacterial and eukaryotic microbiota using the 16S and 28S rRNA genes. We sampled the individuals during early developmental stages in a longitudinal design. Our data revealed that age significantly affected microbial diversity and composition. Nest environment was a notable predictor of microbiota composition, with particularly eukaryotic communities differing between habitats occupied by the hosts. Nestling condition and infection with the blood parasite Leucocytozoon predicted microbial community composition. CONCLUSION: Our findings emphasise the importance of studying microbiome dynamics to capture changes occurring during ontogeny. They highlight the role of microbial communities in reflecting host health and the importance of the nest environment for the developing nestling microbiome. Overall, this study contributes to understanding the complex interplay between microbial communities, host factors, and environmental variables, and sheds light on the ecological processes governing gut microbial colonisation during early-life stages.

High functional allelic diversity and copy number in both MHC classes in the common buzzard.

BACKGROUND: The major histocompatibility complex (MHC), which encodes molecules that recognize various pathogens and parasites and initiates the adaptive immune response in vertebrates, is renowned for its exceptional polymorphism and is a model of adaptive gene evolution. In birds, the number of MHC genes and sequence diversity varies greatly among taxa, believed due to evolutionary history and differential selection pressures. Earlier characterization studies and recent comparative studies suggest that non-passerine species have relatively few MHC gene copies compared to passerines. Additionally, comparative studies that have looked at partial MHC sequences have speculated that non-passerines have opposite patterns of selection on MHC class I (MHC-I) and class II (MHC-II) loci than passerines: namely, greater sequence diversity and signals of selection on MHC-II than MHC-I. However, new sequencing technology is revealing much greater MHC variation than previously expected while also facilitating full sequence variant detection directly from genomic data. Our study aims to take advantage of high-throughput sequencing methods to fully characterize both classes and domains of MHC of a non-passerine bird of prey, the common buzzard (Buteo buteo), to test predictions of MHC variation and differential selection on MHC classes. RESULTS: Using genetic, genomic, and transcriptomic high-throughput sequencing data, we established common buzzards have at least three loci that produce functional alleles at both MHC classes. In total, we characterize 91 alleles from 113 common buzzard chicks for MHC-I exon 3 and 41 alleles from 125 chicks for MHC-IIB exon 2. Among these alleles, we found greater sequence polymorphism and stronger diversifying selection at MHC-IIB exon 2 than MHC-I exon 3, suggesting differential selection pressures on MHC classes. However, upon further investigation of the entire peptide-binding groove by including genomic data from MHC-I exon 2 and MHC-IIA exon 2, this turned out to be false. MHC-I exon 2 was as polymorphic as MHC-IIB exon 2 and MHC-IIA exon 2 was essentially invariant. Thus, comparisons between MHC-I and MHC-II that included both domains of the peptide-binding groove showed no differences in polymorphism nor diversifying selection between the classes. Nevertheless, selection analysis indicates balancing selection has been acting on common buzzard MHC and phylogenetic inference revealed that trans-species polymorphism is present between common buzzards and species separated for over 33 million years for class I and class II. CONCLUSIONS: We characterize and confirm the functionality of unexpectedly high copy number and allelic diversity in both MHC classes of a bird of prey. While balancing selection is acting on both classes, there is no evidence of differential selection pressure on MHC classes in common buzzards and this result may hold more generally once more data for understudied MHC exons becomes available.

Intronic primers reveal unexpectedly high major histocompatibility complex diversity in Antarctic fur seals.

The major histocompatibility complex (MHC) is a group of genes comprising one of the most important components of the vertebrate immune system. Consequently, there has been much interest in characterising MHC variation and its relationship with fitness in a variety of species. Due to the exceptional polymorphism of MHC genes, careful PCR primer design is crucial for capturing all of the allelic variation present in a given species. We therefore developed intronic primers to amplify the full-length 267 bp protein-coding sequence of the MHC class II DQB exon 2 in the Antarctic fur seal. We then characterised patterns of MHC variation among mother-offspring pairs from two breeding colonies and detected 19 alleles among 771 clone sequences from 56 individuals. The distribution of alleles within and among individuals was consistent with a single-copy, classical DQB locus showing Mendelian inheritance. Amino acid similarity at the MHC was significantly associated with genome-wide relatedness, but no relationship was found between MHC heterozygosity and genome-wide heterozygosity. Finally, allelic diversity was several times higher than reported by a previous study based on partial exon sequences. This difference appears to be related to allele-specific amplification bias, implying that primer design can strongly impact the inference of MHC diversity.

Tolerability of Atovaquone-Proguanil Application in Common Buzzard Nestlings.

Differences in drug tolerability among vertebrate groups and species can create substantial challenges for wildlife and ex situ conservation programmes. Knowledge of tolerance in the use of new drugs is, therefore, important to avoid severe toxicity in species, which are both commonly admitted in veterinary clinics and are of conservation concern. Antimalarial drugs have been developed for use in human medicine, but treatment with different agents has also long been used in avian medicine, as haemosporidian infections play a major role in many avian species. This study investigates the effects of the application of atovaquone-proguanil (Malarone®, GlaxoSmithKline) in common buzzards (Buteo buteo). The potential effects of treatment on body condition, growth rate, and chemical blood parameters of nestlings were assessed. All individuals survived the treatment, and no effects on body condition, growth rate, and chemical blood parameters were observed. Our results suggest the tolerability of Malarone® in common buzzards at a single dose of on average 11 mg/kg body weight. For its safe use, we recommend further studies to determine pharmacokinetics in different avian species as well as to assess the effects of repeated treatment.

The prevalence of Leucocytozoon spp. in nestlings of three wild raptor species including implications on haematological and blood chemistry values.

Leucocytozoonosis is a vector-borne infection of birds, caused by members of the haemosporidian genus Leucocytozoon. The clinical presentation may range from asymptomatic to severe disease. Consequences of Leucocytozoon infection on blood profiles remain to be described, especially for different host species in the wild. In the current study, the prevalence of Leucocytozoon infection was determined in wild nestlings of three European raptor species, the common buzzard (Buteo buteo, n = 464), red kite (Milvus milvus, n = 46) and northern goshawk (Accipiter gentilis, n = 18). Among 528 nestlings, 51.9% (n = 274) were infected with Leucocytozoon spp., whereby the highest prevalence was found in common buzzards (54.9%), followed by red kites (32.6%) and northern goshawks (22.2%). For a subset of 87 individuals (50 common buzzards, 29 red kites, 8 northern goshawks), a detailed analysis of differential leukocyte counts and several blood chemistry parameters in response to infection was conducted: AP (alkaline phosphatase), AST (aspartate aminotransferase), GLDH (glutamate dehydrogenase), LDH (lactate dehydrogenase), GGT (gamma glutamyl transferase), CK (creatine kinase), BuChE (butyrylcholinesterase), BA (bile acids), ALB (albumin) and TP (total protein). Even though in the physiological range, infected nestlings displayed significantly increased levels of heterophils, aspartate aminotransferase, lactate dehydrogenase, bile acids and butyrylcholinesterase, but decreased lymphocyte and monocyte values compared to uninfected ones. Furthermore, significant species differences with regard to blood parameters, but no sex differences were found. Overall, obtained results show a high prevalence, but a low pathogenicity of Leucocytozoon spp. in wild raptor chicks, presumably resulting from coevolutionary adaptation, but show signatures of infection in the haematological and blood chemistry profiles.

Territorial behaviour of buzzards versus random matrix spacing distributions.

A deeper understanding of the processes underlying the distribution of animals in space is crucial for both basic and applied ecology. The Common buzzard (Buteo buteo) is a highly aggressive, territorial bird of prey that interacts strongly with its intra- and interspecific competitors. We propose and use random matrix theory to quantify the strength and range of repulsion as a function of the buzzard population density, thus providing a novel approach to model density dependence. As an indicator of territorial behaviour, we perform a large-scale analysis of the distribution of buzzard nests in an area of 300 square kilometres around the Teutoburger Wald, Germany, as gathered over a period of 20 years. The nearest and next-to-nearest neighbour spacing distribution between nests is compared to the two-dimensional Poisson distribution, originating from uncorrelated random variables, to the complex eigenvalues of random matrices, which are strongly correlated, and to a two-dimensional Coulomb gas interpolating between these two. A one-parameter fit to a time-moving average reveals a significant increase of repulsion between neighbouring nests, as a function of the observed increase in absolute population density over the monitored period of time, thereby proving an unexpected yet simple model for density-dependent spacing of predator territories. A similar effect is obtained for next-to-nearest neighbours, albeit with weaker repulsion, indicating a short-range interaction. Our results show that random matrix theory might be useful in the context of population ecology.

Heterozygosity at neutral and immune loci is not associated with neonatal mortality due to microbial infection in Antarctic fur seals.

Numerous studies have reported correlations between the heterozygosity of genetic markers and fitness. These heterozygosity-fitness correlations (HFCs) play a central role in evolutionary and conservation biology, yet their mechanistic basis remains open to debate. For example, fitness associations have been widely reported at both neutral and functional loci, yet few studies have directly compared the two, making it difficult to gauge the relative contributions of genome-wide inbreeding and specific functional genes to fitness. Here, we compared the effects of neutral and immune gene heterozygosity on death from bacterial infection in Antarctic fur seal (Arctocephalus gazella) pups. We specifically developed a panel of 13 microsatellites from expressed immune genes and genotyped these together with 48 neutral loci in 234 individuals, comprising 39 pups that were classified at necropsy as having most likely died of bacterial infection together with a five times larger matched sample of healthy surviving pups. Identity disequilibrium quantified from the neutral markers was positive and significant, indicative of variance in inbreeding within the study population. However, multilocus heterozygosity did not differ significantly between healthy and infected pups at either class of marker, and little evidence was found for fitness associations at individual loci. These results support a previous study of Antarctic fur seals that found no effects of heterozygosity at nine neutral microsatellites on neonatal survival and thereby help to refine our understanding of how HFCs vary across the life cycle. Given that nonsignificant HFCs are underreported in the literature, we also hope that our study will contribute toward a more balanced understanding of the wider importance of this phenomenon.

GCalignR: An R package for aligning gas-chromatography data for ecological and evolutionary studies.

Chemical cues are arguably the most fundamental means of animal communication and play an important role in mate choice and kin recognition. Consequently, there is growing interest in the use of gas chromatography (GC) to investigate the chemical basis of eco-evolutionary interactions. Both GC-MS (mass spectrometry) and FID (flame ionization detection) are commonly used to characterise the chemical composition of biological samples such as skin swabs. The resulting chromatograms comprise peaks that are separated according to their retention times and which represent different substances. Across chromatograms of different samples, homologous substances are expected to elute at similar retention times. However, random and often unavoidable experimental variation introduces noise, making the alignment of homologous peaks challenging, particularly with GC-FID data where mass spectral data are lacking. Here we present GCalignR, a user-friendly R package for aligning GC-FID data based on retention times. The package was developed specifically for ecological and evolutionary studies that seek to investigate similarity patterns across multiple and often highly variable biological samples, for example representing different sexes, age classes or reproductive stages. The package also implements dynamic visualisations to facilitate inspection and fine-tuning of the resulting alignments and can be integrated within a broader workflow in R to facilitate downstream multivariate analyses. We demonstrate an example workflow using empirical data from Antarctic fur seals and explore the impact of user-defined parameter values by calculating alignment error rates for multiple datasets. The resulting alignments had low error rates for most of the explored parameter space and we could also show that GCalignR performed equally well or better than other available software. We hope that GCalignR will help to simplify the processing of chemical datasets and improve the standardization and reproducibility of chemical analyses in studies of animal chemical communication and related fields.