Rapid adaptation to a novel pathogen through disease tolerance in a wild songbird.

Animal hosts can adapt to emerging infectious disease through both disease resistance, which decreases pathogen numbers, and disease tolerance, which limits damage during infection without limiting pathogen replication. Both resistance and tolerance mechanisms can drive pathogen transmission dynamics. However, it is not well understood how quickly host tolerance evolves in response to novel pathogens or what physiological mechanisms underlie this defense. Using natural populations of house finches (Haemorhous mexicanus) across the temporal invasion gradient of a recently emerged bacterial pathogen (Mycoplasma gallisepticum), we find rapid evolution of tolerance (<25 years). In particular, populations with a longer history of MG endemism have less pathology but similar pathogen loads compared with populations with a shorter history of MG endemism. Further, gene expression data reveal that more-targeted immune responses early in infection are associated with tolerance. These results suggest an important role for tolerance in host adaptation to emerging infectious diseases, a phenomenon with broad implications for pathogen spread and evolution.

Bacterial communities along parrot digestive and respiratory tracts: the effects of sample type, species and time.

Digestive and respiratory tracts are inhabited by rich bacterial communities that can vary between their different segments. In comparison with other bird taxa with developed caeca, parrots that lack caeca have relatively lower variability in intestinal morphology. Here, based on 16S rRNA metabarcoding, we describe variation in microbiota across different parts of parrot digestive and respiratory tracts both at interspecies and intraspecies levels. In domesticated budgerigar (Melopsittacus undulatus), we describe the bacterial variation across eight selected sections of respiratory and digestive tracts, and three non-destructively collected sample types (faeces, and cloacal and oral swabs). Our results show important microbiota divergence between the upper and lower digestive tract, but similarities between respiratory tract and crop, and also between different intestinal segments. Faecal samples appear to provide a better proxy for intestinal microbiota composition than the cloacal swabs. Oral swabs had a similar bacterial composition as the crop and trachea. For a subset of tissues, we confirmed the same pattern also in six different parrot species. Finally, using the faeces and oral swabs in budgerigars, we revealed high oral, but low faecal microbiota stability during a 3-week period mimicking pre-experiment acclimation. Our findings provide a basis essential for microbiota-related experimental planning and result generalisation in non-poultry birds.

Selection Balancing at Innate Immune Genes: Adaptive Polymorphism Maintenance in Toll-Like Receptors.

Balancing selection is a classic mechanism for maintaining variability in immune genes involved in host-pathogen interactions. However, it remains unclear how widespread the mechanism is across immune genes other than the major histocompatibility complex (MHC). Although occasional reports suggest that balancing selection (heterozygote advantage, negative frequency-dependent selection, and fluctuating selection) may act on other immune genes, the current understanding of the phenomenon in non-MHC immune genes is far from solid. In this review, we focus on Toll-like receptors (TLRs), innate immune genes directly involved in pathogen recognition and immune response activation, as there is a growing body of research testing the assumptions of balancing selection in these genes. After reviewing infection- and fitness-based evidence, along with evidence based on population allelic frequencies and heterozygosity levels, we conclude that balancing selection maintains variation in TLRs, though it tends to occur under specific conditions in certain evolutionary lineages rather than being universal and ubiquitous. Our review also identifies key gaps in current knowledge and proposes promising areas for future research. Improving our understanding of host-pathogen interactions and balancing selection in innate immune genes are increasingly important, particularly regarding threats from emerging zoonotic diseases.

Adaptation and Cryptic Pseudogenization in Penguin Toll-Like Receptors.

Penguins (Sphenisciformes) are an iconic order of flightless, diving seabirds distributed across a large latitudinal range in the Southern Hemisphere. The extensive area over which penguins are endemic is likely to have fostered variation in pathogen pressure, which in turn will have imposed differential selective pressures on the penguin immune system. At the front line of pathogen detection and response, the Toll-like receptors (TLRs) provide insight into host evolution in the face of microbial challenge. TLRs respond to conserved pathogen-associated molecular patterns and are frequently found to be under positive selection, despite retaining specificity for defined agonist classes. We undertook a comparative immunogenetics analysis of TLRs for all penguin species and found evidence of adaptive evolution that was largely restricted to the cell surface-expressed TLRs, with evidence of positive selection at, or near, key agonist-binding sites in TLR1B, TLR4, and TLR5. Intriguingly, TLR15, which is activated by fungal products, appeared to have been pseudogenized multiple times in the Eudyptes spp., but a full-length form was present as a rare haplotype at the population level. However, in vitro analysis revealed that even the full-length form of Eudyptes TLR15 was nonfunctional, indicating an ancestral cryptic pseudogenization prior to its eventual disruption multiple times in the Eudyptes lineage. This unusual pseudogenization event could provide an insight into immune adaptation to fungal pathogens such as Aspergillus, which is responsible for significant mortality in wild and captive bird populations.

Effect of population size and selection on Toll-like receptor diversity in populations of Galápagos mockingbirds.

The interactions of evolutionary forces are difficult to analyse in free-living populations. However, when properly understood, they provide valuable insights into evolutionary biology and conservation genetics. This is particularly important for the interplay of genetic drift and natural selection in immune genes that confer resistance to disease. The Galápagos Islands are inhabited by four closely related species of mockingbirds (Mimus spp.). We used 12 different-sized populations of Galápagos mockingbirds and one population of their continental relative northern mockingbird (Mimus polyglottos) to study the effects of genetic drift on the molecular evolution of immune genes, the Toll-like receptors (TLRs: TLR1B, TLR4 and TLR15). We found that neutral genetic diversity was positively correlated with island size, indicating an important effect of genetic drift. However, for TLR1B and TLR4, there was little correlation between functional (e.g., protein) diversity and island size, and protein structural properties were largely conserved, indicating only a limited effect of genetic drift on molecular phenotype. By contrast, TLR15 was less conserved and even its putative functional polymorphism correlated with island size. The patterns observed for the three genes suggest that genetic drift does not necessarily dominate selection even in relatively small populations, but that the final outcome depends on the degree of selection constraint that is specific for each TLR locus.

Understanding the evolution of immune genes in jawed vertebrates.

Driven by co-evolution with pathogens, host immunity continuously adapts to optimize defence against pathogens within a given environment. Recent advances in genetics, genomics and transcriptomics have enabled a more detailed investigation into how immunogenetic variation shapes the diversity of immune responses seen across domestic and wild animal species. However, a deeper understanding of the diverse molecular mechanisms that shape immunity within and among species is still needed to gain insight into-and generate evolutionary hypotheses on-the ultimate drivers of immunological differences. Here, we discuss current advances in our understanding of molecular evolution underpinning jawed vertebrate immunity. First, we introduce the immunome concept, a framework for characterizing genes involved in immune defence from a comparative perspective, then we outline how immune genes of interest can be identified. Second, we focus on how different selection modes are observed acting across groups of immune genes and propose hypotheses to explain these differences. We then provide an overview of the approaches used so far to study the evolutionary heterogeneity of immune genes on macro and microevolutionary scales. Finally, we discuss some of the current evidence as to how specific pathogens affect the evolution of different groups of immune genes. This review results from the collective discussion on the current key challenges in evolutionary immunology conducted at the ESEB 2021 Online Satellite Symposium: Molecular evolution of the vertebrate immune system, from the lab to natural populations.

Cannabinoid receptor 2 evolutionary gene loss makes parrots more susceptible to neuroinflammation.

In vertebrates, cannabinoids modulate neuroimmune interactions through two cannabinoid receptors (CNRs) conservatively expressed in the brain (CNR1, syn. CB1) and in the periphery (CNR2, syn. CB2). Our comparative genomic analysis indicates several evolutionary losses in the CNR2 gene that is involved in immune regulation. Notably, we show that the CNR2 gene pseudogenized in all parrots (Psittaciformes). This CNR2 gene loss occurred because of chromosomal rearrangements. Our positive selection analysis suggests the absence of any specific molecular adaptations in parrot CNR1 that would compensate for the CNR2 loss in the modulation of the neuroimmune interactions. Using transcriptomic data from the brains of birds with experimentally induced sterile inflammation we highlight possible functional effects of such a CNR2 gene loss. We compare the expression patterns of CNR and neuroinflammatory markers in CNR2-deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2-intact passerines (represented by the zebra finch, Taeniopygia guttata). Unlike in passerines, stimulation with lipopolysaccharide resulted in neuroinflammation in the parrots linked with a significant upregulation of expression in proinflammatory cytokines (including interleukin 1 beta (IL1B) and 6 (IL6)) in the brain. Our results indicate the functional importance of the CNR2 gene loss for increased sensitivity to brain inflammation.

Inter-annual repeatability and age-dependent changes in plasma testosterone levels in a longitudinally monitored free-living passerine bird.

While seasonal trends in testosterone levels are known from cross-cohort studies, data on testosterone inter-annual individual repeatability in wild birds are rare. Also, our understanding of hormonal age-dependent changes in testosterone levels is limited. We assessed plasma testosterone levels in 105 samples originating from 49 repeatedly captured free-living great tits (Parus major) sampled during the nesting to investigate their relative long-term repeatability and within-individual changes. Furthermore, we examined the inter-annual repeatability of condition-related traits (carotenoid- and melanin-based plumage ornamentation, ptilochronological feather growth rate, body mass, and haematological heterophil/lymphocyte ratio) and their relationships to testosterone levels. We show that testosterone levels are inter-annually repeatable in females, with a non-significant pattern in males, both in absolute values and individual ranks (indicating the maintenance of relative status in a population). In males, we found a quadratic dependence of testosterone levels on age, with a peak in midlife. In contrast, female testosterone levels showed no age-dependent trends. The inter-annual repeatability of condition-related traits ranged from zero to moderate and was mostly unrelated to plasma testosterone concentrations. However, males with elevated testosterone had significantly higher carotenoid-pigmented yellow plumage brightness, a trait presumably involved in mating. Showing inter-annual repeatability in testosterone levels, this research opens the way to further understanding the causes of variation in condition-related traits. Based on a longitudinal dataset, this study demonstrates that male plasma testosterone undergoes age-related changes that may regulate resource allocation. Our results thus suggest that, unlike females, male birds undergo hormonal senescence similar to mammals.

Positive selection and convergent evolution shape molecular phenotypic traits of innate immunity receptors in tits (Paridae).

Despite widespread variability and redundancy abounding animal immunity, little is currently known about the rate of evolutionary convergence (functionally analogous traits not inherited from a common ancestor) in host molecular adaptations to parasite selective pressures. Toll-like receptors (TLRs) provide the molecular interface allowing hosts to recognize pathogenic structures and trigger early danger signals initiating an immune response. Using a novel combination of bioinformatic approaches, here we explore genetic variation in ligand-binding regions of bacteria-sensing TLR4 and TLR5 in 29 species belonging to the tit family of passerine birds (Aves: Paridae). Three out of the four consensual positively selected sites in TLR4 and six out of 14 positively selected positions in TLR5 were located on the receptor surface near the functionally important sites, and based on the phylogenetic pattern evolved in a convergent (parallel) manner. This type of evolution was also seen at one N-glycosylation site and two positively selected phosphorylation sites, providing the first evidence of convergence in post-translational modifications in evolutionary immunology. Finally, the overall mismatch between phylogeny and the clustering of surface charge distribution demonstrates that convergence is common in overall TLR4 and TLR5 molecular phenotypes involved in ligand binding. Our analysis did not reveal any broad ecological traits explaining the convergence observed in electrostatic potentials, suggesting that information on microbial symbionts may be needed to explain TLR evolution. Adopting state-of-the-art predictive structural bionformatics, we have outlined a new broadly applicable methodological approach to estimate the functional significance of positively selected variation and test for the adaptive molecular convergence in protein-coding polymorphisms.

Comparative analysis of the chicken IFITM locus by targeted genome sequencing reveals evolution of the locus and positive selection in IFITM1 and IFITM3.

BACKGROUND: The interferon-induced transmembrane (IFITM) protein family comprises a class of restriction factors widely characterised in humans for their potent antiviral activity. Their biological activity is well documented in several animal species, but their genetic variation and biological mechanism is less well understood, particularly in avian species. RESULTS: Here we report the complete sequence of the domestic chicken Gallus gallus IFITM locus from a wide variety of chicken breeds to examine the detailed pattern of genetic variation of the locus on chromosome 5, including the flanking genes ATHL1 and B4GALNT4. We have generated chIFITM sequences from commercial breeds (supermarket-derived chicken breasts), indigenous chickens from Nigeria (Nsukka) and Ethiopia, European breeds and inbred chicken lines from the Pirbright Institute, totalling of 206 chickens. Through mapping of genetic variants to the latest chIFITM consensus sequence our data reveal that the chIFITM locus does not show structural variation in the locus across the populations analysed, despite spanning diverse breeds from different geographic locations. However, single nucleotide variants (SNVs) in functionally important regions of the proteins within certain groups of chickens were detected, in particular the European breeds and indigenous birds from Ethiopia and Nigeria. In addition, we also found that two out of four SNVs located in the chIFITM1 (Ser36 and Arg77) and chIFITM3 (Val103) proteins were simultaneously under positive selection. CONCLUSIONS: Together these data suggest that IFITM genetic variation may contribute to the capacities of different chicken populations to resist virus infection.

Toll-Like Receptor Evolution in Birds: Gene Duplication, Pseudogenization, and Diversifying Selection.

Toll-like receptors (TLRs) are key sensor molecules in vertebrates triggering initial phases of immune responses to pathogens. The avian TLR family typically consists of ten receptors, each adapted to distinct ligands. To understand the complex evolutionary history of each avian TLR, we analyzed all members of the TLR family in the whole genome assemblies and target sequence data of 63 bird species covering all major avian clades. Our results indicate that gene duplication events most probably occurred in TLR1 before synapsids diversified from sauropsids. Unlike mammals, ssRNA-recognizing TLR7 has duplicated independently in several avian taxa, while flagellin-sensing TLR5 has pseudogenized multiple times in bird phylogeny. Our analysis revealed stronger positive, diversifying selection acting in TLR5 and the three-domain TLRs (TLR10 [TLR1A], TLR1 [TLR1B], TLR2A, TLR2B, TLR4) that face the extracellular space and bind complex ligands than in single-domain TLR15 and endosomal TLRs (TLR3, TLR7, TLR21). In total, 84 out of 306 positively selected sites were predicted to harbor substitutions dramatically changing the amino acid physicochemical properties. Furthermore, 105 positively selected sites were located in the known functionally relevant TLR regions. We found evidence for convergent evolution acting between birds and mammals at 54 of these sites. Our comparative study provides a comprehensive insight into the evolution of avian TLR genetic variability. Besides describing the history of avian TLR gene gain and gene loss, we also identified candidate positions in the receptors that have been likely shaped by direct molecular host-pathogen coevolutionary interactions and most probably play key functional roles in birds.

Signatures of diversifying selection and convergence acting on passerine Toll-like receptor 4 in an evolutionary context.

Positive selection acting on Toll-like receptors (TLRs) has been recently investigated to reveal evolutionary mechanisms of host-pathogen molecular co-adaptation. Much of this research, however, has focused mainly on the identification of sites predicted to be under positive selection, bringing little insight into the functional differences and similarities among species and a limited understanding of convergent evolution in the innate immune molecules. In this study, we provide evidence of phenotypic variability in the avian TLR4 ligand-binding region (LBR), the direct interface between host and pathogen molecular structures. We show that 55 passerine species vary substantially in the distribution of electrostatic potential on the surface of the receptor, and based on these distinct patterns, we identified four species clusters. Seven of the 34 evolutionarily nonconservative and positively selected residues correspond topologically to sites previously identified as being important for lipopolysaccharide, lipid IVa or MD-2 binding. Five of these positions codetermine the identity of the charge clusters. Groups of species that host-related communities of pathogens were predicted to cluster based on their TLR4 LBR charge. Despite some evidence for convergence among taxa, there were no clear associations between the TLR4 LBR charge distribution and any of the general ecological characteristics compared (migration, latitudinal distribution and diet). Closely related species, however, mostly belonged to the same surface charge cluster indicating that phylogenetic constraints are key determinants shaping TLR4 adaptive evolution. Our results suggest that host innate immune evolution is consistent with Fahrenholz's rule on the cospeciation of hosts and their parasites.

Characterisation of Toll-like receptors 4, 5 and 7 and their genetic variation in the grey partridge.

Toll-like receptors (TLRs) are a cornerstone of vertebrate innate immunity. In this study, we identified orthologues of TLR4, TLR5 and TLR7 (representing both bacterial- and viral-sensing TLRs) in the grey partridge (Perdix perdix), a European Galliform game bird species. The phylogeny of all three TLR genes follows the known phylogeny of Galloanserae birds, placing grey partridge TLRs (PePeTLRs) in close proximity to their turkey and pheasant orthologues. The predicted proteins encoded by the PePeTLR genes were 843, 862-863 and 1,047 amino acids long, respectively, and clearly showed all TLR structural features. To verify functionality in these genes we mapped their tissue-expression profiles, revealing generally high PePeTLR4 and PePeTLR5 expression in the thymus and absence of PePeTLR4 and PePeTLR7 expression in the brain. Using 454 next-generation sequencing, we then assessed genetic variation within these genes for a wild grey partridge population in the Czech Republic, EU. We identified 11 nucleotide substitutions in PePeTLR4, eight in PePeTLR5 and six in PePeTLR7, resulting in four, four and three amino acid replacements, respectively. Given their locations and chemical features, most of these non-synonymous substitutions probably have a minor functional impact. As the intraspecific genetic variation of the three TLR genes was low, we assume that either negative selection or a bottleneck may have reduced TLR population variability in this species.

Protein evolution of Toll-like receptors 4, 5 and 7 within Galloanserae birds.

BACKGROUND: Toll-like receptors (TLR) are essential activators of the innate part of the vertebrate immune system. In this study, we analysed the interspecific variability of three TLR (bacterial-sensing TLR4 and TLR5 and viral-sensing TLR7) within the Galloanserae bird clade, investigated their phylogeny, assessed their structural conservation and estimated site-specific selection pressures. RESULTS: Physiochemical properties varied according to the TLR analysed, mainly with regards to the surface electrostatic potential distribution. The predicted ligand-binding features (mainly in TLR4 and TLR5) differed between the avian proteins and their fish and mammalian counterparts, but also varied within the Galloanserae birds. We identified 20 positively selected sites in the three TLR, among which several are topologically close to ligand-binding sites reported for mammalian and fish TLR. We described 26, 28 and 25 evolutionarily non-conservative sites in TLR4, TLR5 and TLR7, respectively. Thirteen of these sites in TLR4, and ten in TLR5 were located in functionally relevant regions. The variability appears to be functionally more conserved for viral-sensing TLR7 than for the bacterial-sensing TLR. Amino-acid positions 268, 270, 343, 383, 444 and 471 in TLR4 and 180, 183, 209, 216, 264, 342 and 379 in TLR5 are key candidates for further functional research. CONCLUSIONS: Host-pathogen co-evolution has a major effect on the features of host immune receptors. Our results suggest that avian and mammalian TLR may be differentially adapted to pathogen-derived ligand recognition. We have detected signatures of positive selection even within the Galloanserae lineage. To our knowledge, this is the first study to depict evolutionary pressures on Galloanserae TLR and to estimate the validity of current knowledge on TLR function (based on mammalian and chicken models) for non-model species of this clade.

Natural selection directing molecular evolution in vertebrate viral sensors.

Diseases caused by pathogens contribute to molecular adaptations in host immunity. Variety of viral pathogens challenging animal immunity can drive positive selection diversifying receptors recognising the infections. However, whether distinct virus sensing systems differ across animals in their evolutionary modes remains unclear. Our review provides a comparative overview of natural selection shaping molecular evolution in vertebrate viral-binding pattern recognition receptors (PRRs). Despite prevailing negative selection arising from the functional constraints, multiple lines of evidence now suggest diversifying selection in the Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs) and oligoadenylate synthetases (OASs). In several cases, location of the positively selected sites in the ligand-binding regions suggests effects on viral detection although experimental support is lacking. Unfortunately, in most other PRR families including the AIM2-like receptor family, C-type lectin receptors (CLRs), and cyclic GMP-AMP synthetase studies characterising their molecular evolution are rare, preventing comparative insight. We indicate shared characteristics of the viral sensor evolution and highlight priorities for future research.

Are there consistent effects of gut microbiota composition on performance, productivity and condition in poultry?

Microbiome of the gastrointestinal tract (GIT) has been identified as one of the crucial factors influencing the health and condition of domestic animals. The global poultry industry faces the challenge of understanding the complex relationship between gut microbiota composition and performance-related traits in birds. Considerable variation exists in the results of correlational studies using either 16S rRNA profiling or metagenomics to identify bacterial taxa associated with performance, productivity, or condition in poultry (e.g., body weight, growth rate, feeding efficiency, or egg yield). In this review, we survey the existing reports, discuss variation in research approaches, and identify bacterial taxa consistently linked to improved or deteriorated performance across individual poultry-focused studies. Our survey revealed high methodological heterogeneity, which was in contrast with vastly uniform focus of the research mainly on the domestic chicken (Gallus gallus) as a model. We also show that the bacterial taxa most frequently used in manipulative experiments and commercial probiotics intended for use in poultry (e.g., species of Lactobacillus, Bacillus, Enterococcus, or Bifidobacterium) do not overlap with the bacteria consistently correlated with their improved performance (Candidatus Arthromitus, Methanobrevibacter). Our conclusions urge for increased methodological standardization of the veterinary research in this field. We highlight the need to bridge the gap between correlational results and experimental applications in animal science. To better understand causality in the observed relationships, future research should involve a broader range of host species that includes both agricultural and wild models, as well as a broader range of age groups.

Nearly (?) sterile avian egg in a passerine bird.

During early ontogeny, microbiome affects development of the gastrointestinal tract, immunity, and survival in vertebrates. Bird eggs are thought to be (1) initially sterile (sterile egg hypothesis) and (2) colonized after oviposition through horizontal trans-shell migration, or (3) initially seeded with bacteria by vertical transfer from mother oviduct. To date, however, little empirical data illuminate the contribution of these mechanisms to gut microbiota formation in avian embryos. We investigated microbiome of the egg content (day 0; E0-egg), embryonic gut at day 13 (E13) and female faeces in a free-living passerine, the great tit (Parus major), using a methodologically advanced procedure combining 16S rRNA gene sequencing and microbe-specific qPCR assays. Our metabarcoding revealed that the avian egg is (nearly) sterile, but acquires a slightly richer microbiome during the embryonic development. Of the three potentially pathogenic bacteria targeted by qPCR, only Dietzia was found in E0-egg (yet also in negative controls), E13 gut and female samples, which might indicate possible vertical transfer. Unlike in poultry, we have shown that major bacterial colonization of the gut in passerines does not occur before hatching. We emphasize that protocols that carefully check for environmental contamination are critical in studies with low-bacterial biomass samples.

Subclinical peripheral inflammation has systemic effects impacting central nervous system proteome in budgerigars.

Regulation of neuroimmune interactions varies across avian species. Little is presently known about the interplay between periphery and central nervous system (CNS) in parrots, birds sensitive to neuroinflammation. Here we investigated the systemic and CNS responses to dextran sulphate sodium (DSS)- and lipopolysaccharide (LPS)-induced subclinical acute peripheral inflammation in budgerigar (Melopsittacus undulatus). Three experimental treatment groups differing in DSS and LPS stimulation were compared to controls. Individuals treated with DSS showed significant histological intestinal damage. Through quantitative proteomics we described changes in plasma (PL) and cerebrospinal fluid (CSF) composition. In total, we identified 180 proteins in PL and 978 proteins in CSF, with moderate co-structure between the proteomes. Between treatments we detected differences in immune, coagulation and metabolic pathways. Proteomic variation was associated with the levels of pro-inflammatory cytokine mRNA expression in intestine and brain. Our findings shed light on systemic impacts of peripheral low-grade inflammation in birds.

Varying conjunctival immune response adaptations of house finch populations to a rapidly evolving bacterial pathogen.

Pathogen adaptations during host-pathogen co-evolution can cause the host balance between immunity and immunopathology to rapidly shift. However, little is known in natural disease systems about the immunological pathways optimised through the trade-off between immunity and self-damage. The evolutionary interaction between the conjunctival bacterial infection Mycoplasma gallisepticum (MG) and its avian host, the house finch (Haemorhous mexicanus), can provide insights into such adaptations in immune regulation. Here we use experimental infections to reveal immune variation in conjunctival tissue for house finches captured from four distinct populations differing in the length of their co-evolutionary histories with MG and their disease tolerance (defined as disease severity per pathogen load) in controlled infection studies. To differentiate contributions of host versus pathogen evolution, we compared house finch responses to one of two MG isolates: the original VA1994 isolate and a more evolutionarily derived one, VA2013. To identify differential gene expression involved in initiation of the immune response to MG, we performed 3'-end transcriptomic sequencing (QuantSeq) of samples from the infection site, conjunctiva, collected 3-days post-infection. In response to MG, we observed an increase in general pro-inflammatory signalling, as well as T-cell activation and IL17 pathway differentiation, associated with a decrease in the IL12/IL23 pathway signalling. The immune response was stronger in response to the evolutionarily derived MG isolate compared to the original one, consistent with known increases in MG virulence over time. The host populations differed namely in pre-activation immune gene expression, suggesting population-specific adaptations. Compared to other populations, finches from Virginia, which have the longest co-evolutionary history with MG, showed significantly higher expression of anti-inflammatory genes and Th1 mediators. This may explain the evolution of disease tolerance to MG infection in VA birds. We also show a potential modulating role of BCL10, a positive B- and T-cell regulator activating the NFKB signalling. Our results illuminate potential mechanisms of house finch adaptation to MG-induced immunopathology, contributing to understanding of the host evolutionary responses to pathogen-driven shifts in immunity-immunopathology trade-offs.

Peripheral inflammation-induced changes in songbird brain gene expression: 3' mRNA transcriptomic approach.

Species-specific neural inflammation can be induced by profound immune signalling from periphery to brain. Recent advances in transcriptomics offer cost-effective approaches to study this regulation. In a population of captive zebra finch (Taeniopygia guttata), we compare the differential gene expression patterns in lipopolysaccharide (LPS)-triggered peripheral inflammation revealed by RNA-seq and QuantSeq. The RNA-seq approach identified more differentially expressed genes but failed to detect any inflammatory markers. In contrast, QuantSeq results identified specific expression changes in the genes regulating inflammation. Next, we adopted QuantSeq to relate peripheral and brain transcriptomes. We identified subtle changes in the brain gene expression during the peripheral inflammation (e.g. up-regulation in AVD-like and ACOD1 expression) and detected co-structure between the peripheral and brain inflammation. Our results suggest benefits of the 3'end transcriptomics for association studies between peripheral and neural inflammation in genetically heterogeneous models and identify potential targets for the future brain research in birds.