Contacts with environmental biodiversity affect human health: links revealed during the initial waves of the COVID-19 pandemic.

The gradual decrease in the prevalence of serious infectious diseases over the last century has been followed by increase in so called "modern" diseases, including allergies, chronic inflammatory conditions, psychiatric, and metabolic disorders. Between 2019 and 2022, public awareness of the threat of infectious diseases in humans was renewed by the global pandemic of a new type of a coronavirus, the SARS-COV-2. This public interest opened improved possibilities to test hypotheses on the factors associated with inter-individual variation in susceptibility to infectious and "modern" diseases. Based on the Hygiene hypothesis and Biodiversity hypothesis, we predicted that contacts with natural environment and wildlife in childhood and/or in adulthood can improve general health and decrease the risks of severe COVID-19 progression or prevalence of the "modern" diseases, namely the allergies. Here we report the results of an online, self-evaluating questionnaire survey conducted in the Czech Republic, where we contrasted selected health issues, and linked them to the living environment, including the level of contacts with biodiversity. In a sample of 1188 respondents, we revealed a significant association of time spent in nature or contacts with biodiversity with physical and mental health, or incidence of allergies. This is unlike the COVID-19 progression, which was related to age, physical health, smoking, allergies, and interaction of age with smoking, but not to contacts with the natural environmental diversity. Our findings regarding to physical and mental health and allergies are in agreement with the Biodiversity hypothesis of allergy and, linking human and environmental health, they urge for One Health approach application.

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.

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.

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.

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.

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.

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.

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.

Genome sequence of parvovirus from budgerigar (Melopsittacus undulatus).

Here, we report a parvovirus genome identified in Melopsittacus undulatus. The genome is 4,547 bp long and codes for two major open reading frames (ORFs): the non-structural replicase protein 1 (NS1) and the structural capsid gene (VP1). Phylogenetic analysis shows that this virus belongs to the genus Chaphamaparvovirus.

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.

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.

Divergent evolution drives high diversity of toll-like receptors (TLRs) in passerine birds: Buntings and finches.

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.

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.

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.

Proteomic-based evidence for adult neurogenesis in birds and mammals as indicated from cerebrospinal fluid.

Adult neurogenesis is the life-long process of neural stem cell proliferation, differentiation into neurons, migration, and incorporation into the existing neuronal circuits. After decades of research, it is now widely accepted that mammals and birds retain the capacity to regenerate neurons even after their subadult ontogeny. Cerebrospinal fluid participates in the regulation of the neurogenic niches of the vertebrate brain through signaling pathways not fully elucidated. Proteomic studies of cerebrospinal fluid have the potential to allow the in-depth characterization of its molecular composition. Comparative studies help to delineate those pathways that are universally critical for the regulation of neurogenesis in adulthood. In this review, we performed literature-based data mining in studies using liquid chromatography-tandem mass spectroscopy that analyzed cerebrospinal fluid samples from healthy adult humans (Homo sapiens); mice (Mus musculus); sheep (Ovis aries); chickens (Gallus gallus); and two parrot species, the budgerigar (Melopsittacus undulatus) and cockatiel (Nymphicus hollandicus). We identified up to 911 proteins represented in cerebrospinal fluid, involved in various pathways regulating adult neurogenesis. However, only 196 proteins were common across humans, mice, and birds. Pathway components involved in nervous system development, cell migration, and axonal guidance were commonly evident in all species investigated so far. Extensive bioinformatic analysis revealed that the universally over-represented pathways involved L1 cell adhesion molecule protein interactions, cell-adhesion molecules, signals regulating extracellular matrix remodeling, regulation of insulin growth factor signaling, axonal guidance, programmed cell death, immune signaling, and post-translational modifications. Most of the reported proteins are part of extracellular vesicles enriched in cerebrospinal fluid. However, the information presently available is still highly fragmentary, and far more questions persist than are answered. Technological advances will allow cerebrospinal fluid comparative proteomic research to delve into the fundamental processes of adult neurogenesis and eventually translate this research into any regenerative interventions.

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.

Dynamic Evolution of Avian RNA Virus Sensors: Repeated Loss of RIG-I and RIPLET.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are key RNA virus sensors belonging to the RIG-I-like receptor (RLR) family. The activation of the RLR inflammasome leads to the establishment of antiviral state, mainly through interferon-mediated signaling. The evolutionary dynamics of RLRs has been studied mainly in mammals, where rare cases of RLR gene losses were described. By in silico screening of avian genomes, we previously described two independent disruptions of MDA5 in two bird orders. Here, we extend this analysis to approximately 150 avian genomes and report 16 independent evolutionary events of RIG-I inactivation. Interestingly, in almost all cases, these inactivations are coupled with genetic disruptions of RIPLET/RNF135, an ubiquitin ligase RIG-I regulator. Complete absence of any detectable RIG-I sequences is unique to several galliform species, including the domestic chicken (Gallus gallus). We further aimed to determine compensatory evolution of MDA5 in RIG-I-deficient species. While we were unable to show any specific global pattern of adaptive evolution in RIG-I-deficient species, in galliforms, the analyses of positive selection and surface charge distribution support the hypothesis of some compensatory evolution in MDA5 after RIG-I loss. This work highlights the dynamic nature of evolution in bird RNA virus sensors.

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.

Repeated MDA5 Gene Loss in Birds: An Evolutionary Perspective.

Two key cytosolic receptors belonging to the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family sense the viral RNA-derived danger signals: RIG-I and melanoma differentiation-associated protein 5 (MDA5). Their activation establishes an antiviral state by downstream signaling that ultimately activates interferon-stimulated genes (ISGs). While in rare cases RIG-I gene loss has been detected in mammalian and avian species, most notably in the chicken, MDA5 pseudogenization has only been detected once in mammals. We have screened over a hundred publicly available avian genome sequences and describe an independent disruption of MDA5 in two unrelated avian lineages, the storks (Ciconiiformes) and the rallids (Gruiformes). The results of our RELAX analysis confirmed the absence of negative selection in the MDA5 pseudogene. In contrast to our prediction, we have shown, using multiple dN/dS-based approaches, that the MDA5 loss does not appear to have resulted in any compensatory evolution in the RIG-I gene, which may partially share its ligand-binding specificity. Together, our results indicate that the MDA5 pseudogenization may have important functional effects on immune responsiveness in these two avian clades.