Summary: 1. The phytohaemagglutinin (PHA) skin‐swelling test is widely used in immunoecology and ecotoxicology to estimate cell‐mediated immunity. Although often presumed, the involvement of T cells in generating an immune response to PHA in vivo remains unclear. 2. To investigate the mechanism triggering this response we have compared in zebra finch (Taeniopygia guttata) the immune responses to two PHA isolectins differing in their biological properties and one control protein. 3. In Experiment I, we applied PHA‐P (the commonly used L and E isolectin mixture) into one wing‐web and bovine serum allbumin (BSA) into the other. The swelling response to PHA‐P was significantly stronger than to BSA confirming that the reaction is governed by PHA‐specific properties. 4. In Experiment II, purified PHA‐L (the T‐cell‐stimulating isolectin) and PHA‐E (the erythroagglutinating isolectin) were compared. Contrary to our expectations, PHA‐E induced a significantly stronger swelling response than PHA‐L. Histological analysis revealed significantly higher quantities of erythrocytes and thrombocytes in PHA‐E‐treated patagia. Nevertheless, there was a positive correlation between the magnitudes of these swellings. 5. In Experiment III, we tested whether the results obtained by the application of PHA‐P and pure PHA‐L differ. Here, we failed to find any significant difference between these two preparations in their immunostimulatory activity. Magnitudes of the PHA‐L‐ and PHA‐P‐induced swellings were positively correlated. 6. To the best of our knowledge this is the first study to compare the biological activity of purified PHA fractions in vivo and also the first to show the importance of erythroagglutination in the development of an inflammatory response to PHA‐P. 7. Our results indicate that the skin‐swelling test using PHA‐P reliably mirrors the individual general proinflammatory potential. However, the immunological background of the test is highly complex and the test results cannot be interpreted as measurements of the adaptive immunity or T‐cell activity. This interpretational change importantly alters our view on the test results regarding the costs of the response or the evolutionary immunological adaptations.

Immunity exhibits extraordinarily high levels of variation. Evolution of the immune system in response to host-pathogen interactions in particular ecological contexts appears to be frequently associated with diversifying selection increasing the genetic variability. Many studies have documented that immunologically relevant polymorphism observed today may be tens of millions years old and may predate the emergence of present species. This pattern can be explained by the concept of trans-species polymorphism (TSP) predicting the maintenance and sharing of favourable functionally important alleles of immune-related genes between species due to ongoing balancing selection. Despite the generality of this concept explaining the long-lasting adaptive variation inherited from ancestors, current research in TSP has vastly focused only on major histocompatibility complex (MHC). In this review we summarise the evidence available on TSP in human and animal immune genes to reveal that TSP is not a MHC-specific evolutionary pattern. Further research should clearly pay more attention to the investigation of TSP in innate immune genes and especially pattern recognition receptors which are promising candidates for this type of evolution. More effort should also be made to distinguish TSP from convergent evolution and adaptive introgression. Identification of balanced TSP variants may represent an accurate approach in evolutionary medicine to recognise disease-resistance alleles.

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. Abstract Reviewing current advances in our understanding of molecular evolution underpinning vertebrate immunity, we propose hypotheses to explain differences in selection modes across immune genes and discuss supporting evidence.

In vertebrates, it has been repeatedly demonstrated that genes encoding proteins involved in pathogen-recognition by adaptive immunity (e.g. MHC) are subject to intensive diversifying selection. On the other hand, the role and the type of selection processes shaping the evolution of innate-immunity genes are currently far less clear. In this study we analysed the natural variation and the evolutionary processes acting on two genes involved in the innate-immunity recognition of Microbe-Associated Molecular Patterns (MAMPs).