Periodic Reporting for period 4 - EcoImmuneCosts (Immunity in Ecology and Evolution: 'Hidden' costs of disease, immune function and their consequences for Darwinian fitness) Reporting period: 2022-02-01 to 2023-01-31 Summary of the context and overall objectives of the project Is there such a thing as a harmless parasite infection? Why does the immune system induce costs and sometimes even attack our own body, leading to devastating outcomes? Why are we ageing, and why some faster than others? These are fundamental questions in biology and medicine - and key aspects of this ERC project.There is currently an immense interest in factors (incl. sex-specific patterns) that mediate and shape disease resistance, immune genes and immune response-related diseases (i.e. immunopathology), and how this relates to ageing and reduced lifespan. We clearly need to know more about how the immune system has evolved to optimize protection and minimize immunopathology (incl. autoimmune) costs. For this, a whole-organism approach is necessary that integrates concepts and methods of evolutionary ecology, immunology and molecular biology (a so-called ‘immunoecology approach’) – this lies at the core of my ERC AdG project ‘EcoImmuneCosts’ (EIC). This cross-disciplinary project has generated results of interest to evolutionary, behavioral and functional ecologists, but also to researchers in e.g. medicine and gerontology.A primary target of EIC is to study the long-term consequences of low-virulent pathogens causing mild infections (previously assumed harmless), on ageing, lifetime reproductive success and lifespan - so called Darwinian fitness costs. However, the mechanisms mediating these costs are still unknown. One possible mechanism is degradation of telomeres (repetitive, non-coding DNA sequences essential for chromosome integrity and cell survival). EIC has combined correlational and experimental studies to investigate if accelerated telomere degradation can be a causative mechanism through which small immune costs (and other stressors) can accumulate and be translated into accelerated senescence and reduced Darwinian fitness. Specifically, we investigated whether there are signs of a lower ‘critical threshold’ in telomere length and patterns of TL elongation both over short and long term. Costs of infection may also be difficult to measure if there are sexually antagonistic effects on immune gene variation and regulation. The project therefore specifically targeted sex-specific immune gene variation and expression to investigate ‘hidden' immune costs of infection. In conclusion, the EIC project has been very successful in fulfilling its project goals running large-scale long-term experiments and field studies and analysing samples. These studies have already generated exciting and novel results and I anticipate that many more will accumulate as we analyse more data. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Research problem 1: Are correlations between telomere shortening and ageing processes merely a result of telomere length (TL) being a marker of biological age or is there evidence for TL playing a causal role in physiological deterioration and (immune-)senescence? In a large long-term experiment on canaries, we tested the separate (and combined) effects of a mild chronic infection on disease progress and physiological senescence. We used qPCR to measure TL on 800 samples from >160 individuals. Analyses on oxidative damage and antioxidant capacity are being completed (>450 samples). This allows us to study how telomere shortening and oxidative stress covary across treatments. Results from our two other study systems (great reed warbler (GRW) and zebra finch (ZF)) show strong indications of a lower critical threshold in TL and that individuals with TL close to this threshold are more likely to elongate TL (despite potentially substantial costs involved in telomere elongation). This implies that there is a lower limit of short TL where cell senescence sets in, jeopardizing organism function and accelerating ageing. Research problem 2: Is telomere length an important agent mediating transgenerational effects on immunity?We studied the potential connections between malaria infection and TL in longitudinal studies of wild GRWs. One year old individuals that had become infected with malaria had lower mean early-life TL (eTL) compared to uninfected. This result appear to be a selective disappearance effect because infected birds did not elongate their telomeres, exhibited faster TL shortening, and showed disappearance of a larger fraction of individuals with short eTL before 1 year of age. We created parental ZF pairs with either short or long eTL (as nestlings) and let these pairs produce offspring . This allowed us to test the assumption that individual telomere (and, hence, ageing) trajectories are already set very early in life. While offspring eTL matched their parents eTL, they did so only after birth but not at the embryo stage. Our studies provide new evidence that TL is not set for life at conception, but may instead be modulated by environmental factors and individual-specific (possibly genetically inherited) TL trajectories.Research problem 3: Can sexually antagonistic selection explain patterns in immune gene diversity and risk of immunopathology/autoimmunity? We have found that immune gene diversity at the MHC class I (MHC-I) genes in GRWs is under sexually antagonistic selection: males with a higher number of different MHC-I alleles produce offspring with superior return rate, whereas the opposite pattern is true for females. Moreover, that haplotypes found in wild GRW show higher variability than expected by chance thus favoring a broader immunity recognition system. This data is being complemented with data from a long-term experiment on sex-specific effects of chronic mild disease. Captive GRW (40 birds) infected with malaria were sampled for TL measurements and immune gene expression studies. So far, these projects have resulted in three PhD theses and 10 international publications. Nine further manuscripts have been produced (several now submitted). Results have been disseminated in 10 international conference presentations (lower than expected due to Covid). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The EIC project has yielded unexpected results from different datasets that in combination provide strong circumstantial support for the existence of a lower ‘critical threshold’ in TL of blood cells. Moreover, parallel results obtained in multiple study systems suggest that changes in TL across life are much more dynamic (elongation more common) than previously thought. These are novel results that have the potential to move the field forward and provide new avenues for research. Moreover, publication of two conceptual articles (incl. a major review of all hypotheses in telomere ecology and evolution) is a major contribution to the research field from the EIC project and I expect that these articles will help advance research beyond the current state of knowledge. Finally, the 36 year study of GRWs at Lake Kvismaren, providing multi-generational DNA samples and data of breeding and life history parameters, has given us unprecedented data of how immune gene diversity can affect Darwinian fitness under natural conditions and that sexually antagonistic selection plays an important role. These results may help explain the increasing incidence of autoimmune diseases in modern societies and why women generally are more affected than men. To catch great reed warblers, mist nets are set up in the reed beds. Singing reat reed warbler male at Lake Kvismaren. All birds are marked with colour rings. A great reed warbler is blood sampled.