Periodic Reporting for period 1 - MATHORMAL (From maternal hormones to malaria susceptibility and accelerated ageing: new insights from wild birds.)
Reporting period: 2023-09-01 to 2025-08-31
Emerging infectious diseases are becoming more frequent, complex, and difficult to control in a scenario of global change. In the past decades, outbreaks such as malaria, SARS-CoV2, West Nile virus, and Ebola have disrupted public health systems, threatened biodiversity conservation, and imposed substantial economic burdens worldwide. These crises highlight the urgent need for integrative health management, with the "One-World, One Health" initiative providing a key framework for addressing the interconnected health of humans, animals, and the environment through global collaboration. Malaria represents a longstanding and devastating global health challenge but, despite intensive research efforts, malaria remains a persistent issue in developing countries, exacerbated by its impacts on wildlife, including the decline and extinction of avian populations. The MATHORMAL project seeks to address a fundamental gap in our understanding of malaria susceptibility by exploring how prenatal hormonal effects shape individual physiological traits that influence infection susceptibility. Maternal effects, particularly those mediated by androgens, play a pivotal role in shaping offspring phenotypes, influencing their growth, immune responses, and life-history traits. Avian eggs contain various amounts of these maternally derived hormones that could adjust the offspring development to post-hatching environment. The project was guided by three overarching research objectives:
1. Investigating Immune Costs of Hormone-Mediated Maternal Effects: To determine whether high prenatal exposure to hormones, such as testosterone (T), induces delayed immune costs that increase susceptibility to malaria infection in adulthood.
2. Linking Telomere Dynamics to Malaria Susceptibility: To explore whether early-life telomere length predicts individual vulnerability to malaria infection and whether malaria susceptibility explains delayed telomere shortening caused by maternal hormonal effects.
3. Examining the Role of the Uropygial Gland Microbiome: To assess whether hormone-mediated maternal effects alter the uropygial gland’s microbial community, influencing the susceptibility to malaria through changes in vector-host dynamics.
Regarding the pathway to impact, MATHORMAL addresses critical gaps in understanding the physiological and ecological mechanisms that underpin individual variability in malaria susceptibility. By linking maternal hormonal effects, immune function, telomere dynamics, and the microbiome, this project aims to deliver novel insights that could be applied across several domains such as Scientific Impact, Public Health Applications, Wildlife Conservation and Biodiversity, and Global Health Policy and Strategy. Our findings will have wide-reaching implications, from advancing basic science to informing applied health and conservation strategies. By elucidating the role of prenatal hormonal programming in disease susceptibility, MATHORMAL could transform how we approach the prevention and management of malaria and other vector-borne diseases. The insights generated will contribute to a more holistic and proactive framework for global health, directly supporting efforts to mitigate the interconnected challenges of emerging diseases, biodiversity loss, and environmental change.
1. Investigating Immune Costs of Hormone-Mediated Maternal Effects: To determine whether high prenatal exposure to hormones, such as testosterone (T), induces delayed immune costs that increase susceptibility to malaria infection in adulthood.
2. Linking Telomere Dynamics to Malaria Susceptibility: To explore whether early-life telomere length predicts individual vulnerability to malaria infection and whether malaria susceptibility explains delayed telomere shortening caused by maternal hormonal effects.
3. Examining the Role of the Uropygial Gland Microbiome: To assess whether hormone-mediated maternal effects alter the uropygial gland’s microbial community, influencing the susceptibility to malaria through changes in vector-host dynamics.
Regarding the pathway to impact, MATHORMAL addresses critical gaps in understanding the physiological and ecological mechanisms that underpin individual variability in malaria susceptibility. By linking maternal hormonal effects, immune function, telomere dynamics, and the microbiome, this project aims to deliver novel insights that could be applied across several domains such as Scientific Impact, Public Health Applications, Wildlife Conservation and Biodiversity, and Global Health Policy and Strategy. Our findings will have wide-reaching implications, from advancing basic science to informing applied health and conservation strategies. By elucidating the role of prenatal hormonal programming in disease susceptibility, MATHORMAL could transform how we approach the prevention and management of malaria and other vector-borne diseases. The insights generated will contribute to a more holistic and proactive framework for global health, directly supporting efforts to mitigate the interconnected challenges of emerging diseases, biodiversity loss, and environmental change.
During the 2023 field season, I conducted a field experiment increasing prenatal yolk T levels using a dose-dependent experiment in a wild great tit (Parus major) population breeding in nest-boxes installed in Ruissalo Island (Turku, Finland). Treatments were randomly assigned between clutches and consisted of either: (1) Low dose: 12.8 ng of T (2 SD) dissolved in 5 µl of sesame oil, or (2) High dose: 25.6 ng T (4 SD) in 5 µl of sesame oil. Within each control clutch, all eggs received 5 μl of sesame oil. In the spring of 2024 and based on the results obtained in 2023, we decided to use the higher dose (4 SD) in another yolk T-injection experiment and combine it with a postnatal treatment with TA-65 (a telomerase activator and telomere extender). Here, half of the chicks hatched from each experimental nest were treated every other day with sterile water (control), whereas the other half was treated with TA-65 (0.5 mg / g body weight). This 2x2 experimental design enabled to separate the direct effect of telomere length on malaria susceptibility from the indirect effect mediated by maternal testosterone.
In both experiments, nestlings were measured on day 7 and 14 post-hatching. At these ages, we recorded biometric measurements, and all birds were banded with unique alphanumeric codes for individual identification. Blood samples were collected twice during postnatal development for immunological and aging markers quantification. Breathing rate was also measured for at least 2 chicks/nest. The sex of the chicks was determined by PCR-based methods (primers P2-P8). During autumn 2023 and 2024, juveniles from both experiments were captured using mist nets placed close to feeders allowing estimating malaria infection status, physiological status and apparent survival. Biometric measurements and another blood sample were also taken to compare the effect of treatment on the immune system, telomere length, and malaria parasite prevalence. In addition, we measured the uropygial gland size and took a sample of its oily secretion. The 2 innermost tertiary feathers were taken to count the number of feather mites. In all cases, a drop of blood was smeared on individually marked microscope slides, air-dried, and stained with Quick Panoptic and Giemsa methods.
With regard to the 2023 samples, and through the supervision of 2 Master students from Université Claude Bernard Lyon 1 and Sorbonne Université (France) at UTU, we were able to assess the immune system of chicks and juveniles through differential leucocyte counts in blood smears, while calculating the prevalence and intensity of avian malaria parasite infection. The other immune assays performed on plasma samples, such as immunoglobulin G and haptoglobin concentration, or the levels of natural antibodies and complement (haemolysis-hemaggluttination assays), were carried out at the University of Jyväskylä (Finland) with the support of Suvi Ruuskanen, a key collaborator in achieving the objectives described in MATHORMAL. Finally, to assess the effect of androgen treatment on ageing (measuring telomere length by qPCR); I did a research stay at the Institut Pluridisciplinaire Hubert Curien (IPHC) in Strasbourg (France) with Antoine Stier (my MSCA supervisor).
Because I have obtained a permanent teaching position at the University of Cordoba (Spain), MATHORMAL had to be finished earlier than expected (after 14 months of its start). However, the rest of the analyses linked to the samples of the 2024 experiment will be carried out during 2025, even if I do not have European funding, as I am really interested in extracting the data and writing at least a couple of scientific papers to share the MATHORMAL findings with the rest of the scientific community. My current contract at the University of Cordoba could allow me to undertake research stays to carry out these objectives, at least to cover transport and accommodation. Regarding the analysis of microbiota in faeces or uropygial secretion, it will be carried out at the University of Jyväskylä by Suvi Ruuskanen's team as they have the necessary equipment and funding.
In both experiments, nestlings were measured on day 7 and 14 post-hatching. At these ages, we recorded biometric measurements, and all birds were banded with unique alphanumeric codes for individual identification. Blood samples were collected twice during postnatal development for immunological and aging markers quantification. Breathing rate was also measured for at least 2 chicks/nest. The sex of the chicks was determined by PCR-based methods (primers P2-P8). During autumn 2023 and 2024, juveniles from both experiments were captured using mist nets placed close to feeders allowing estimating malaria infection status, physiological status and apparent survival. Biometric measurements and another blood sample were also taken to compare the effect of treatment on the immune system, telomere length, and malaria parasite prevalence. In addition, we measured the uropygial gland size and took a sample of its oily secretion. The 2 innermost tertiary feathers were taken to count the number of feather mites. In all cases, a drop of blood was smeared on individually marked microscope slides, air-dried, and stained with Quick Panoptic and Giemsa methods.
With regard to the 2023 samples, and through the supervision of 2 Master students from Université Claude Bernard Lyon 1 and Sorbonne Université (France) at UTU, we were able to assess the immune system of chicks and juveniles through differential leucocyte counts in blood smears, while calculating the prevalence and intensity of avian malaria parasite infection. The other immune assays performed on plasma samples, such as immunoglobulin G and haptoglobin concentration, or the levels of natural antibodies and complement (haemolysis-hemaggluttination assays), were carried out at the University of Jyväskylä (Finland) with the support of Suvi Ruuskanen, a key collaborator in achieving the objectives described in MATHORMAL. Finally, to assess the effect of androgen treatment on ageing (measuring telomere length by qPCR); I did a research stay at the Institut Pluridisciplinaire Hubert Curien (IPHC) in Strasbourg (France) with Antoine Stier (my MSCA supervisor).
Because I have obtained a permanent teaching position at the University of Cordoba (Spain), MATHORMAL had to be finished earlier than expected (after 14 months of its start). However, the rest of the analyses linked to the samples of the 2024 experiment will be carried out during 2025, even if I do not have European funding, as I am really interested in extracting the data and writing at least a couple of scientific papers to share the MATHORMAL findings with the rest of the scientific community. My current contract at the University of Cordoba could allow me to undertake research stays to carry out these objectives, at least to cover transport and accommodation. Regarding the analysis of microbiota in faeces or uropygial secretion, it will be carried out at the University of Jyväskylä by Suvi Ruuskanen's team as they have the necessary equipment and funding.
High prenatal T levels could carry short-term benefits (i.e. accelerated metabolism and growth), but carry long-term costs including accelerated telomere shortening (i.e. a hallmark of ageing) and depressed immunity that could both increase susceptibility to malaria. Our results from the experiment conducted in 2023 show no effects of T-treatment on body size or condition of nestlings, but chicks treated with the higher T-dose had higher breathing rates. Nestling survival to fledging was positively influenced by prenatal T (higher T-dose > control and lower T-dose), while we found no evidence for an effect on hatching success or post-fledging survival. The lowest dose had a higher effect on some components of the immune system like the total leukocyte count and the proportion of monocytes. Immunological analyses did not reveal any negative effect of prenatal testosterone on the immune system, irrespective of age. However, the highest dose of testosterone did lead to higher malaria prevalence in the juveniles, but only in males. Our results suggest that exposure to high concentrations of testosterone during the prenatal period may predispose male chicks to increased susceptibility to malaria. With regard to postnatal telomere dynamics, our findings showed that telomeres shortened with age and, contrary to our hypotheses, were longer in individuals hatched from testosterone-treated eggs—non-significantly for the low dose (p = 0.064) and significantly for the high dose (p = 0.049). Contrary to expectations, uropygial gland volume or secretion did not increase with higher testosterone doses; instead, we observed a slight decrease in gland size in chicks treated with the lowest dose. However, our results demonstrated that feather mite load in juveniles decreased as testosterone dose increased, while mite load also decreased with an increase in the volume of secretion. Future studies should carefully consider a range of testosterone concentrations, as the balance of costs and benefits of prenatal hormones may be dose-dependent.
Regarding the data from the spring 2024 experiment, we do not yet have consistent results as I had to terminate my MSCA contract at the end of October when the autumn capture of juveniles was in progress. Hatching success did not differ between testosterone-treated eggs (56.4%) and controls (50.0%). Fortunately, we were able to capture 52 experimental juveniles in autumn (27 controls and 25 hatchlings from testosterone-injected eggs), detecting no differences in survival due to androgen action. We also did not detect differences in survival derived from TA-65 administration, where in juveniles hatched from T-eggs we captured 14 and 11 individuals supplemented with TA-65 or water respectively, and in those hatched from control eggs we captured 16 and 11 individuals supplemented with TA-65 or water respectively. Beyond the possible effect of TA-65 or sex, by visually exploring the results, it could be intuited that those chicks hatched from T-eggs have a higher body weight, as well as a larger uropygial gland and faster respiratory rates than controls, which could have a direct effect on telomere length and immunity, possibly affecting susceptibility to malaria. To analyse these data more precisely we need more time, as in the following months (still without the MSCA budget) we plan to do DNA extraction (using blood samples) for nestling sexing, telomere length measurement (ageing) and malaria parasite detection and sequencing.
Regarding the data from the spring 2024 experiment, we do not yet have consistent results as I had to terminate my MSCA contract at the end of October when the autumn capture of juveniles was in progress. Hatching success did not differ between testosterone-treated eggs (56.4%) and controls (50.0%). Fortunately, we were able to capture 52 experimental juveniles in autumn (27 controls and 25 hatchlings from testosterone-injected eggs), detecting no differences in survival due to androgen action. We also did not detect differences in survival derived from TA-65 administration, where in juveniles hatched from T-eggs we captured 14 and 11 individuals supplemented with TA-65 or water respectively, and in those hatched from control eggs we captured 16 and 11 individuals supplemented with TA-65 or water respectively. Beyond the possible effect of TA-65 or sex, by visually exploring the results, it could be intuited that those chicks hatched from T-eggs have a higher body weight, as well as a larger uropygial gland and faster respiratory rates than controls, which could have a direct effect on telomere length and immunity, possibly affecting susceptibility to malaria. To analyse these data more precisely we need more time, as in the following months (still without the MSCA budget) we plan to do DNA extraction (using blood samples) for nestling sexing, telomere length measurement (ageing) and malaria parasite detection and sequencing.