In vertebrates, disease susceptibility often varies with age. Older individuals may be more susceptible than younger individuals due to senescent declines in immune function. Alternatively, disease susceptibility may decrease with age if older individuals are more likely to have had prior exposures to parasites and acquired adaptive immune responses that allowed them to resist future infections. Disease susceptibility can also vary with reproductive state, and reproductive hormones have been shown to increase infection susceptibility. Here we investigated how age and experimentally elevated levels of the reproductive hormone testosterone affected haemosporidian infections in free-living adult male dark-eyed juncos (Junco hyemalis). Samples were collected before and at least one year after experimental treatment with either a testosterone implant or empty control implant. The probability of being infected with haemosporidians in the Plasmodium/Haemoproteus group increased with host age but was unaffected by experimental testosterone elevation. Our longitudinal sampling design allowed us to determine that the increase in infections with host age was driven by variation within individuals, such that an individual’s probability of being infected increased with age. This suggests that haemosporidian susceptibility increases with host age, and/or that haemosporidian-infected juncos sustain long-term chronic infections that are rarely completely cleared, and that the probability of being infected is higher in older juncos because they have experienced higher cumulative exposure risk.

Haemosporidian parasite infections in the junco blood samples were measured in the Center for the Integrative Study of Animal Behavior Laboratory at Indiana University, Bloomington. To determine haemosporidian infection status, the haemosporidian parasite cytochrome B (cytb) gene was amplified using published nested polymerase chain reaction (PCR) protocols (Hellgren et al. 2004, Waldenström et al. 2004). Each gDNA sample was amplified using the external PCR primers HAEMNF and HAEMNR2. Following the first round of amplification, PCR product was amplified again using the nested or internal primers HAEMF and HAEMR2 (Hellgren et al. 2004), which have been shown to very efficiently amplify Plasmodium and Haemoproteus cytb sequences, and additionally with the nested or internal primers HAEMFL and HAEMR2L, for amplification of Leucocytozoon sequences (Hellgren et al. 2004).

Slowinski et al. 2021 JAB Dryad data key

Data sheets:

• Plas.Haem: Data sheet for the Plasmodium/Haemoproteus infection status of birds, as determined using the nested or internal primers HAEMF and HAEMR2 (Hellgren et al. 2004). This sheet includes only birds for which the Plasmodium/Haemoproteus infection status could be determined at the time of first implant and in at least one following year.
• Leucocytozoon: Data sheet for Leucocytozoon infection statust of birds, as determined using the nested or internal primers HAEMFL and HAEMR2L (Hellgren et al. 2004). This sheet includes only birds for which the Leucocytozoon infection status could be determined at the time of first implant and in at least one following year.
• First.samples: Data sheet for Plasmodium/Haemoproteus and Leucocytozoon infection status of birds at the time of their first implant. This sheet includes only birds for which both the Plasmodium/Haemoproteus infection status and the Leucocytozoon infection status could be determined at the time of first implant

Column headings

• Blood_sample_date: date at which blood sample was collected
• Implant_in: Closest date to blood sample collection date on which bird was given a testosterone or control implant
• BloodIDnoletter: Unique ID number associated with each blood sample
• Ring: Unique ring number associated with each bird
• Age: age of bird at time blood sample was collected
• Age_at_first_implant: Age of the focal bird when it received its first testosterone or control implant
• Mean age: Mean age of the focal bird across all of the ages at which blood samples were collected
• Age_minus_mean_age: Age of the focal bird at the time the present blood sample was collected, minus the mean age of the focal bird across samples 
• Implant_number: Number of times the focal bird has been implanted (including the implant associated with the present sample).
• First_implant_year: Year in which the focal bird was first implanted
• Years_since_first_implant: Years between the time at which the focal bird was first implanted, and the present sample
• Treatment_0C_1T: Implant treatment. Control implants represented by zeroes. Testosterone implants represented by ones.
• Plas.Haem.Infections: Plasmodium/Haemoproteus infection status, as determined by nested PCR and gel electrophoresis. Zeroes represent negative infections. Ones represent positive infections
• Leuc.0n_1p: Leucocytozoon infection status. Zeroes represent negative infections. Ones represent positive infections.