Pathogen infection has been progressively recognized as a potential driver of animal migration, and the infection pattern in a certain host may be related to its migration status. Nestling viability can be a crucial life stage in the evolution of the migration cycle but has been irregularly studied to date. In this study, we tested the ‘migratory exposure’ hypothesis by comparing the prevalence and diversity of avian haemosporidian parasites in the breeding area between two dominant species including adults and nestlings: yellow-rumped flycatcher (Ficedula zanthopygia), which is a summer migratory species, and Japanese tit (Parus minor), which is a local resident species. The yellow-rumped flycatcher harboured more diverse parasite lineages than the Japanese tit, which is in line with the expectation of the ‘migratory exposure’ hypothesis, while nestlings present a similar but more applicable pattern. Among the 40 unique identified parasite lineages, only one was shared between host species and three between age classes. Nestlings suffered a high parasite diversity with plenty of age-specific lineages in the breeding area, mainly Leucocytozoon, which may suggest an extraordinary selection pressure in the life-stages not just for adults during migration. However, the most common lineages in adults were not detected in nestlings, and the prevalence of infection is significantly lower in nestlings than in adults. Our results suggest that migratory birds may have suffered from more frequent parasite infections during migration, but escaped from more virulent lineages at the same time. The difference in susceptibility and parasite assembly between adults and nestlings in migrants may result from an intricate interaction along the evolution of life history. Our investigations have revealed the significance of avian haemosporidian parasite infection patterns in nestlings in breeding areas, and provide a novel insight into the driving forces of migration.

Blood samples were collected from the brachial vein and stored in absolute ethanol. DNA extraction was performed using TIANamp Genomic DNA kits (Tiangen Biotech Ltd., Beijing, China) following the manufacturer’s protocol.

The identification of haemosporidian parasites was conducted following a general nested PCR protocol (Hellgren et al., 2004). To exclude false negatives and reduce the effect of amplification randomness in mixed infections, three repeats were required in the polymerase chain reaction (PCR) analysis of each sample. In addition, to avoid false positives, negative controls (adding double distilled (dd)H₂O as the template instead of DNA samples) were included in each run. Positive samples were distinguished by 1% agarose gel scanning, and products were sequenced bidirectionally using a 3730XL automatic sequencer (Applied Biosystems, Waltham, MA, USA).

The obtained sequences were assembled and end-trimmed in Geneious Prime v.2021.2.4 (http://www.geneious.com/), and sequences containing one or more ambiguous nucleotides were considered coinfections. To avoid overestimation of lineage diversity, coinfections including lineages that could not be distinguished were eliminated from the dataset. A total of 307 effective samples were analysed, including 205 Japanese tits (36 adults and 169 nestlings) and 102 yellow-rumped flycatchers (28 adults and 74 nestlings). Parasite taxa identification was conducted using the basic local alignment search tool (BLAST) module in the MalAvi database (http://130.235.244.92/Malavi/blast.html, accessed in August 2022).