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In vertebrates, myoglobin (Mb) and cytoglobin (Cygb) are closest relatives in the family of globins, which are heme-containing proteins that can bind gaseous molecules. Mb acts not only as an O2 transporter but also a nitric oxide (NO) scavenger in cardiac and striated muscle. Cygb has been suggested to play important functions in lipid-based signaling processes, defense against reactive oxygen species (ROS), and nitric oxide (NO) metabolism, and it is present in a variety of cell types. However, little information about the structures and functions of Mb and Cygb is known in birds. Here, we cloned the full-length open reading frames (ORFs) of the two globins in Eurasian Tree Sparrow (Passer montanus). The Mb ORF cDNA contains 465 base pairs (bp) encoding 154 amino acids (aa), and the Cygb ORF cDNA contains 540 bp encoding 179 aa. Our results show that the amino acid sequences and three-dimensional (3D) structures of Mb and Cygb are highly conserved in vertebrate species. Interestingly, two specific substitutions were detected in Cygb compared with other vertebrates, which resulted in slight variation of the 3D conformation (e.g., distance between Tyr H16 and Lys G8, the strength of hydrogen bonds, and angles between the G–H helices). Our results may contribute to further understanding the structures, properties, and functions of Mb and Cygb as well as the potential mechanisms of oxygen utilization pathways in vertebrates.

Life-history theory assumes that the fitness costs of immunity may have negative effects on reproductive success. Similarly, the immunocompetence handicap hypothesis is based on findings that testosterone (T) has immunosuppressive effects, although the basis of this hypothesis has recently been challenged. As much of the work examining the relationship between T levels and immune function has been carried out in captive-housed species, these results may not accurately reflect the situation of animals living in natural environments. To better understand the relationship between plasma T levels and immune function, studies focusing on free-living animals are needed. A previous study by our group determined the changes in both baseline and stress-induced T levels in free-living Eurasian Tree Sparrows (Passer montanus) across different annual cycle stages. In this study, we further report the phytohaemagglutinin skin-swelling (PHA) immune response in this multi-brooded species during different breeding sub-stages, and then determine the relationships between the PHA response and both baseline and stress-induced T levels. Our results show that the PHA response varied across the different sub-stages and differed significantly between the first and second brood stage. Furthermore, T levels in male Tree Sparrows are positively correlated with the PHA response during the breeding season, whereas this relationship is negative in females, suggesting that the biological function of T differs between the sexes. Therefore, our results suggest that free-living animals have evolved the ability to orchestrate trade-offs between reproduction and immune functions based on changes in physiology and the environment, which should provide further opportunities to study the flexibility and plasticity of physiological and ecological adaptations in natural environments.