Fuel stores and time of day account for variation in serum metabolomes of passerine migrants stopping over

Migratory birds excel in phenotypic flexibility, adapting physiologically as their life histories and environments require. Discerning the metabolic processes underlying migrants' physiology, an emergent property of multiple continuous and dynamic organism–environment interactions, is therefore challenging, particularly under natural conditions. Accordingly, analyses of snapshot-sampled serum-circulating metabolites, versatile and readily applicable for migrating birds, have increasingly become the method of choice for such physiologic inference. However, the atemporal nature of single sampling might obscure the links between observed metabolite concentrations and the processes producing them, necessitating an analytical decoupling of focal processes from their broader biochemical background. In the present study, we examined how variation in combined fat and muscle fuel stores, traits pivotal in migratory context, relates to the serum-circulating metabolomes of spring-migrating Eurasian blackcaps stopping-over. Our analyses accounted for potential spatiotemporal influences in the form of time past night's fasting and random local conditions across three sites within the Negev Desert. We shifted the focus from compound-level analysis of preselected metabolites towards the level of inclusive metabolome, quantifying serum-circulating lipophilic and polar molecules via UHPLC–MS/MS untargeted metabolomic technique. Our results indicated a general relationship between fuel stores and the metabolome, comprising 16 326 lipophilic and 6923 polar compounds, among which 918 and 44 were annotated, respectively. By applying generalized latent-variable linear modeling (GLLVM) upon concentrations of annotated metabolites, we identified several candidate biomarkers, some novel in migratory context, notably the fuel-associated increase in serum ceramides likely derived from circulating very low-density lipoproteins (VLDLs). Relying on estimated metabolite links with fuel and foraging time and on modeled residual covariations among metabolites, we demonstrate fuel–metabolite associations generally consistent with higher fat- and lower protein mobilization in birds having greater stores and with decreased fuel utilization as ingested nutrients accumulate over time, thus introducing a novel approach for the physiological study of migrating birds.