Toward temporally calibrated biomarkers of heat stress in free-living songbirds

As heatwaves increase in intensity and frequency, more birds are exposed to sublethal heat, which can affect many elements of the phenotype, from growth to cognition to reproduction. These widespread performance-related effects of heat, coupled with the rapid declines seen in many bird populations in recent decades, underscore the urgency of detecting recent heat exposure and its downstream physiological effects in the wild. To develop minimally invasive biomarkers of past heat, we experimentally elevated nest temperatures for free-living nestling Tree Swallows (Tachycineta bicolor) for four hours on their twelfth day of life. Twenty-four hours later, we returned to collect a small blood sample and quantify carryover effects of prior sublethal heat on the blood transcriptome. By comparing these carryover effects to those that occur in the immediate aftermath of heat, we identify biomarkers of heat that reflect distinct and time-dependent processes. Candidate biomarkers include four upregulated genes with connections to stress and disease (LAMA3, ATP1B1, RASGEF1A, TMEM181) and two additional down-regulated genes. By incorporating the sex of each nestling into our analyses, we also unveiled marked sexual dimorphism in the blood transcriptome, even among autosomal genes and including pathways that imply inherent sex differences in heat tolerance. When these sex differences are controlled, we see that the sexes respond to heat with overwhelming similarity, further grounding the utility of our suggested transcriptomic biomarkers. Though these biomarkers will require further validation to be used across bird species, our collective results uncover temporally calibrated targets can be measured with just one drop of blood, improving our understanding of climate impacts on wild birds. Lay summaryO_LIAs global temperatures rise, many birds experience bouts of heat stress, but we do not have simple biomarkers that reliably reflect this past exposure in the wild. C_LIO_LIWe tested whether a small blood sample could reveal recent heat stress through changes in gene activity. C_LIO_LIOur experiment exposed nestling Tree Swallows to a non-lethal heat stressor and measured how their gene activity changed during and after the heat event. C_LIO_LISome genes reacted quickly but returned to normal within a day, while others showed longer-lasting effects. C_LIO_LIMales and females responded to heat in similar ways, even though their baseline gene activity differed substantially. C_LIO_LISix genes responded consistently across the sexes and in relation to temperature, making them promising biomarkers of past heat. C_LIO_LIThese results can help scientists better track heat exposure in wild birds and improve predictions on how populations respond to continued climate change. C_LI