Supplementary methods from Difference in control between spring and autumn migration in birds: insight from seasonal changes in hypothalamic gene expression in captive buntings

We hypothesized differences in molecular strategies for similar journeys that migrants undertake to reproduce in spring and to overwinter in autumn. We tested this, in redheaded buntings (<i>Emberiza bruniceps</i>) photoinduced into spring and autumn migratory states, with winter and summer non-migratory states as controls. Compared to controls, buntings fattened, gained weight and showed <i>Zugunruhe</i> (nocturnal migratory restlessness) in the migratory state. Spring migration was associated with greater fat and body mass, and higher intensity of <i>Zugunruhe</i>, compared to the autumn migration. Circulating corticosterone levels were higher in spring, while T3 levels were higher in autumn. Hypothalamic expression of thyroid hormone-responsive (<i>dio2</i>, <i>dio3</i>), light-responsive (<i>per2</i>, <i>cry1</i>, <i>adcyap1</i>) and <i>th</i> (tyrosine hydroxylase, involved in dopamine biosynthesis) genes showed significant changes with transition from non-migratory to the migratory state. There were significantly higher mRNA expressions in autumn, except for higher <i>th</i> levels in the spring. Furthermore, the expression patterns of <i>dnmt3a</i> (not <i>dnmt3b</i>) and <i>tet2</i> genes suggested an epigenetic difference between the non-migrant and migrant periods, and the spring and autumn migrant periods. These results demonstrate for the first time seasonal transition in hypothalamic gene expressions, and suggest differences in regulatory strategies at the transcriptional level for spring and autumn migrations in songbirds.