title: Brain gene expression associated with sex-reversed parental care in biparental poison frogs
design: To investigate the molecular mechanisms associated with flexible transport behavior (or lack thereof) across the sexes in Ranitomeya imitator, N=21 female and N=10 male frogs were selected for whole brain gene expression analysis. Based on observed individual variation in female transport propensity, sex-reversed females were categorized a priori as either Flexible (i.e., transported tadpole in a male removal trial) or Inflexible (i.e., failed to transport tadpole in a male removal trial). Male partners were removed on Day 9 to create the Sex-Reversed social condition. Individuals were sacrificed at three timepoints: (1) during egg care, but before hatching; (2) after eggs hatched and tadpole transport was initiated (within one day of hatching); and (3) after tadpoles had been hatched for three days without transport. This sampling scheme yielded four behavioral groups: Egg Care (i.e., males (N=5) and females (N=5) performing egg care only, sampled 9 days post-oviposition), Sex-Typical (i.e., male transporting (N=5) and female observing (N=5), sampled 01 days post-hatching), Sex-Reversed-Flexible (i.e., female transporting in absence of male (N=5), sampled 01 days post-hatching), and Sex-Reversed-Inflexible (i.e., non-transporting female in absence of male (N=5), sampled 3 days post-hatching). Because hatching times varied between clutches, timing of sampling varied slightly across behavioral groups. Relative to oviposition day (Day 0), sex-typical pairs were sampled between day 10 and day 15 and sex-reversed females were sampled between day 14 and day 20 (Flexible females: day 1420; Inflexible females: day 1517). Immediately upon capture, frogs were rapidly decapitated, and tissue was added to cryotubes pre-filled with ceramic beads (Omni International, Kennesaw, GA, USA) and then flash-frozen in liquid nitrogen. Brain tissue was stored at -80C until further processing. This full process took <5 minutes.