Failure to reproduce period-dependent song cycles in Drosophila is due to poor automated pulse-detection and low-intensity courtship.

Stern has criticized a body of work from several groups that have independently studied the so-called "Kyriacou and Hall" courtship song rhythms of male Drosophila melanogaster, claiming that these ultradian ∼60-s cycles in the interpulse interval (IPI) are statistical artifacts that are not modulated by mutations at the period (per) locus [Stern DL (2014) BMC Biol 12:38]. We have scrutinized Stern's raw data and observe that his automated song pulse-detection method identifies only ∼50% of the IPIs found by manual (visual and acoustic) monitoring. This critical error is further compounded by Stern's use of recordings with very little song, the large majority of which do not meet the minimal song intensity criteria which Kyriacou and Hall used in their studies. Consequently most of Stern's recordings only contribute noise to the analyses. Of the data presented by Stern, only perL and a small fraction of wild-type males sing vigorously, so we limited our reanalyses to these genotypes. We manually reexamined Stern's raw song recordings and analyzed IPI rhythms using several independent time-series analyses. We observe that perL songs show significantly longer song periods than wild-type songs, with values for both genotypes close to those found in previous studies. These per-dependent differences disappear when the song data are randomized. We conclude that Stern's negative findings are artifacts of his inadequate pulse-detection methodology coupled to his use of low-intensity courtship song records.