(A) Definition of forward-locomotion wave in freely moving larvae. During forward locomotion (run), tail speed and body length exhibit wave-like patterns. During the initiation of the forward wave, tail speed peaks due to the contraction of the most posterior segment (left panel, black arrow in tail speed). At the end of the wave of peristaltic contraction, larvae extend their body to its maximal length. This extension translates into a peak in the body length (left panel, black arrow in body length). (Left panel) We define the forward wave cycle as the time window between a peak in tail speed and the next peak in body length. (Right panel) Approximate mapping of phases of wave cycle on the abdominal segments (a full cycle spans 360 degrees). The initial phase of the wave corresponds to the contraction of the most posterior segments (initial phase, blue). As the wave is half-finished, segmental contractions reach the middle part of the body (green, hinge phase). The most anterior segments contract at the last phase of the wave (red, late phase). (B–C) Activation of the PDM-DN neuron at different phases of wave propagation. Optogenetic activation of the PDM-DN neuron during the initial, hinge and late phases. (B) PDM-DN activation evokes a drop in tail speed irrespective of the phase of the forward wave (left panel). The inset shows the mean tail speed throughout the flash. During the light flash (6 s), there is no significant difference in the minimum tail speed reached upon PDM-DN activation between the three phases considered in this analysis (right panel, Wilcoxon rank-sum test, p>0.05 upon Bonferroni correction). Each point in the background represents a single trial. Horizontal lines represent the median for each condition. Semi-transparent boxes represent the 25th and 75th percentiles. The whiskers extend to the most extreme data points excluding the outliers (default settings of the ‘boxplot’ function of Matlab_R2015b). Different letters indicate statistically significant differences. (C) Optogenetic activation of the PDM-DN neuron does not cease the wave propagation immediately. Upon light onset, the body length increases during the first second of the optogenetic activation of PDM-DN (dashed box to be compared to the dashed box of panel B). The inset shows the mean body length throughout the flash. During the light flash (6 s), there is no significant difference in the maximum body length reached upon PDM-DN activation between phases (Wilcoxon rank-sum test, p>0.05 upon Bonferroni correction). Each point represents a single trial. Horizontal lines represent the median for each condition. Semi-transparent boxes represent the 25th and 75th percentiles. The whiskers extend to the most extreme data points excluding the outliers (default settings of the ‘boxplot’ function of Matlab_R2015b). Different letters indicate statistically significant differences. Sample sizes: n = 45 trials for initial phase, n = 44 trials for hinge phase and n = 25 trial for the end phase. (D–E) Analysis of the PDM-DN activation phenotype at the level of segmental contractions using immobilized larvae. (D) To observe the effect of PDM-DN activation on the wave propagation at the level of segmental contractions, we immobilized the larva by pinning it down from both ends (tip of the head and the tail) on a transparent PDMS slab (left panel). Although the larva could generate forward peristaltic waves, it could not move forward, making it easier to track the segmental contractions (see Video 2). In this preparation, sequential contractions of the abdominal segments were clearly visible and quantified as displacement of each segment (∆x) along the anterior-posterior axis (right panel). (E) The effect of PDM-DN activity on the segmental contractions was observed by activating the PDM-DN neuron at different stages of the wave of peristaltic contraction. Prior to the light flash, larvae generated forward waves (tilted dashed lines) by sequentially contracting the abdominal segments starting at segment A7. The time course of segmental displacements is illustrated for conditions where the light onset coincides with contractions at segments A7 (Eiii), A4 (Eii) or A2 (Ei) (red arrows). (F) Different effects of PDM-DN activation on the ongoing peristaltic wave are reported as proportions of the total number of observations. For activation of PDM-DN while the wave was travelling through both the anterior and posterior segments, the ongoing wave reached and terminated at A1, but subsequent waves could not be initiated (gray). For posterior segments,~7% of the ongoing wave terminated immediately (purple) and a new wave could not be initiated throughout the flash. However no statistical difference is observed when compared to the anterior segments (Z-test, p=0.2076). About ~20% of the ongoing wave propagated up to the hinge point (segment A4) where they terminated (green color). In addition, new waves could not be initiated in this condition throughout the flash. This condition was never observed for the anterior segments (Z-test, p<0.05). The proportions of the failure to stop the wave is not significantly different for anterior and posterior segments (Z-test, p=0.6672). Sample size: 136 flashes (five preparations). Starts indicate statistically significant differences (**: p<0.01, ***p<0.001). (G) Calcium imaging of fictive locomotion patterns upon PDM-DN activation. We used isolated CNS preparations to analyze the effect of PDM-DN activation on fictive waves of forward locomotion. To record the motor neuron activity, we expressed the genetically-encoded calcium indicator GCamP6f in glutamate-expressing neurons using the VGlut-LexA driver line. (Left panel) The picture sequence corresponds to snapshots at different time points during forward wave propagation (time interval between pictures: 1 s). Note the sequential increase in fluorescence intensity from A7 to A1. (Right panel) Prior to the light flash, larvae generated fictive forward waves (tilted dashed lines) manifested by sequential increase in the fluorescence levels starting from the posterior-most segment A7. The increase in fluorescence was symmetrically coordinated between the left and the right segments (left in green, right in blue). Upon optogenetic activation of the PDM-DN neuron, a fictive wave of locomotion starting from the posterior segments (A7–A5) terminated before reaching A4 (truncated dashed line). (H) To quantify the effects of the light-driven activity on PDM, we computed the number of forward waves before and during the light flash. While PDM-DN activation (magenta) leads to a significant decrease in the number of waves that were initiated and completed (n = 19 trials, Wilcoxon signed-rank test, p<0.05), wild-type controls (black) do not display a significant change (n = 12 trials). Each dot represents an independent trial. Different letters indicate statistically significant differences. For more information about the statistics, see Supplementary file 1.