IP₃R function is required from 16 to 32 hours after puparium formation to regulate flight.

<p><b>A</b>) Quantification of wing posture defects by knockdown of the IP₃R (<i>dsitpr</i>) at specific stages during pupal development indicated above the bars using the <i>GAL4/GAL80ts</i> system (<i>TARGET</i>), are shown. Mixed populations of normal (grey bar), mild wing posture defect (pink bar) and severe wing posture defective (blue bar) flies were obtained when <i>dsitpr</i> is expressed from 16 h, 24 h or 32 h after puparium formation (APF) to adulthood, but not when expression is induced at 48 h APF and onwards. Animals were either moved from 18°C to 29°C, at the appropriate pupal phases to induce RNAi expression or maintained at a constant temperature post egg-laying (PEL) as indicated. Percentage of animals with varying wing posture defects is shown as a stacked histogram for males (M) and females (F). <b>B</b>) Quantification of flight duration in single flight assays (mean ± SEM) from female animals of the indicated genotypes. Open circles within each bar represent percentage flight time for each fly. Mean flight times (± SEM) were obtained for three batches of 10 tethered flies of each genotype after an air-puff stimulus. All the knockdowns were compared to 29°C <i>dsitpr/+</i> control (**p<0.01, one-way ANOVA). Color codes of the histograms are the same as in (A) above. <b>C</b>) Representative traces of electrophysiological recordings from DLMs of the indicated genotypes in response to a manual air-puff stimulus (arrow). The number of flies that showed the given response upon the number of flies tested is given below each trace. Adult female flies were sorted on the basis of the severity of their wing defects as shown. Loss of rhythmic flight patterns accompanied by severe wing posture defects is observed due to pan-neuronal depletion of IP₃R during early pupal development (16 h APF). Knockdown at later time points shows reduced flight duration in flies with mild wing posture defect and normal flight in animals with normal wings. <b>D</b>) Quantification of the frequency of spontaneous firing as recorded from DLMs of the indicated genotypes. As with flight deficits and air-puff induced electrical firing patterns, spontaneous firing frequencies are higher in animals with wing posture defects (color coded as in A) and the time of transfer to 29°C. (**p<0.01, *p<0.05, one-way ANOVA, N = 15). Firing frequencies were calculated by counting the number of spikes over 2 min. Individual data points are represented by open circles. <b>E</b>) Representative traces for (D) showing increased spontaneous firing in animals with severe wing posture defect due to early knockdown of the IP₃R.