Fig. 4: The global ICWs are generated by the periodic release of AKH in the circulating hemolymph.

A, B 3rd instar larvae were immobilized on the glass slide and imaged before and after the application of chloroform. Filter paper containing ~10 ul chloroform was placed close to the larvae, which temporarily stopped the heartbeat of the larvae for about 30 min. Data was pooled from 3 independent experiments. N = 3, 3 larvae (B). C Ca²⁺ imaging of the larvae treated with and without chloroform. The result was repeated in 3 independent experiments. D Fluorescent beads were injected into the 3rd instar larvae and traced under a microscope. The trajectory of one traced bead was shown with velocity coded in color. The result was repeated in 3 independent experiments. E The velocities of beads transported in the anterograde direction (from head to tail) and retrograde direction (from tail to head) were quantified. Data was pooled from 3 independent experiments. N = 19, 13 beads. F–H 3rd instar larvae were immobilized on a glass slide which is placed at the center of a CO₂ fly anesthetizing pad. Neuron activity in the APCs was revealed using Akh::GCaMP7S-T2A-mRuby3. Ca²⁺ activity before and after CO₂ application was quantified. Time courses of average Ca²⁺ signal from multiple larvae were plotted as mean ± S.E.M. (shaded region). Data was pooled from 5 independent experiments. N = 5, 5 larvae (G). I–L The Ca²⁺ waves of fat bodies are rapidly (t½ ≈ 35 s) and completely blocked by CO₂ treatment, and the fat bodies of Akh overexpression still present Ca²⁺ waves in the treatment of CO₂. A representative trace of average Ca²⁺ intensity from a CO₂ treated larva was plotted in J. Ca²⁺ intensity was expressed as arbitrary units (a.u.) (J). Time courses of average Ca²⁺ intensity from multiple larvae were plotted as mean ± S.E.M. (shaded region) in (K). Data was pooled from 6 independent experiments. N = 6, 6, 6, 6 larvae (L). Ca²⁺ intensity (a.u.) was presented using a linear colour scale (minimum = 0, maximum = 255) (C, I). Paired two-tailed Student’s t-test is utilized in (B, G), and ordinary two-way ANOVA with uncorrected Fisher’s LSD is utilized in (L). Data were plotted as mean ± S.E.M (H, K), or mean ± SD (others). Scale bars, 500 μm (C, I), 1000 μm (D), 50 μm (F). Source data are provided as a Source Data file.