(A) Calcium responses of Fdg to optogenetic activation of water (blue, ppk28-LexA), sugar (green, Gr5a-LexA), or bitter (red, Gr66a-LexA) GRNs in food-deprived flies. To examine Fdg responses, GCaMP7b was expressed using SS46913. Fluorescence traces are shown on the left of the panel (ΔF/F), while ΔF/F area for each trace is shown on the right. Stimulation with 660 nm light is indicated with vertical gray bars. Kruskal Wallace test with Dunn’s test using Holm’s correction to adjust for multiple comparisons, n=5-7 flies per genotype, ns p>0.05, *p≤0.05, **p≤0.01. (B–C) Calcium responses of water gustatory sensory neurons and feeding initiation neurons to taste stimulation of the proboscis. GCaMP6s fluorescence traces are shown on the left of each panel (ΔF/F), while ΔF/F area for each trace is shown on the right. n=5-10 flies per genotype, ns p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. (B) Taste responses to water in fed (purple) versus 2 hr desiccated (2 hr dess, teal) flies (left). Taste responses to water in fed flies allowed to rest for 1 hr in low osmolality artificial hemolymph after dissection before imaging (1 hr fed, purple) versus pseudodessicated, thirsty-like flies (p-dess, blue) (right). (C) Taste responses in pseudodessicated, thirsty-like flies. The proboscis of each tested individual was stimulated with water (green), sugar (blue), and bitter (red) tastants in sequential trials during the indicated period (thick black line). Thin black lines indicate sample pairing. The following split-GAL4 lines were imaged for each cell type: Clavicle; SS48947, G2N-1; SS47082, Usnea; SS37122, Rattle; SS50091, Roundup; SS47744. Quade’s test with Quade’s All Pairs test, using Holm’s correction to adjust for multiple comparisons. (D) GRN synapes onto second-order neurons, with GRN categories based on Engert et al., 2021.