Starvation alters the behavior and olfactory responses of B. dorsalis.

(A) Schematic illustration of the experimental design. (B) Schematic illustration of the mating behavior assay device. (C) Representative foraging trajectories in the 100 mm diameter arenas within a 15-min observation period of flies starved for different durations. (D) Cumulative successful foraging within a 15-min observation period of male flies starved for different durations. Data are means ± SEM, n = 60 flies for each condition. Different letters above the error bars indicate significant differences (one-way ANOVA followed by Tukey’s multiple comparisons test; P < 0.05). (E‒F) EAG responses to artificial food in fed and starved male (E) and female (F) flies (unpaired t-test). (Top) Representative EAG recordings. (G) Cumulative courtship rate within a 15-min observation period of flies starved for different durations (n = 78, 76, 78, 84 and 91, respectively, from fed to 12 h). (H) Cumulative copulation rate within a 15-min observation period of the flies starved for different durations (n = 78, 76, 79, 83 and 93, respectively, from fed to 12 h). (G–H) Kruskal-Wallis and post hoc Mann-Whitney U tests were applied. (I‒J) EAG responses to body extracts in fed and starved male (I) and female (J) flies. (Top) Representative EAG recordings. Data are means ± SEM, n = 10–12 antennae per genotype (unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001).

Starvation-induced expression of neuropeptide system components and phenotypes of null mutant flies.

(A) Changes in the expression of neuropeptide system components in the antennae of starved flies. The relative expression values are the fold-changes compared to the control. Data are means ± SEM, n = 3 (unpaired t-test; *P < 0.05, **P < 0.01). (B) Altered amino acid sequence and mature peptides in the null mutants sk−/− and skr1−/− compared to wild-type (WT) flies. (C) Mean velocity of flies representing each genotype during a 15-min observation period. Data are means ± SEM (Kruskal–Wallis test). (D) Knockout of sulfakinin and SkR1 increases food consumption in B. dorsalis at different times of day. Each experiment consisted of one fly, and at least 15 flies were contained for the assay. Different lower-case letters indicate significant differences between treatments (one-way ANOVA followed by Tukey’s multiple comparisons test; P < 0.05).

Sk-SkR1 signaling is required for changes in behavior and olfactory sensitivity induced by starvation.

(A‒B) Cumulative successful foraging within a 15-min observation period of fed and starved flies with different genotypes. The successful foraging of females (A) and males (B) was measured separately. Data are means ± SEM, n = 60 flies for each condition. Different letters above the error bars indicated significant differences (one-way ANOVA followed by Tukey’s multiple comparisons test; P < 0.05). (C) Cumulative copulation rates within a 15-min observation period of fed and starved flies with different genotypes (n = 92, 86, 88, 106, 80 and 76, respectively, from fed WT to starved skr1−/−). (D) Cumulative courtship rates within a 15-min observation period of fed and starved flies with different genotypes (n = 91, 85, 86, 98, 80 and 77, respectively, from fed WT to starved skr1−/−). (C–D) Kruskal-Wallis and post hoc Mann-Whitney U tests were applied (*P < 0.05, **P < 0.01, ***P < 0.001). (E‒F) EAG responses to artificial food in fed and starved flies of different genotypes. The EAG responses to artificial food in females (E) and males (F) were measured separately. (Top) Representative EAG recordings. (G‒H) EAG responses to body extracts in fed and starved flies of different genotypes. The EAG responses to body extracts of females (G) and males (H) were measured separately. (Top) Representative EAG recordings. Data are means ± SEM, n = 10–12 antennae for each condition. Different letters above the error bars indicated significant differences (one-way ANOVA followed by Tukey’s multiple comparisons test; P < 0.05).

The transition of olfactory responses is associated with the expression of different sets of ORs in OR neurons induced by Sk-SkR1 signaling.

(A) Principal component analysis (PCA) using differentially expressed genes obtained from pairwise comparisons between different treatments. (B) The expression profiles of the candidate OR genes in wild-type (WT) and sk−/− flies determined by qRT-PCR. The OR names with red fonts represent the set of ORs for food odor and blue fronts represent the set of ORs for sex pheromone. Data are mean relative expression levels ± SEM. R1−R3 represent biological replicates. Different lower-case letters indicate significant differences between treatments (one-way ANOVA followed by Tukey’s multiple comparisons test; P < 0.05). (C) Quantification of calcium levels following the response of candidate ORs to sex pheromones and food odors at a concentration of 10-4 M (n = 3‒6). Different lower-case letters indicate significant differences between treatments (one-way ANOVA followed by Tukey’s multiple comparisons test; P < 0.05). (D) Dose–response curves of candidate ORs to sex pheromone components such as ethyl laurate (EL), ethyl palmitate (EP), 2,3,5-trimethyl pyrazine (TMP), and 2,3,5,6-tetramethylpyrazine (TTMP). (E) Dose–response curves of candidate ORs to food odorants such as ethyl benzoate (EBE), ethyl butyrate (EBU), diethyl maleate (DM), and methyl eugenol (ME). In both cases, n = 5. (F) Co-localization of Orco (red) and SkR1 (green) neurons in B. dorsalis antennae. Scale bars = 50 μm.

Schematic representation of peripheral olfactory remodeling in OR neurons of the antenna that arbitrate between mating and foraging behavior in B. dorsalis.

Starvation increases the abundance of SkR1 in the OR neurons (red arrows) and SkR1 signaling induces the expression of genes encoding ORs that sense food odors, resulting in successful foraging. Satiation suppresses SkR1 expression and induces the expression of genes encoding ORs that sense opposite-sex pheromones, leading to successful mating (blue arrows). Dashed arrows represent the additional possible pathways that have not been tested in this study, but not excluded in the model. Please see the discussion for details of additional possible factors modulating odorant sensitivity relevant to satiety.