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 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). (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. 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. 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. (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. 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. 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).

The cumulative successful foraging within a 15-min observation period of female flies with different starvation durations. Data are means ± SEM. n = 60 flies for each condition. Different letters above the error bars indicated significant differences (P < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test).

The removal of antennae significantly inhibits sexual behavior. (A) The cumulative courtship rate of flies with different removal of antennae during a 15-min observation period. n = 93, 109, 98, and 101, respectively (from Normal to All removed). (B) The cumulative copulation rate of flies with different removal of antennae. n = 95, 105, 100, and 93, respectively (from Normal to All removed). MA, male antenna; FA, female antenna. Kruskal-Wallis and post hoc Mann-Whitney U tests, *P < 0.05, **P < 0.01, ***P < 0.001.

EAG responses of fed and starved flies to food relevant odors and sex pheromones at three concentrations. Data are means ± SEM. n = 10–12 antennae for each condition. Unpaired t test, n.s. indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.

Generation of the BdSk null mutant via CRISPR-Cas9 system. (A) Schematic of the Sk gene, showing the single guide RNA (sgRNA) target site. The Sk gene has only one exon, shown as an orange box. A 7-bp deletion was chosen for generation of sk-/- flies. The protospacer adjacent motif (PAM) is highlighted in green. (B) The screening strategy for Sk and SkR1 mutants. (C) Suppression of Sk expression in Sk mutants.

Generation of the BdSkR1 null mutant via CRISPR-Cas9 system. (A) Schematic of the SkR1 gene, showing the single guide RNA (sgRNA) target sites. The SkR1 gene has five exons, shown as orange boxes. (B) A 187-bp deletion and 2-bp substitution was chosen for generation of skr1-/-flies. The protospacer adjacent motif (PAM) is highlighted in green. (C) The predicted seven transmembrane domains of BdSkR1. Disruption of the first transmembrane domain by a frameshift is shown in purple. (D) Suppression of SkR1 expression in SkR1 mutants.

Analysis of off-target effects of mutants. (A) Prediction of potential off-target sites of the Sk and SkR1 genes. (B) PCR amplification and sequencing of potential off-target sites of the Sk gene. (C) PCR amplification and sequencing of potential off-target sites of the SkR1 gene.

Phylogenetic tree of sulfakinin receptors constructed using the neighbour-joining (NJ) method

EAG responses of starved flies with different genotypes to food relevant odors and sex pheromones at three concentrations. Data are means ± SEM. n = 10 antennae for each condition. Unpaired t test, n.s. indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.

Primer sequences used in this study

Chemicals used for electroantennograms