Electrophysiological responses of larval and adult taste sensilla in Helicoverpa armigera to sugars.

(A) The representative spike traces of the responses of lateral sensilla styloconica in larval maxillary galea to seven sugars at 10 mM (left), and quantifications of the firing rates (right) (n = 13). (B) Dose–responses of lateral sensilla styloconica in larval maxillary galea to sucrose and fucose (sucrose: n = 8; fucose: n = 6). (C) The representative spike traces of the responses of sensilla chaetica in adult antennae to seven sugars at 10 mM (left), and quantifications of the firing rates (right) (n = 21). (D) Dose–responses of sensilla chaetica in adult antennae to sucrose, fucose, and fructose (n = 21). (E) The representative spike traces of the responses of sensilla trichodea in adult tarsi to seven sugars at 10 mM (left), and quantifications of the firing rates (right) (n = 15). (F) Dose–responses of sensilla trichodea in adult tarsi to sucrose, fucose, and fructose (n = 21). (G) The representative spike traces of the responses of sensilla styloconica in adult proboscis to seven sugars at 10 mM (left), and quantifications of the firing rates (right) (n = 21). (H) Dose–responses to sucrose (n = 21), fucose (n = 21), and fructose (fructose 0 mM, n = 18; fructose 0.01 mM-100 mM, n = 21) of sensilla styloconica in adult proboscis. (A to H) Data are mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001. (A, C, E, G) Data were analyzed by independent-samples t test (compared with control). (B, D, F, H) Data were analyzed by one-way ANOVA with Tukey’s HSD test.

Behavioral responses of Helicoverpa armigera larvae and adults to sugars.

(A) Feeding responses and the preference index (PI) value of 5th instar larvae to seven sugars painted on the cabbage leaf discs at 10 mM in two-choice tests. ** P < 0.01; ns indicates no significance, P ≥ 0.05 (paired t test, n = 20). (B) Feeding responses and the PI value of 5th instar larvae to different concentrations of sucrose painted on the cabbage leaf discs in two-choice tests. ** P < 0.01, *** P < 0.001; ns indicates no significance, P ≥ 0.05 (paired t test, n = 20). (C) Proboscis extension reflex (PER) in adult females upon antennal stimulation by seven sugars at 100 mM. ** P < 0.01; *** P < 0.001 (independent-samples t test compared with control, n = 3). (D) PER in adult females upon tarsal stimulation by seven sugars at 100 mM. * P < 0.05; ** P < 0.01 (independent-samples t test, n = 3). (E) PER in adult females upon antennal stimulation by different concentrations of sucrose, fucose, and fructose. * P < 0.05 (one-way ANOVA with Tukey’s HSD test, n = 3). (F) PER in adult females upon tarsal stimulation by different concentrations of sucrose, fucose, and fructose (n = 3). * P < 0.05 (one-way ANOVA with Tukey’s HSD test, n = 3). (A to F) Data are mean ± SEM.

The phylogenetic relationship and the expression level of sugar GRs in larval maxilla and female adult antennae, tarsi and proboscis of Helicoverpa armigera.

(A) The phylogenetic tree of insect sugar GRs. Diptera (orange): Aaeg, Aedes aegypti; Agam, Anopheles gambiae; Dmel, Drosophila melanogaster. Hymenoptera (blue): Am, Apis mellifera. Lepidoptera (black): Bmor, Bombyx mori; Dple, Danaus plexippus; Hmel, Heliconius melpomene; Pxyl, Plutella xylostella. Harm (red), Helicoverpa armigera. Numbers above branches indicate ultrafast bootstrap approximation (UFBoot). (B) Relative expression levels of sugar GRs in the maxillary galea of 5th instar larvae of H. armigera determined by qRT-PCR. (C) Relative expression levels of sugar GRs in the female adult antennae. (D) Relative expression levels of sugar GRs in female adult tarsi. (E) Relative expression levels of sugar GRs in female adult proboscis. (B to E) Data are mean ± SEM. One-way ANOVA was used, and different letters labeled indicate significant difference (Tukey’s HSD test, P < 0.05, n = 3).

The inward current responses of the Xenopus oocytes expressing sugar GRs of Helicoverpa armigera to sugar compounds.

(A) The representative traces of the oocytes expressing Gr10, Gr6, and Gr10 + Gr6 to 11 sugar compounds at 100 mM. (B) The responses of the oocytes expressing Gr10, Gr6, and Gr10 + Gr6 to sugars at 100 mM (Gr10, n = 14. Gr6, n = 20. Gr10 + Gr6, n = 7). (C) The representative trace of the oocytes expressing Gr10 to sucrose (the upper), the representative trace of the oocytes expressing Gr6 to sucrose, fucose, and fructose (the middle), the representative trace of the oocytes expressing Gr10 + Gr6 to sucrose and fucose (the lower). (D) The dose-responses of the oocytes expressing Gr10 to sucrose (the upper, n = 13); the dose-responses of the oocytes expressing Gr6 to sucrose, fucose, and fructose (the middle, sucrose: n = 6; fucose: n = 6; fructose: n = 3); the dose-responses of the oocytes expressing Gr10 + Gr6 to sucrose and fucose (the lower, sucrose: n = 4; fucose: n = 3). (B and D) Data are mean ± SEM, and were analyzed by one-way ANOVA with Tukey’s HSD test (P < 0.05). Different letters labeled indicate significant differences, * P < 0.05. (A to D) Ara, arabinose; Fru, fructose; Fuc, fucose; Gal, galactose; Glu, glucose; Lac, lactose; Mal, maltose; Man, mannose; Suc, sucrose; Tre, trehalose; Xyl, xylose; Rin, ringer solution.

Establishment of Gr10 and Gr6 homozygous mutants (Gr10−/− and Gr6−/−) of Helicoverpa armigera via CRISPR/Cas9.

(A) The cross process of obtaining Gr10−/−. (B) The genomic structure of Gr10, the single-guide RNA (sgRNA) targeting sequence (in green), and representative chromatograms of direct sequencing of the PCR products obtained from wild types (WT) and Gr10−/−, in which 4-bp of the Gr10 sequence were deleted. (C) The predicted secondary structures of the Gr10 protein in WT and the truncated Gr10 protein in Gr10−/−. (D) The cross process of obtaining Gr6−/−. (E) The genomic structure of Gr6, the single-guide RNA (sgRNA) targeting sequence (in green), and representative chromatograms of direct sequencing of the PCR products obtained from WT and Gr6−/−, in which 17-bp of the Gr6 sequence were deleted. (F) The predicted secondary structures of the Gr6 protein in WT and the truncated Gr6 protein in Gr6−/−. (B and D) Boxes represent exons, black lines represent introns, the green arrowhead indicates the direction of sgRNA; the protospacer adjacent motif (PAM) is in blue. (C and F) The secondary structure of Gr10 and Gr6 in WT, Gr10−/− and Gr6−/− was predicted by https://dtu.biolib.com/DeepTMHMM, and the image was constructed by TOPO2 software (http://www.sacs.ucsf.edu/TOPO2).

Electrophysiological responses of larval and adult taste sensilla in WT, Gr10−/− and Gr6−/− of Helicoverpa armigera to sucrose and other compounds.

(A) The representative spike traces of lateral sensilla styloconica in larval maxillary galea. (B) Quantifications of the firing rates of lateral sensilla styloconica in larval maxillary galea (mean ± SEM; WT, n = 12; Gr6−/−, n = 13; Gr10−/−, n = 13). (C) The representative spike traces of sensilla chaetica in female antennae. (D) Quantifications of the firing rates of sensilla chaetica in female antennae (mean ± SEM; WT, n = 20. Gr6−/−: nicotine, n = 17; other compounds, n = 18. Gr10−/−, n = 20). (E) The representative spike traces of sensilla trichodea in female tarsi. (F) Quantifications of the firing rates of sensilla trichodea in female tarsi (mean ± SEM; WT, n = 18; Gr6−/−: nicotine, n = 14. other compounds, n = 18. Gr10−/−, n = 18). (G) The representative spike traces of sensilla styloconica in female proboscis. (H) Quantifications of the firing rates of sensilla styloconica in female proboscis (mean ± SEM; WT: sucrose 100 mM, n = 17; other compounds, n = 18. Gr6−/−: sucrose 100 mM, n = 17; other compounds, n = 18. Gr10−/−, n = 18). (B, D, F, and H) Two-way ANOVA with post hoc Tukey’s multiple comparison was used separately for sucrose and fucose, and one-way ANOVA with Tukey’s HSD test was used for KCl, sinigrin, fructose, and nicotine. Different letters labeled indicate significant differences (P < 0.05). Suc: sucrose; Fuc: fucose; Sin: sinigrin; Fru: fructose; Nic: nicotine.

Behavioral responses of WT, Gr10−/− and Gr6−/− larvae and adults of Helicoverpa armigera to sugars and plant tissues.

(A) Feeding area of 5th instar larvae in two-choice tests and the PI value to 10 mM sucrose (n = 20). ** P < 0.01; ns indicates no significance, P ≥ 0.05 (paired t test). Suc, sucrose. (B) Feeding area of 5th instar larvae in two-choice tests and the PI value to 100 mM sucrose (n = 20). * P < 0.05; ** P < 0.01; *** P < 0.001 (paired t test). Suc, sucrose. (C) Feeding amount of 5th instar larvae on cabbage leaves, corn kernels, pea seeds, pepper fruits, and tomato fruits in no-choice tests (n = 20). Data were analyzed by one-way ANOVA with Tukey’s HSD test, and different letters labeled on the data of WT, Gr10−/− and Gr6−/−for each plant tissue indicate significant differences (P < 0.05). (D) Proboscis extension reflex (PER) in adult females upon antennal stimulation by sucrose concentrations (n = 3). (E) PER in adult females upon antennal stimulation by fucose concentrations (n = 3). (F) PER in adult females upon antennal stimulation by fructose concentrations (n = 3). (G) PER in adult females upon tarsal stimulation by sucrose concentrations (n = 3). (H) PER in adult females upon tarsal stimulation by fucose concentrations (n = 3). (I) PER in adult females upon tarsal stimulation by fructose concentrations (n = 3). (A to I) Data are mean ± SEM. (D to I) Data were analyzed by two-way ANOVA with post hoc Tukey’s multiple comparison. * P < 0.05.