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. 2024 May 30:12:RP91711.
doi: 10.7554/eLife.91711.

The larva and adult of Helicoverpa armigera use differential gustatory receptors to sense sucrose

Shuai-Shuai Zhang #  1   2 , Pei-Chao Wang #  1   2 , Chao Ning  1   2 , Ke Yang  1   2 , Guo-Cheng Li  1   2 , Lin-Lin Cao  1   2 , Ling-Qiao Huang  1 , Chen-Zhu Wang  1   2
Affiliations

The larva and adult of Helicoverpa armigera use differential gustatory receptors to sense sucrose

Shuai-Shuai Zhang et al. Elife. .

Abstract

Almost all herbivorous insects feed on plants and use sucrose as a feeding stimulant, but the molecular basis of their sucrose reception remains unclear. Helicoverpa armigera as a notorious crop pest worldwide mainly feeds on reproductive organs of many plant species in the larval stage, and its adult draws nectar. In this study, we determined that the sucrose sensory neurons located in the contact chemosensilla on larval maxillary galea were 100-1000 times more sensitive to sucrose than those on adult antennae, tarsi, and proboscis. Using the Xenopus expression system, we discovered that Gr10 highly expressed in the larval sensilla was specifically tuned to sucrose, while Gr6 highly expressed in the adult sensilla responded to fucose, sucrose and fructose. Moreover, using CRISPR/Cas9, we revealed that Gr10 was mainly used by larvae to detect lower sucrose, while Gr6 was primarily used by adults to detect higher sucrose and other saccharides, which results in differences in selectivity and sensitivity between larval and adult sugar sensory neurons. Our results demonstrate the sugar receptors in this moth are evolved to adapt toward the larval and adult foods with different types and amounts of sugar, and fill in a gap in sweet taste of animals.

Keywords: CRISPR/Cas9; Xenopus oocyte expression and two-electrode voltage-clamp; cotton bollworm; ecology; gustatory receptor; sucrose.

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Conflict of interest statement

SZ, PW, CN, KY, GL, LC, LH, CW No competing interests declared

Figures

Figure 1.
Figure 1.. Electrophysiological responses of larval and adult contact chemosensilla in Helicoverpa armigera to sugars.
(A) The maxilla of the larva (left), the representative spike traces of the responses of lateral sensilla styloconica on larval maxillary galea to eight sugars at 10 mM (middle), and quantifications of the firing rates (right) (n=13). The red arrow marks the recorded lateral sensillum styloconicum. Scale bar represents 20 μm. (B) Dose–responses of the lateral sensilla styloconica on larval maxillary galea to sucrose and fucose (sucrose: n=8; fucose: n=6). (C) The antenna terminal of the female (left), the representative spike traces of the responses of the contact chemosensilla on female antennae to eight sugars at 10 mM (middle), and quantifications of the firing rates (right) (n=21). The red arrows mark the recorded contact chemosensilla. Scale bar represents 100 μm. (D) Dose–responses of the contact chemosensilla on female antennae to sucrose, fucose, and fructose (n=21). (E) The fifth tarsomere of the female (left) (Zhang et al., 2010), the representative spike traces of the responses of contact chemosensilla on female tarsi to eight sugars at 10 mM (middle), and quantifications of the firing rates (right) (n=15). The red arrows mark the recorded contact chemosensilla. Scale bar represents 100 μm. (F) Dose–responses of the contact chemosensilla on female tarsi to sucrose, fucose, and fructose (n=21). (G) The proboscis terminal of the female (left), the representative spike traces of the responses of contact chemosensilla on female proboscis to eight sugars at 10 mM (middle), and quantifications of the firing rates (right) (n=21). The red arrows mark the recorded contact chemosensilla. Scale bar represents 200 μm. (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 the contact chemosensilla on the female 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.
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. Electrophysiological responses of contact chemosensilla in Helicoverpa armigera to sugars.
(A) The representative spike traces and waveforms of lateral sensilla styloconica on larval maxillary galea and contact chemosensilla on female antennae, tarsi and proboscis responding to test compounds. (B) Representative dose–response spike traces of the lateral sensilla styloconica on larval maxillary galea and contact chemosensilla on female antennae, tarsi and proboscis to sucrose, fucose, and fructose. (C) The representative spike traces of the responses of medial sensilla styloconica on larval maxillary galea to eight sugars at 10 mM (left), and quantifications of the firing rates (right) (n=13). (D) The representative spike traces of the responses of lateral sensilla styloconica on larval maxillary galea (left), and quantifications of the firing rates to 0.05 mM sucrose, 5 mM fucose, and the mixture of those (right) (n=6). (A–B) The dotted lines indicate the spike trace of the first 200ms. (C–D) Data are mean ± SEM; * p<0.05; ** p<0.01; *** p<0.001; ns indicates no significance (independent-samples t test).
Figure 2.
Figure 2.. Behavioral responses of Helicoverpa armigera larvae and adults to sugars.
(A) Feeding responses and the preference index (PI) value of 5th instar larvae to eight 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 eight 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 eight 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 red arrow indicates the stimulating site.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Feeding responses and the PI value of 5th instar larvae of Helicoverpa armigera to different concentrations of fructose painted on the cabbage leaf discs in two-choice tests.
Data are mean ± SEM; ** p<0.01; ns indicates no significance (paired t test, n=20).
Figure 3.
Figure 3.. 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).
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Expression patterns of sugar GRs in taste organs of Helicoverpa armigera male adults.
(A) Relative expression levels of sugar GRs in male antennae determined by qRT-PCR. (B) Relative expression levels of sugar GRs in male tarsi. (C) Relative expression levels of sugar GRs in male proboscis. (A–C) 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).
Figure 4.
Figure 4.. The inward current responses of the Xenopus oocytes expressing sugar GRs of Helicoverpa armigera to sugars.
(A) The representative traces of the oocytes expressing Gr10, Gr6, and Gr10 +Gr6 to 11 sugars 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.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. The inward current responses and representative traces of Xenopus oocytes expressing sugar GRs of Helicoverpa armigera.
(A) Heat-map signal indicates the mean of the responses to eleven sugars at 100 mM of the oocytes expressing H. armigera sugar GRs (H2O: mannose, n=5; other sugars and ringer solution: n=6. Gr4: arabinose, n=3; other sugars and ringer solution: n=7. Gr5: n=7. Gr6: mannose, n=15; other sugars and ringer solution: n=20. Gr7: n=5. Gr8: n=5. Gr10: maltose, n=13; other sugars and ringer solution: n=14. Gr11: n=6. Gr12: n=6. Gr10 +Gr6: n=7). Gray and red signals represent lower and higher response levels, respectively. (B) The representative two-electrode voltage-clamp traces of oocytes expressing Gr4, Gr5, Gr7, Gr8, Gr11, and Gr5 +Gr6 to eleven sugars at 100 mM and the control (H2O). (A–B) 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. (C) The responses of the oocytes expressing Gr5 +Gr6 to sugars at 100 mM (n=10). Data (mean ± SEM) were analyzed by one-way ANOVA with Tukey’s HSD test, and different letters labeled indicate significant differences (p<0.05).
Figure 5.
Figure 5.. 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 E) 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).
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Confirmation of the deletion of Gr10 and Gr6 in Gr10-/- and Gr6-/- mutants at mRNA level.
(A) The alignment of the nucleic acid sequences based on Gr10 transcripts in WT and Gr10-/-. (B) The alignment of the nucleic acid sequences based on Gr6 transcripts in WT and Gr6-/-. The green rectangle corresponds to the position of the sgRNA targeting site, the blue rectangle corresponds to the position of the PAM site.
Figure 5—figure supplement 2.
Figure 5—figure supplement 2.. The potential off-target effects detection.
(A) Representative chromatograms of potential off-target PCR products obtained from the wild type (WT) and Gr6-/-. (B) The representative spike traces (the left) and quantifications of the firing rate (the right) of medial sensilla styloconica on larval maxillary galea to 2 mM KCl and 10 mM xylose (n=8). (C) The larval body weight at the beginning of the fifth instar (the upper, n=16), the adult lifespan (the middle, n=16), and the number of eggs laid (the lower, n=5). (B–C) Data are mean ± SEM. Data were analyzed by one-way ANOVA with Turkey’s HSD test, and different letters labeled indicate significant differences (p<0.05).
Figure 6.
Figure 6.. Electrophysiological responses of larval and adult contact chemosensilla in WT, Gr10-/- and Gr6-/- of Helicoverpa armigera to sucrose and other compounds.
(A) The representative spike traces of lateral sensilla styloconica on larval maxillary galea. (B) Quantifications of the firing rates of the lateral sensilla styloconica on larval maxillary galea (mean ± SEM; WT, n=12; Gr6-/-, n=13; Gr10-/-, n=13). (C) The representative spike traces of contact chemosensilla on female antennae. (D) Quantifications of the firing rates of the contact chemosensilla on female antennae (mean ± SEM; WT, n=20. Gr6-/-: nicotine, n=17; other compounds, n=18. Gr10-/-, n=20). (E) The representative spike traces of contact chemosensilla on female tarsi. (F) Quantifications of the firing rates of the contact chemosensilla on female tarsi (mean ± SEM; WT, n=18; Gr6-/-: nicotine, n=14. other compounds, n=18. Gr10-/-, n=18). (G) The representative spike traces of contact chemosensilla on female proboscis. (H) Quantifications of the firing rates of the contact chemosensilla on 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.
Figure 7.
Figure 7.. 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). Proboscis extension reflex (PER) in adult females upon (D) antennal stimulation by sucrose concentrations (n=3), (E) antennal stimulation by fucose concentrations (n=3), (F) antennal stimulation by fructose concentrations (n=3), (G) tarsal stimulation by sucrose concentrations (n=3), (H) tarsal stimulation by fucose concentrations (n=3), and (I) 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. The red arrow indicates the stimulating site.
Figure 7—figure supplement 1.
Figure 7—figure supplement 1.. Feeding responses and the PI value of 5th instar larvae of WT, Gr10-/- and Gr6-/- of Helicoverpa armigera to fructose painted on the cabbage leaf discs in two-choice tests.
(A) Feeding area and the PI value to 10 mM fructose. (B) Feeding area and the PI value to 100 mM fructose. Data are mean ± SEM, n=20, paired t test, ** p<0.01. Fru, fructose. Supplementary file 1. GenBank accession numbers for sugar gustatory receptors used in this study.
Figure 8.
Figure 8.. Two gustatory receptors in the cotton bollworm, Helicoverpa armigera mainly mediate taste sensation of sugars in the larval and adult foods.
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Update of

  • doi: 10.1101/2023.09.19.558417
  • doi: 10.7554/eLife.91711.1
  • doi: 10.7554/eLife.91711.2

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