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Insects
2019 Aug 26;109:. doi: 10.3390/insects10090272.
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A Gustatory Receptor GR8 Tunes Specifically to D-Fructose in the Common Cutworm Spodoptera litura.
Liu XL
,
Yan Q
,
Yang YL
,
Hou W
,
Miao CL
,
Peng YC
,
Dong SL
.
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Gustatory receptors (GRs) are crucial in the peripheral coding of the non-volatile compounds in insects, and thus play important roles in multiple behaviors including feeding, mating, and oviposition. However, little research has been done on GRs in lepidopteran pests. In the current work with Spodoptera litura, an important worldwide crop's pest, a candidate fructose GR gene (SlitGR8) was cloned in full length, and its spatial and temporal expression profiles were determined by quantitative real-time PCR (qPCR). It revealed that SlitGR8 was highly expressed in antennae of both male and female adults, as well as in larva of first, fifth and sixth instar. Functional analyses were further conducted using the Xenopus oocyte system. SlitGR8 responded specifically to D-fructose among 12 tested sugar compounds. In addition, the behavioral assay demonstrated that both female and male moths could respond with proboscis extension behavior to D-fructose applied onto the antenna, but females showed higher sensitivity than males. The results provide an important base for further elucidation of molecular mechanisms of gustation, and a potential target for development of feeding interfering technique in S. litura.
Figure 1. Alignment of the amino acid sequences of four lepidopteran fructose GR genes, showing positions of seven predicted transmembrane domains (TM1–TM7). Identical amino acids are marked with black shading. Insect species and GenBank IDs of the GR genes are same as in Table 1.
Figure 2. Phylogenetic tree of the amino acid sequences of SlitGR8 and other GRs, including all functionally characterized fructose GRs from other species of D. melanogaster, B. mori, and H. armigera. The tree was constructed by neighbor joining method with a bootstrap of 1000. Fructose receptor clad is marked in blue color. SlitGR8 is in red color.
Figure 3. Relative expression levels of SlitGR8 mRNA at different development stages (A) and in different tissues (B). (A) L1–L6: 1–6 instar larva; MP: Male pupae; FP: Female pupae; MM: Male moths; FM: Female moths. (B) A: Antennae; P: Proboscis; La: Labial palpi; H: Heads; T: Thoraxes; Ab: Abdomens; L: Legs; W: Wings. Different lowercase letters indicate significant differences (p < 0.05) based on one-way ANOVA, followed by Tukey’s HSD test for multiple comparisons. Means± standard error from three replicates are shown.
Figure 4. Responses of Xenopus oocytes expressed with SlitGR8 to stimulation of sugar components. (A) Inward currents of Xenopus oocytes injected with SlitGR8 cRNA (upper) and buffer (lower) to sugar components (0.1 M). (B) Response profile of Xenopus oocytes expressed with SlitGR8. Error bars indicate SEM (n = 5). (C) Response of SlitGR8 expressing Xenopus oocytes to D-fructose of different concentrations. (D) Dose–response curve of SlitGR8 expressing Xenopus oocytes to D-fructose, with EC50 = 0.217 M. Error bars indicate SEM (n = 5).
Figure 5. Proboscis extension reflex (PER) resulted from adult male and female antennae stimulation by 0.001 M, 0.01 M, 0.1 M and 1 M fructose. Double distilled water (the solvent) was used as the control. Bars represent standard error form three replications. Different letters (a–c) indicate significant differences (p < 0.05) of male or female responses among different concentrations based on one-way ANOVA followed by Tukey’s HSD test for multiple comparisons. * and ** mean significant differences (p < 0.05 and <0.01, respectively), while NS indicates no significant difference between male and female responses by T-test.
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