Takada T et al. (2017), A mannitol/sorbitol receptor stimulates dietary...

XB-ART-54135

PLoS One 2017 Jan 01;1210:e0186420. doi: 10.1371/journal.pone.0186420.

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A mannitol/sorbitol receptor stimulates dietary intake in Tribolium castaneum.

Takada T , Sato R , Kikuta S .

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In insects, perception of chemical stimuli is involved in the acceptance or rejection of food. Gustatory receptors (Grs) that regulate external signals in chemosensory organs have been found in many insects. Tribolium castaneum, a major pest of stored products, possesses over 200 Gr genes. An expanded repertoire of Gr genes appears to be required for diet recognition in species that are generalist feeders; however, it remains unclear whether T. castaneum recognizes a suite of chemicals common to many products or whether its feeding is activated by specific chemicals, and whether its Grs are involved in feeding behavior. It is difficult to determine the food preferences of T. castaneum based on dietary intake due to a lack of appropriate methodology. This study established a novel dietary intake estimation method using gypsum, designated the TribUTE (Tribolium Urges To Eat) assay. For this assay, T. castaneum adults were fed a gypsum block without added organic compounds. Sweet preference was determined by adding sweeteners and measuring the amount of gypsum in the excreta. Mannitol was the strongest activator of T. castaneum dietary intake. In a Xenopus oocyte expression, TcGr20 was found to be responsible for mannitol and sorbitol responses, but not for responses to other tested non-volatile compounds. The EC50 values of TcGr20 for mannitol and sorbitol were 72.6 mM and 90.6 mM, respectively, suggesting that TcGr20 is a feasible receptor for the recognition of mannitol at lower concentrations. We used RNAi and the TribUTE assay to examine whether TcGr20 expression was involved in mannitol recognition. The amounts of excreta in TcGr20 dsRNA-injected adults decreased significantly, despite the presence of mannitol, compared to control adults. Taken together, our results indicate that T. castaneum adults recognized mannitol/sorbitol using the TcGr20 receptor, thereby facilitating the dietary intake of these compounds.

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Species referenced: Xenopus
Genes referenced: mal zbtb22

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	Fig 1. Establishment of feeding assay in T. castaneum adults using gypsum block.The starved T. castaneum adults show in a series of the behavior from recognition, eating to excretion the gypsum diet. Scale bars: 1 mm. A. The adult beetle bit gypsum. The gypsum was stained with Coomassie Brilliant Blue (CBB). B, B’. Digestive tract of adult beetle with the CBB staining gypsum (B) and without the CBB staining (B’). C. Excreta of the CBB staining gypsum by T. castaneum (arrowheads). D. The assessment of the effect of sexual differences in gypsum intake. T. castaneum adult beetles were individually fed on sweetener-free gypsum blocks for 48 h. The amount of excreta was measured using microbalance. Each plot represents the amount of excreta of adult beetles in individuals (n = 10). Standard error bars show S.E.M. Statistical analyses were performed Mann-Whitney U test (P = 0.26). “ns” not significant. Total numbers are 20. The results presented are representative of three separate experiments. E. The effect of gypsum intake with sugar or sugar alcohol mixture. Each sugar/sugar alcohol at 200 mM was contained in the gypsum block. T. castaneum adults fed on the artificial diet for 48 h. Each plot represents the amount of excreta of adult beetles in individuals (n = 10). Standard error bars show S.E.M. The results presented are representative of several separate experiments. Statistical analyses were performed Kruskal-Wallis test and the Dunn’s multiple comparison test (“*” P ≤ 0.001, “**” P ≤ 0.0001, “ns” not significant). Sorbitol, Sor; Trehalose. Tre; Glucose, Glu; Maltose, Mal; Sucrose, Suc; Fructose, Fru; Mannitol. Man. Total numbers are 80, and number of comparison family is 28.
	Fig 2. Phylogenetic analysis of deduced amino acid sequences of T. castaneum Gr genes.Amino acid sequence alignment was generated using ClustalW, and the unrooted tree of T. castaneum Grs was conducted by neighbor-joining method in MEGA ver. 7 [25]. The percentage of replicate trees are shown in the associated taxa clustered together in the bootstrap test of 500 replicates. The scale bar represents 0.2 substitutions per amino acid site. Amino acid sequences of B. mori and T. castaneum Grs were obtained from [9, 14], respectively. The amino acid sequences of fructose receptor, HarmGr4 from Helicoverpa armigera, DmGr43 derived from Drosophila melanogaster, were obtained from the NCBI public database.
	Fig 3. Current recording of Xenopus oocytes expressing TcGr20.A. Inward current response of Xenopus oocytes expressing TcGr20 to candidate tastants (arrowheads). Tastants were tested at 200 mM. B. The current of water-injected oocytes to same tastants were also recorded. The current data are representative of recordings independently performed in several times.
	Fig 4. Ligand dose-dependent response of TcGr20.Two-electrode voltage clamp recordings of TcGr20-expressing Xenopus oocytes. A–B. Inward current response of TcGr20-expressing oocytes with a range of 20 to 400 mM mannitol (A), and 40 to 230 mM sorbitol (B). Each arrowhead represents various concentrations. C–D. Curves were fitted with a standard slope, and EC50 values were calculated for mannitol (C) and sorbitol (D), respectively. Data are shown as mean ± S.E.M. (n = 3).
	Fig 5. Tissue expression of TcGr20.The relative expression level of the TcGr20 in different tissues was determined by quantitative RT-PCR. TcGr20 expression in male (A) and female (B). An, antennae; H, head; T, thorax; Ab, abdomen and L, legs. Relative expression was calculated using the 2-ΔΔCt method. Ribosomal protein S3 (RpS3) in T. castaneum was used as the control to normalize the amount of templates. Data are shown as mean ± S.E.M. (n = 3). Statistical analyses were performed one-way ANOVA (A. F (DFn, DFd) F (4, 10) = 14.17, P = 0.0004; B. F (DFn, DFd) F (4, 10) = 163.8, P < 0.0001) and the post hoc Tukey’s multiple comparison test. Columns labeled with the same letters indicate not significant difference (P > 0.05).
	Fig 6. Concentration response of mannitol and sorbitol in TribUTE assay.The dose effect of gypsum intake with sugar alcohol mixture. A. mannitol; B. sorbitol. Sugar alcohols at various concentrations were contained in the gypsum block. T. castaneum adults fed on the gypsum for 48 h. Scatter plot represents the amount of excretion of adult beetles in individuals (n = 6). Standard error bars show S.E.M. The results presented are representative of three separate experiments. Statistical analyses were performed Kruskal-Wallis test (A. “*” P = 0.0002, n = 24; B. “” P = 0.0002, n = 24) and Dunn's multiple comparisons test (“” P ≤ 0.05, “” P ≤ 0.01, “*” P ≤ 0.001, “ns” not significant).
	Fig 7. TribUTE assay in TcGr20-silencing T. castaneum.A. Knockdown of TcGr20 using the injection of the dsRNA into the starved adult beetles. The Eluc-dsRNA was injected as a control. TcGr20 expression levels of whole body were examined at 48 h after the dsRNA injection. Standard error bars show S.E.M. Statistical significance was determined by t-test (P = 0.0006). B. Effect of gypsum intake supplemented with 100 mM mannitol. The gypsum in the presence of 100 mM mannitol was given to the TcGr20- and Eluc-dsRNA-injected adult beetle individuals at 48 h after dsRNA injection, respectively for 48 h. The amount of excreta was measured using microbalance. Each plot represents the amount of excreta of adult beetles in individuals (n = 24). Standard error bars show S.E.M. Statistical significance was determined by t-test (“****” P ≤ 0.0001).

References [+] :

Abdel-Latief, A family of chemoreceptors in Tribolium castaneum (Tenebrionidae: Coleoptera). 2007, Pubmed