Gustatory Sensing Mechanism Coding for Multiple Oviposition Stimulants in the Swallowtail Butterfly, Papilio Xuthus

Typical electrophysiological recordings of L1 sensilla (left column) and L2 or S sensilla (right column) on the fifth tarsomere in response to eight oviposition stimulants during the first 500 ms after stimulation. Vertical bar indicates 1.0 mV. Arrow indicates the onset of stimulation. To the left of each trace, the spikes are superimposed. Open circles, open squares, and triangles indicate the positions of high (1.0–1.5 mV), intermediate (0.6–0.8 mV), and low (0.2–0.4 mV) spikes, respectively. Gray squares indicate spikes elicited by water (0.6–0.8 mV). Recordings show responses to A, 2.5 and 10 mm synephrine; B, 0.5 and 10 mm 5-hydroxy-Nω-methyltryptamine; C, 10 mm stachydrine; D, 10 mm chiro-inositol; E, 5 mm narirutin; F, 10 mm adenosine; G, 33 μm hesperidin; and H, 55 μm rutin. I, Electrophysiological responses of L1 (left column) and L2 or S sensilla (right column) to increasing concentrations of six oviposition stimulants in females (mean ± SE spike frequencies; n = 7–88 for synephrine, n = 5–70 for 5-hydroxy-Nω-methyltryptamine, n = 7–80 for stachydrine, n = 6–19 for adenosine, n = 6–53 for chiro-inositol, and n = 5–54 for narirutin). Gray boxes indicate the estimated concentrations of oviposition stimulants in intact leaves of C. unshiu, as described previously (Nishida et al., 1987; Ohsugi et al., 1991). These concentrations were as follows: synephrine: 2.4 mm; 5-hydroxy-Nω-methyltryptamine: 0.04 mm; stachydrine: 5.1 mm; adenosine: 0.02 mm; chiro-inositol: 8.3 mm; and narirutin: 0.03 mm. The number of spikes was calculated during the first second. Different letters (a–c) indicate significant differences of water spikes in L2 and S sensilla and low spikes of 5-hydroxy-Nω-methyltryptamine in L1 sensilla among concentrations by the Tukey–Kramer test (p < 0.05). *p < 0.05, **p < 0.01, ***p < 0.0001 versus 0.01 mm synephrine, 0.00005 mm 5-hydroxy-Nω-methyltryptamine, 0.01 mm stachydrine, 0.01 mm adenosine, 0.1 mm chiro-inositol, and 0.001 mm narirutin, respectively. J, Percentage of L1 sensilla responding to increasing concentrations of five oviposition stimulants (mean ± SE the percentage of responding sensilla; n = 7–14 for synephrine, n = 3–6 for 5-hydroxy-Nω-methyltryptamine, n = 4–18 for stachydrine, n = 5–7 for chiro-inositol, and n = 4 or 5 for narirutin). Each value of independent recording was the average response of 10–20 sensilla per individual. *p < 0.05; **p < 0.01, versus 1 mm synephrine, 0.005 mm 5-hydroxy-Nω-methyltryptamine, 1 mm stachydrine, 2.5 mm chiro-inositol, and 0.01 mm narirutin, respectively.

Responses of L1 sensilla on the fifth tarsomere to mixtures of two oviposition stimulants. A, Sample responses of L1 sensilla to 10 mm stachydrine plus 10 mm chiro-inositol. Oviposition spike 1 (os1, circles) and os2 (squares) spikes are shown. Arrowheads indicate the doublet spikes. B, Total spikes and doublets. Total spikes indicate superimposed spikes of trace A. Doublet spikes 1–5 are shown by arrowheads. C, Response to 2.5 mm synephrine and 10 mm chiro-inositol. Arrowheads indicate doublets of os2 and os3 (triangles). D, Response to 10 mm synephrine and 10 mm chiro-inositol. The mixture elicited three types of spikes simultaneously. Arrowheads 1 and 2 indicate doublets consisting of os1 + os2 and os1 + os3, respectively. E, Response to 10 mm synephrine and 5 mm narirutin. The mixture elicited three types of spikes. Arrowhead indicates a doublet of os1 and os2. F, Response to 10 mm stachydrine and 1.0 mm 5-hydroxy-Nω-methyltryptamine. The mixture elicited two types of spikes. Arrowheads indicate four doublets of os1 and os3. G, Response to 10 mm stachydrine and 2.5 mm synephrine. H, Response to 10 mm stachydrine and 10 mm 5-hydroxy-Nω-methyltryptamine. I, Response to 10 mm chiro-inositol and 5 mm narirutin. J, Response to 0.05 mm 5-hydroxy-Nω-methyltryptamine and 2.5 mm synephrine. Mixtures G–J each elicited a single type of spike. K, Responses to 25 mm synephrine and L, 10 mm 5-hydroxy-Nω-methyltryptamine. Arrowheads indicate doublets of os1 and os3. Arrows indicate the onset of stimulation. Trace A lasted 200 ms and traces C–L 500 ms.

Typical recordings of L1 (left column) and L2 or S sensilla (right column) in response to A, 100 mm sucrose; B, 100 mm fructose; C, 100 mm glucose; D, 1 mm KCl; E, 100 mm KCl; F, 50 mm NaCl, G, 200 mm NaCl, H, 10 mm caffeine, and I, 0.1 mm quinine. Gray squares, open circles, open squares, closed squares, and triangles indicate water, sugar, salt (low amplitude-spikes), salt (high amplitude-spikes), and bitter responses, respectively. J, Dose dependency of responses to sucrose, fructose, glucose, KCl, NaCl, caffeine, and quinine. (mean ±SD spike frequencies; n = 5–50 for sucrose, n = 6–21 for fructose, n = 7–13 for glucose, n = 11–60 for KCl, n = 6–66 for NaCl, n = 5–33 for caffeine, and n = 6–15 for quinine). Different letters (a–d) indicate significant differences of water spikes and salt spikes (low amplitude) in L2 and S sensilla among concentrations (Tukey–Kramer test, p < 0.05). *p < 0.01, **p < 0.001, ***p < 0.0001 versus 0.1 mm sucrose, 1 mm fructose, 1 mm glucose, 0.01 mm KCl, 0.1 mm NaCl, 0.01 mm caffeine, and 0.01 mm quinine, respectively.

Electrophysiological and oviposition responses to whole-leaf citrus extract and mixtures of oviposition stimulants. A, Typical response to 1.0× citrus extract (left). Scale bar, 1.0 mV. Arrows indicate the onset of stimulation. The first trace showed impulses during the first second after stimulation; the 0.5 s trace below it expands the boxed region in the 1.0 s trace. Circles, squares, and triangles indicate high, intermediate, and low spikes, respectively. The 0.1–0.001× citrus extracts also elicited three types of spikes in L1 sensilla during the first second of stimulation (right). B, Typical responses of L1 sensilla to mixtures of oviposition stimulants. The mixtures of oviposition stimulants were based on the estimated concentrations of each compound in whole-leaf extracts of C. unshiu (see Fig. 2). The five oviposition stimulants were numbered as follows: synephrine, 1; stachydrine, 2; 5-hydroxy-Nω-methyltryptamine, 3; chiro-inositol, 4; and narirutin, 5. Circles, squares, and triangles indicate oviposition spike 1 (os1), os2, and os3, respectively. C, Assay of oviposition behavior (oviposition activities; n = 9 for 1.0× citrus extract, n = 5 for 1 + 2 + 3 + 4 + 5, n = 10 for 1 + 2 + 3 + 4, n = 15 for 2 + 3 + 4 + 5, n = 7 for 2 + 3 + 4, n = 9 for 1 + 2 + 4, n = 12 for 3 + 4 + 5, n = 12 for 2 + 4 + 5, n = 15 for 1 + 4 + 5, n = 12 for 1 + 5, n = 20 for 1 + 4, n = 3 for 2 + 3, n = 3 for 1 + 3, n = 3 for 1 + 2, n = 4 for 3 + 4, n = 4 for 2 + 4, n = 5 for 5, n = 7 for 4, n = 4 for 3, n = 4 for 2, n = 7 for 1, and n = 7 for water). Each value represents the mean ± SE of 3–20 independent determinations, each of which was the average of 7–10 individuals. The gray, white, and black bars indicate whether three, two, or one GRN was fired by each solution, except for water. *p < 0.0001 versus oviposition activity induced by water alone (Tukey–Kramer test; F_(22,157) = 48.947). D, Correlation between number of spikes and oviposition activity. Gray circles, closed circles, squares, triangles, open circles, open square, open diamond, gray square, gray diamond, closed triangle, and closed triangle (downward) indicate citrus extract and mixtures of 1 + 2 + 3 + 4 + 5, 1 + 2 + 3 + 4, 1 + 2 + 4, 1 + 3 + 4, 2 + 3 + 4 + 5, 1 + 4 + 5, 1 + 4, 1 + 5, 2 + 4 + 5, and 3 + 4 + 5, respectively. Each value represents oviposition activity on the vertical axis (mean ± SE n = 4–11 for citrus extract, n = 3–5 for 1 + 2 + 3 + 4 + 5, n = 3–10 for 1 + 2 + 3 + 4, n = 3–7 for 1 + 2 + 4, n = 3–9 for 1 + 3 + 4, n = 15 for 2 + 3 + 4 + 5, n = 15 for 1 + 4 + 5, n = 20 for 1 + 4, n = 12 for 1 + 5, n = 12 for 2 + 4 + 5, and n = 12 for 3 + 4 + 5) and the number of spikes on the horizontal axis (mean ± SE n = 12–20 for citrus extract, n = 17–30 for 1 + 2 + 3 + 4 + 5, n = 7–26 for 1 + 2 + 3 + 4, n = 12–21 for 1 + 2 + 4, n = 11–28 for 1 + 3 + 4, n = 40 for 2 + 3 + 4 + 5, n = 10 for 1 + 4 + 5, n = 13 for 1 + 4, n = 12 for 1 + 5, n = 40 for 2 + 3 + 4, and n = 7 for 3 + 4 + 5). The results of post hoc multiple comparisons of these data are described in Tables 2 and 3, respectively.