Quantification of contact oviposition stimulants for black swallowtail butterfly, Papilio polyxenes, on the leaf surfaces of wild carrot, Daucus carota

Ovipositing black swallowtail butterflies, Papilio polyxenes, make their final host-selection decisions on the basis of compounds present on the leaf surface. Little information is available, however, on the chemistry of leaf surfaces. The purpose of this study was to develop a technique to extract and quantify the concentrations of compounds from the leaf surfaces of Daucus carota, one of the main host species for P. polyxenes, with particular reference to compounds already identified as contact oviposition stimulants, namely trans-chlorogenic acid (CA) and luteolin-7-O-(6''-O-malonyl)-β-D- glucopyranoside (L7MG), as well as its degradation product luteolin-7 glucoside (L7G). Plant surfaces were extracted by dipping leaves sequentially in pairs of solvents: (1) CHCl₃-MeOH, (2) near-boiling H₂O. (3) CHCl₃- near-boiling H₂O, and (4) CH₂Cl₂-CH₂Cl₂. The resulting extracts were fractionated and analyzed using high-performance liquid chromatography. The leaf-surface concentrations of each compound were calculated using regressions relating leaf surface area to leaf weight that were obtained from measurements of field-collected carrot plants. All four methods removed the three compounds from carrot leaf surfaces, but the solvent systems differed in effectiveness. The chloroform-near-boiling water solvent system performed better than the other solvent combinations, but not significantly so. This system also extracted the highest number of polar, UV-absorbing compounds. Methylene chloride was significantly less efficient than the other methods. An additional test confirmed that the chloroform-near-boiling water method removed compounds from the surface alone and probably not from the apoplast or symplast. Surface concentrations of CA (up to 600 ng/cm² leaf surface) were substantially greater than those of the two flavonoid compounds. No clear seasonal trend in concentrations was evident from the limited number of sampling dates.