Disulfooxy fatty acids from the American bird grasshopper Schistocerca americana, elicitors of plant volatiles

Abstract

A previously unidentified class of compounds has been isolated from the regurgitant of the grasshopper species Schistocerca americana. These compounds (named here "caeliferins") are composed of saturated and monounsaturated sulfated alpha-hydroxy fatty acids in which the omega-carbon is functionalized with either a sulfated hydroxyl or a carboxyl conjugated to glycine via an amide bond. The regurgitant contains a series of these compounds with fatty acid chains of 15-20 carbons and in varying proportions. Of these, the 16-carbon analogs are predominant and are also most active in inducing release of volatile organic compounds when applied to damaged leaves of corn seedlings. Caeliferins are nonlepidopteran elicitors identified in insect herbivores. This adds a category of insect herbivore-produced elicitors of plant responses, providing further evidence of the ability of plants to detect and respond to a broad range of insect herbivore-produced compounds.

Fig. 1.

HPLC separation and bioassay results of collected fractions. (A) Chromatographic trace (using 200-nm UV detection and pH 4.5 solvent) and 1-ml fraction collection of protein-precipitated and solid-phase extraction (SPE)-purified S. Americana regurgitant. (B) Bioassay results (n = 20) of combined fractions showing strong activity in the 15- to 17-min region and some activity in the 13- to 14-min fraction. (C) Bioassay result (n = 12) of the combined fractions 15–17 min from B after repeated HPLC. Separation was achieved using a neutral (pH 7) solvent and a slow gradient. The active 13- to 14-min fraction was assigned active fraction 1 (AF1) and the 15- to 16-min fraction was assigned AF2. The excised plant assay was used for all bioassays, and the induced volatile release was normalized and analyzed statistically (P < 0.01) as described in Methods.

Fig. 2.

Electro-spray LC/MS total negative ion trace of assigned active fraction 1 (AF1) (A) and AF2 (B) (in Fig. 1C). Negative ion analyses gave a strong (M-1)⁻ ion at m/z 445 (MW 446 amu) for AF1 and (M-1)⁻ ion at m/z 447 (MW 448 amu) for AF2. Both compounds gave strong double-charged ions (M-2)²⁻ at m/z 222 and m/z 223, respectively. Positive ion analyses gave three characteristic ions at MZ 464, 481, and 498 for AF1 and at MZ 466, 483, and 499 for AF2, indicating compounds with a MW of 446 and 448 amu containing three acidic sites forming ammonium salts by the solvent buffer.

Fig. 3.

The structures of caeliferin A16:1, and A16:0 and caeliferin B16:1 and B16:0. The H-NMR chemical shifts were obtained for isolated natural compounds dissolved in D₂O. Caeliferin A16:1 = (E)-2,16 disulfooxy-6-hexadecenoic acid; caeliferin A16:0 = 2,16 disulfooxyhexadecanoic acid; caeliferin B16:1 = N-[(E)-15-sulfooxy,15-carboxy-10-pentadecenoyl] glycine; and caeliferin B16:0 = N-(15-sulfooxy,15-carboxy pentadecanoyl) glycine.

Fig. 4.

Negative ion LC/MS separation and bioassay results of collected fractions. (A) Total ion trace of regurgitant from laboratory-reared S. Americana. The numbers indicate fractions that were collected for intact plant bioassays. Fraction 2, caeliferin B16:1; fraction 4, caeliferin B16:0; fraction 7, caeliferin A16:1; fraction 9, caeliferin A16:0; fraction 10, caeliferin A17:0; and fraction 11, caeliferin A18:0. (B) Normalized result of intact seedling bioassay at natural relative concentrations (0.5-μl regurgitant equivalents), n = 12. (C) Bioassay of selected fractions with all of the concentrations adjusted to that of caeliferin A16:1 (100 pmol in 0.5-μl regurgitant equivalents). Different letters indicate significant differences (P < 0.01).