Chemical Attraction between Adults of Nippostrongylus brasiliensis: Characterization of the Substance Which Attracts Females

Abstract

Females of Nippostrongylus brasiliensis were attracted to the lipid fraction of excretory and secretory (ES) products of both males and females. Thin layer chromatography revealed several components in the lipid fraction, one of which (designated Band II) attracted adult females in vitro. The substance cochromatographed by TLC with cholesterol and p-sitosterol but neither authentic sterol was attractive when tested in vitro. Band II was detected in ES from both sexes of normal and immunedamaged worms but that from damaged females was not attractive, thereby correlating with the previously reported nonattractiveness of live damaged females and their whole ES. As determined by quantitative TLC, damaged female ES contained a significantly lower concentration of Band II material than that of other worm types although qualitative differences between damaged female Band II and that of other worm types were not ruled out. Chemical attraction between adults has been demonstrated for 21 species of nematodes (reviewed by Anya, 1976) yet little is known of the chemical properties of the substances involved. This is particularly true of zooparasitic species knowledge of which is limited to sexual attraction patterns. Results of a previous study (Roberts and Thorson, in press) indicated that adult males and females of Nippostrongylus brasiliensis possess different pheromone systems. That produced by males specifically attracts females and is not affected by host immunity. Females emit substances which attract both sexes and are host-labile in that those produced by immune-damaged females are not attractive to other worms. The purpose of the present study was to characterize the pheromone which attracts females. MATERIALS AND METHODS Methods of producing normal and immunedamaged adults, aseptic procedures for obtaining excretory and secretory (ES) products, and the basic design of the behavioral assay have been described in detail elsewhere (Roberts and Thorson, in press). Briefly, normal and damaged worms were obtained from 7-day (non-immune) and 14-day infected (immune) rats, respectively. Aliquots of ES material, obtained by incubating normal or damaged males or females in antibioticReceived for publication 4 January 1977. * Supported in part by NIH Training Grant T01 AI00400. t Present address: Department of Tropical Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. supplemented balanced salt solution at 37 C for 24 hr, were extracted in 15 volumes of chloroform: methanol (2:1, v/v) for 18 to 24 hr at 4 C (Folch et al., 1957) and washed twice in 0.04% CaCl2. The aqueous phases from each wash were combined and stored at -20 C. The organic solvent phase was rotoevaporated to dryness, with the residue reconstituted in chloroform at 50% of the initial volume and used for either behavioral analysis or thin layer chromatography. Exposure to air was minimized and when storage was necessary samples were purged with N2 and maintained at -20 C in the dark. To test the attractiveness of ES lipid fractions, filter paper discs, 6 mm in diameter, were impregnated with 10 Aliters of redissolved residue, dried under N2, and placed 2 cm from a live female in 60 by 15 mm plastic petri dishes containing 7 ml balanced salt solution and maintained at 37 C on a slide warmer. Chambers were observed macroscopically at 2, 4, and 6 hr; those with worms within 5 mm of the disc were considered positive. Discs soaked in chloroform served as controls. Thin layer chromatographic analyses of additional lipid fraction samples were conducted using glass plates coated with a 0.25 mm layer of silica gel G (E. Merck, Darmstadt, W. Germany). Plates were reactivated by heating at 110 C for 10 min just before use. Samples of unknowns and lipid standards, including cholesterol, famesol (Calbiochem, Los Angeles, CA), B-sitosterol, stigmasterol, and a mixture containing oleic acid, methyl oleate, triolein, and cholesteryl oleate (Applied Science Laboratories, Inc., State College, PA), were applied 1.5 cm from the bottom edge and developed 13 cm in any of 8 solvent systems. All solvents were lipid negative by TLC analysis. Developed plates were dried in a stream of N2 and placed in a chamber containing iodine crystals. Following detection the iodine was allowed to sublime from the plates and bands of silica gel con-