Identification and field testing of sex-attractant semiochemicals produced by male deer mice, Peromyscus maniculatus

Abstract

Following previous reports that male deer mice, Peromyscus maniculatus, produce chemical signals that attract conspecific females, we analysed and field-tested sex-attractant semiochemicals (message-bearing chemicals) of male deer mice. Field traps baited with urine- and faeces-soiled bedding of male mice captured adult female, but not male, mice, indicating dissemination of sex-attractant semiochemicals from the males' excreta. Analysing excreta headspace volatiles of both males and females by gas chromatography-mass spectrometry revealed that 5-methyl-2-hexanone was male-specific, and that eight other ketones (3-methyl-2-pentanone, 2-hexanone, 4-heptanone, 2-heptanone, 6-methyl-2-heptanone, 3-octanone, 2-octanone, 2-nonanone) were 2.6-5.6 times more abundant in male, than in female, samples. In a field experiment with paired trap boxes, treatment boxes baited with the synthetic ketone lure captured 3.4 times more females (17 : 5) and 1.6 times fewer males (5 : 8) than corresponding unbaited boxes. In a follow-up paired-trap field experiment, treatment boxes baited with both the ketone lure and synthetic testosterone captured 8 times more mature females and 2.3 times more immature females, but 9 times fewer immature males, than control boxes baited only with the ketone lure, all indicating that testosterone is a synergistic sex-attractant semiochemical. As previously shown in house mice, Mus musculus, and brown rats, Rattus norvegicus, sex-attractant semiochemicals of male deer mice comprise both volatile and sex steroid components.

Keywords: ketones; sex-attractant semiochemicals; testosterone; volatiles.

Figure 1.

Photographs illustrating (a) the paired-trap experimental design deployed in field experiments, and (b–d) detailed views of the food bait and semiochemical lure tested in randomly assigned treatment and control traps, as follows: 1 = trap box, 2 = snap trap with food bait [53], 3 = bedding soiled with urine and faeces excreta of male deer mice or clean bedding (control); 4 = glass scintillation vial (20 ml) containing a blend of candidate sex-attractant semiochemicals formulated in mineral oil (10 ml) or mineral oil alone (10 ml; control); and 5 = piece of filter paper treated either with testosterone (750 ng) dissolved in acetonitrile (50 μl) or with acetonitrile alone (50 μl; control).

Figure 2.

Captures of (a) mature deer mice, Peromyscus maniculatus, and (b) eastern meadow voles, Microtus pennsylvanicus, and vagrant shrews, Sorex vagransii, in paired traps in Exp. 1. The randomly assigned treatment trap in each pair was baited with bedding soiled with urine and faeces of male deer mice, whereas the corresponding control trap received clean bedding. The asterisk (*) denotes significantly more captures of females in traps baited with soiled bedding than in traps fitted with clean bedding (binominal tests: female deer mice: p = 3.052 × 10⁻⁵; female shrews: p = 0.04).

Figure 3.

Total ion chromatograms of headspace volatile extracts obtained from (a) bedding soiled with urine and faeces excreta from three male deer mice, (b) bedding soiled with excreta from five female deer mice and (c) a trap lure mineral oil formulation of synthetic ketones (5 days after preparation) that was tested in field experiments with wild deer mice (see §2 for details). Note: (1) 5-methyl-2-hexanone (3) was male-specific, and eight other ketones (3-methyl-2-pentanone (1), 2-hexanone (2), 4-heptanone (4), 2-heptanone (5), 6-methyl-2-heptanone (6), 3-octanone (7), 2-octanone (8), 2-nonanone (9)) were 2.6–5.6 times more abundant, on average, in samples of three males each (n = 5) than in samples of five females each (n = 5) (see table 1); (2) 2-hexanone (2) was not present in this particular female sample but was detectable in other samples; (3) in (c), the two compounds marked by an asterisk are contaminants; (4) the headspace profile of synthetic ketones emanating from the trap lure during 24 h greatly resembles that of natural ketones emanating from soiled bedding of three deer mouse males during 24 h; (5) mass spectrometric analysis: Varian Saturn Ion Trap GC-MS; DB-5 MS GC column; temperature programme: 40°C (5 min), 10°C min⁻¹ to 280°C (5 min).

Figure 4.

Captures of mature female and male deer mice, Peromyscus maniculatus, in paired traps (figure 1a) in Exp. 2. Both boxes in each pair received a glass scintillation vial (20 ml; figure 1b) containing either a 4 mg blend of the candidate sex-attractant semiochemicals (CSSs) (3-methyl-2-pentanone (0.03 mg), 2-hexanone (0.01 mg), 5-methyl-2-hexanone (0.02 mg), 4-heptanone (0.03 mg), 2-heptanone (0.8 mg), 6-methyl-2-heptanone (0.03 mg), 3-octanone (2.1 mg), 2-octanone (0.3 mg), 2-nonanone (0.7 mg)) formulated in mineral oil (10 ml) or a mineral oil (10 ml) control stimulus. The asterisk denotes significantly more captures of females in traps baited with the CSS lure (binomial test, p = 0.02).

Figure 5.

Captures of mature and immature female and male deer mice, Peromyscus maniculatus, in paired traps (figure 1a) in Exp. 3. Both boxes in each pair received the blend of volatile candidate sex-attractant semiochemicals (CSSs) formulated in mineral oil (see caption of figure 4). The treatment box in each pair also received a piece of filter paper (figure 1c) treated with testosterone (750 ng) dissolved in acetonitrile (50 μl), whereas the filter paper in the corresponding control trap received acetonitrile only (50 μl). The asterisks denote significantly more captures of mature females (binomial test, p = 0.04), and fewer captures of immature males (binomial test, p = 0.02), in testosterone-baited traps. Note that the amount of testosterone represents 1.5 times the amount of testosterone present in 1 g of male deer mouse faeces [46].