Sex-specific mating pheromones in the nematode Panagrellus redivivus

Abstract

Nematodes use an extensive chemical language based on glycosides of the dideoxysugar ascarylose for developmental regulation (dauer formation), male sex attraction, aggregation, and dispersal. However, no examples of a female- or hermaphrodite-specific sex attractant have been identified to date. In this study, we investigated the pheromone system of the gonochoristic sour paste nematode Panagrellus redivivus, which produces sex-specific attractants of the opposite sex. Activity-guided fractionation of the P. redivivus exometabolome revealed that males are strongly attracted to ascr#1 (also known as daumone), an ascaroside previously identified from Caenorhabditis elegans hermaphrodites. Female P. redivivus are repelled by high concentrations of ascr#1 but are specifically attracted to a previously unknown ascaroside that we named dhas#18, a dihydroxy derivative of the known ascr#18 and an ascaroside that features extensive functionalization of the lipid-derived side chain. Targeted profiling of the P. redivivus exometabolome revealed several additional ascarosides that did not induce strong chemotaxis. We show that P. redivivus females, but not males, produce the male-attracting ascr#1, whereas males, but not females, produce the female-attracting dhas#18. These results show that ascaroside biosynthesis in P. redivivus is highly sex-specific. Furthermore, the extensive side chain functionalization in dhas#18, which is reminiscent of polyketide-derived natural products, indicates unanticipated biosynthetic capabilities in nematodes.

Fig. 1.

(Left) Select ascaroside-based signaling molecules that regulate development and behavior in C. elegans (black) (14). Ascr#1, ascr#2, ascr#3, and ascr#8 are dauer pheromone components (4, 11, 12); ascr#2, ascr#3, ascr#4, and ascr#8 synergize in male attraction (3, 4), and ascr#3 contributes to hermaphrodite repulsion (6, 7). (Right) Ascarosides discovered in P. redivivus from this study that are produced specifically by females (red) and males (blue).

Fig. 2.

Identification of ascr#1 and dhas#18 as sex pheromones in P. redivivus. (A) Activity-guided fractionation of P. redivivus WW. (B) ¹H NMR spectra of isolated natural dhas#18 (red), synthetic dhas#18 (green), and a mixture of natural and synthetic samples (blue). (C) dqf-COSY spectrum of isolated dhas#18. (D) Section of the dqf-COSY spectrum in C showing cross-peaks diagnostic of the ascarylose ring (black boxes) and the highly functionalized side chain (yellow boxes). (E) Synthesis of dhas#18. a, 1. TiCl₄, 2. allylSn(PPh₃); b, vinylmagnesium bromide, Me₂S-CuBr, tetrahydrofuran (THF); c, BzCl, pyridine; d, t-butyldimethylsilyl chloride (TBDMSCl), imdidazole, dichloromethane (DCM); e, Grubbs II, 1,4-benzochinone, DCM; f, H₂, Pd/C, EtOH; g, trimethylsilyl triflate (TMSOTf), DCM; h, aqueous LiOH, THF, dioxane.

Fig. 3.

Dose–response for male and female attraction to major P. redivivus ascarosides. P. redivivus males and females were separately scored for their response to areas conditioned with different amounts of synthesized ascarosides (10⁰–10⁵ fmol). Their occupancy in the area containing the ascaroside was compared with occupancy in the control region (containing the same concentration of ethanol in water as the ascaroside dilution for each trial) for the duration of 20 min. Experiments in which males or females spent significantly more time in the control region have a negative index score, indicating avoidance of the ascaroside. Experiments in which males or female spent significantly more time in the ascaroside region have a positive index score, indicating attraction to the ascaroside. Error bars are SD. Statistical significance for each value was calculated compared with response to ddH₂O when present in both scoring regions, which was shown first in each set. (One-factor ANOVA followed by Dunnett’s posttest; *P < 0.05, **P < 0.01.)

Fig. 4.

Sex-specific expression of ascarosides in P. redivivus. (A) HPLC-MS/MS screen for precursor ions of m/z = 73 selectively detects ascarosides. (B) SIR for molecular ions of ascarosides detected by MS/MS in P. redivivus samples reveals sex-specific expression of ascarosides. Shown chromatograms are derived from mixed populations (black), females (green), and males (red). (C) Proposed biosynthetic pathway for ascaroside biosynthesis in P. redivivus. (D) Distribution of major ascaroside in male and female WW samples.