Natural variation in dauer pheromone production and sensing supports intraspecific competition in nematodes

Abstract

Dauer formation, a major nematode survival strategy, represents a model for small-molecule regulation of metazoan development [1-10]. Free-living nematodes excrete dauer-inducing pheromones that have been assumed to target conspecifics of the same genotype [9, 11]. However, recent studies in Pristionchus pacificus revealed that the dauer pheromone of some strains affects conspecifics of other genotypes more strongly than individuals of the same genotype [12]. To elucidate the mechanistic basis for this intriguing cross-preference, we compared six P. pacificus wild isolates to determine the chemical composition of their dauer-inducing metabolomes and responses to individual pheromone components. We found that these isolates produce dauer pheromone blends of different composition and respond differently to individual pheromone components. Strikingly, there is no correlation between production of and dauer response to a specific compound in individual strains. Specifically, pheromone components that are abundantly produced by one genotype induce dauer formation in other genotypes, but not necessarily in the abundant producer. Furthermore, some genotypes respond to pheromone components they do not produce themselves. These results support a model of intraspecific competition in nematode dauer formation. Indeed, we observed intraspecific competition among sympatric strains in a novel experimental assay, suggesting a new role of small molecules in nematode ecology.

Figure 1. Introduction to P. pacificus Small Molecules and Strains Used in This Study

(A) Ascarosides and paratosides previously identified from P. pacificus. The shown compounds constitute the most-abundant members (across all strains in this study) of the known families of ascarosides and paratosides, including unmodified ascarosides (ascr#1 and ascr#9), unmodified paratosides (part#9), phenylethanolamine-containing ascarosides (pasc#9), dimeric ascarosides (dasc#1), ureido-isobutyrate ascarosides (ubas#1), and nucleoside-containing paratosides (npar#1). (B) Potential theoretical outcomes of pheromone interactions. Nematodes show the strongest response either to their own pheromones (self-preference) or to pheromones from other strains (cross-preference). (C) Phylogenetic structure of P. pacificus. The six strains are highlighted in the phylogenetic dendrogram from [13].

Figure 2. Ascaroside and Paratoside Profiles of P. pacificus Isolates Used in This Study

(A) Relative abundances of ascarosides and paratosides in the exometabolomes and endometabolomes of exemplary P. pacificus wild isolates derived from HPLC-MS analysis (Figures 1A and S1) [7]. (B) Comparison of abundances of seven major ascarosides and paratosides in the exometabolomes and endometabolomes of six different P. pacificus wild isolates, represented in ng/day and normalized by worm pellet dry weight (see Supplemental Experimental Procedures). Error bars show SD.

Figure 3. Natural Variation in Dauer Formation

(A) Dauer formation of six P. pacificus strains in response to synthetic standards at 1 µM concentration (three to six independent biological replicates). The significance of the variation among strains and small molecules tested was calculated by using an ANOVA (GLM; strain: p = < 0.001, F = 12.51; compound: p = < 0.001, F = 20.73). Each dot represents one replicate. If fewer than three dots are visible, these dots are hidden behind the ones displayed. (B) Concentration dependencies for the three most-potent dauer-inducing small molecules: pasc#9, part#9, and npar#1 (three independent biological replicates). The mean dauer formation of three replicates is displayed for each of the six wild isolates tested. Error bars show exact binomial 95% confidence intervals.

Figure 4. Comparison of Production of and Dauer Response to Ascarosides and Paratosides Supporting Intraspecific Competition in P. pacificus Wild Isolates

(A) Comparative differences between production of and response to major ascarosides and paratosides in P. pacificus isolates used in this study. (B) Dauer pheromone competition assay with RS5380, RS5399, and RSB020 grown in Ussing chambers (see also Figure S3A). In control experiments, both compartments of a chamber were filled with nematodes of the same strain (Control). In the competition experiments, one strain was grown in one compartment of an Ussing chamber, while a different strain was grown in the other compartment of the same chamber. For each strain, the mean dauer formation of eight samples (taken from two independent experiments) is shown in response to being cultured with nematodes of the same (Control) or of a different genotype. Error bars show exact binomial 95% confidence intervals. (C) Model for intraspecific competition among RS5380, RS5399, and RSB020.