C. elegans males optimize mate-preference decisions via sex-specific responses to multimodal sensory cues

Abstract

For sexually reproducing animals, selecting optimal mates is important for maximizing reproductive fitness. In the nematode C. elegans, populations reproduce largely by hermaphrodite self-fertilization, but the cross-fertilization of hermaphrodites by males also occurs. Males' ability to recognize hermaphrodites involves several sensory cues, but an integrated view of the ways males use these cues in their native context to assess characteristics of potential mates has been elusive. Here, we examine the mate-preference behavior of C. elegans males evoked by natively produced cues. We find that males use a combination of volatile sex pheromones (VSPs), ascaroside sex pheromones, surface-associated cues, and other signals to assess multiple features of potential mates. Specific aspects of mate preference are communicated by distinct signals: developmental stage and sex are signaled by ascaroside pheromones and surface cues, whereas the presence of a self-sperm-depleted hermaphrodite is likely signaled by VSPs. Furthermore, males prefer to interact with virgin over mated, and well-fed over food-deprived, hermaphrodites; these preferences are likely adaptive and are also mediated by ascarosides and other cues. Sex-typical mate-preference behavior depends on the sexual state of the nervous system, such that pan-neuronal genetic masculinization in hermaphrodites generates male-typical social behavior. We also identify an unexpected role for the sex-shared ASH sensory neurons in male attraction to ascaroside sex pheromones. Our findings lead to an integrated view in which the distinct physical properties of various mate-preference cues guide a flexible, stepwise behavioral program by which males assess multiple features of potential mates to optimize mate preference.

Keywords: C. elegans; mate choice; mate preference; neurogenetics; pheromones; sexual behavior; sexual dimorphism.

Figure 1.. C. elegans males can determine the sex and developmental stage of potential mates.

(A) The Mate Preference Assay (MPA). Immobilized targets of two classes are placed in two opposing quadrants; after 30 mins, searchers are placed in the center. Their positions are used to calculate a Mate Preference Index (MPI) (see STAR Methods). (B) MPA with control adult male searchers paired with adult male and adult hermaphrodite targets. MPI values at individual time points, as well as their average, are shown. (C) MPA with male searchers paired with L4 and adult hermaphrodite targets. (D) MPA with male searchers paired with male and hermaphrodite targets at L3, L4, and adult stages. (E) MPA with control adult male and hermaphrodite searchers paired with adult male and hermaphrodite targets. In all figures, schematic diagrams depict the cues potentially produced by targets: VSPs are indicated by gradient shading, ascarosides by small hexagons, and surface cues by the outline of the body. Red or “H” indicates hermaphrodite and blue or “M” indicates male. Each data point represents an individual MPI derived from a single assay with ~10 searchers; the vertical gray bar indicates the mean MPI. For each condition, a Wilcoxon signed-rank test was used to ask whether the MPI differed from zero (i.e., that males had no preference for either target class). Results are shown with black squares at the right of each row: ■, p ≤ 0.05; ■■, p ≤ 0.005; ■■■, p ≤ 0.001. To compare MPI values between assays, we used Mann-Whitney tests or Kruskal-Wallis tests with Dunn’s correction. Asterisks indicate the associated p values: *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.001. Comparisons that did not reveal statistical significance are shown with gray dotted lines and are labeled “ns”. See also Figure S1.

Figure 2.. Ascaroside pheromones have central roles in sex and stage discrimination.

(A, F) Schematic depiction of targets. (B, C, E, H, J) MPAs with control male searchers paired with targets of the indicated sexes, stages, and genotypes. (D) Fraction of targets mated assessed using searchers with GFP-labeled sperm. Data points indicate individual assays; mean and standard deviation are also shown. (G, I) Epifluorescence images of mab-3∷GFP in animals of the indicated sex and genotype. Dashed white lines outline the body; yellow arrows indicate the intestine. The break in the vertical axis in (H) is used here and in subsequent figures to separate experiments that were not carried out in parallel and therefore cannot be directly compared. See also Figure S2.

Figure 3.. Morphological and surface cues also contribute to sex discrimination.

(A, D) Schematic depiction of targets. (B, C, E, F, G) MPAs with control male searchers paired with targets of the indicated sexes and genotypes. (H) Contact-response frequency of control males paired with hermaphrodites of the indicated genotypes. (I) Male food-leaving assay measuring retention by different classes of targets (none, control males, control hermaphrodites, and hyp^fem males). Data points show the fraction of males remaining on a food spot harboring the indicated retention cue over 24 hr. Error bars indicate standard deviation. See also Figure S3.

Figure 4.. Volatile sex pheromones (VSPs) that act through srd-1 are powerful mate preference signals.

(A) Schematic depiction of targets. (B-E) MPAs with control or srd-1 mutant male searchers paired with targets of the indicated sexes and genotypes. In (C), targets were placed on small agar plugs attached to the lid of the assay plate. The lid was then inverted to cover the assay plate, such that targets were suspended ~3 mm above the surface on which searchers navigated. See also Figure S4.

Figure 5.. C. elegans males assess target nutritional state and reproductive history in mate-preference decisions.

(A, D) Schematic depiction of targets. (B, C, E-K) MPAs with control or srd-1 mutant male searchers paired with targets of the specified sexes, genotypes, and conditions. In (B) and (C), some targets were deprived of food for 4 or 28 hr prior to the beginning of the assay. In (E-K), targets were either virgin (cultured overnight with sterile males) or mated (cultured overnight with wild-type males). In (J) and (K), some targets were briefly washed with sterile water before placing them on the assay plate.

Figure 6.. Sex-specific features of shared neurons and circuits guide sex-typical mate-choice decisions.

(A, D) Schematic depiction of targets. (B, C, E) MPAs with control males, males bearing neuronal sex-reversal transgenes, or neuronal ablation transgenes, paired with targets of the indicated sex and genotype. (F) Quadrant chemotaxis assay measuring ascr#3 attraction/retention using control or ASH-ablated males. See also Figures S5, S6, and S7.

Figure 7.. C. elegans males assess multiple features of potential mates using a stepwise behavioral strategy that incorporates multimodal sensory cues.

(A) The stepwise nature of mate-preference behavior is determined by the physical properties of diverse mate-preference signals and the behaviors they elicit. In the scenario shown, a hermaphrodite has been migrating to the right and has recently transitioned from exploratory to dwelling behavior. A trail of ascaroside-containing deposits is indicated. As volatile cues,^, VSPs likely attract males from a distance. Less-diffusible ascaroside cues are thought to be deposited as excrement and are likely non-uniform in distribution. This may allow males to identify regions in which hermaphrodites have recently been present and could trigger a transition out of an exploratory state. Detection and evaluation of peri-mating surface cues likely happens during the initial phase of mating behavior, as male-specific neurons in the male tail contact the mate’s body during scanning behavior. Each phase of mate preference behavior allows males to assess multiple features of potential mates. (B) A table summarizing various categories of potential mates, ordered according to male preference. Because we did not explicitly compare all classes of potential mates to each other, cases where the relative preference order is unknown are indicated with a dashed line. For each category, the role of different classes of mate-preference signals is indicated. “—” indicates that a signal is absent or does not play a detectable role. “?” indicates that the status of a signal is unknown.