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

Abstract

For sexually reproducing animals, selecting optimal mates is essential for maximizing reproductive fitness. Because the nematode C. elegans reproduces mostly by self-fertilization, little is known about its mate-choice behaviors. While several sensory cues have been implicated in males' ability to recognize hermaphrodites, achieving an integrated understanding of the ways males use these cues to assess relevant characteristics of potential mates has proven challenging. Here, we use a choice-based social-interaction assay to explore the ability of C. elegans males to make and optimize mate choices. We find that males use a combination of volatile sex pheromones (VSPs), ascaroside pheromones, surface-bound chemical cues, and other signals to robustly assess a variety of features of potential mates. Specific aspects of mate choice are communicated by distinct signals: the presence of a sperm-depleted, receptive hermaphrodite is likely signaled by VSPs, while developmental stage and sex are redundantly specified by ascaroside pheromones and surface-associated cues. Ascarosides also signal nutritional information, allowing males to choose well-fed over starved mates, while both ascarosides and surface-associated cues cause males to prefer virgin over previously mated hermaphrodites. The male-specificity of these behavioral responses is determined by both male-specific neurons and the male state of sex-shared circuits, and we reveal an unexpected role for the sex-shared ASH sensory neurons in male attraction to endogenously produced hermaphrodite ascarosides. Together, our findings lead to an integrated view of the signaling and behavioral mechanisms by which males use diverse sensory cues to assess multiple features of potential mates and optimize mate choice.

Figure 1.. Mate-choice behavior is male-specific and allows males to distinguish the sex and developmental stage of potential mates.

(A) In their natural environment, C. elegans males encounter adult conspecifics of both sexes, larvae, and other nematodes. Multiple classes of chemical and physical signals have been implicated in C. elegans mate recognition, including surface signals (indicated by the outline of the body), ascaroside pheromones (small colored hexagons), and volatile sex pheromones (VSPs, indicated by gradient shading around the animal). (B) The Mate Choice Assay (MCA). Immobilized targets of two classes are placed in two opposing quadrants; after 30 mins, searchers are placed in the center. Their positions over the next 90 mins are used to calculate a Mate Choice Index (MCI) (see Materials and Methods). (C-E) MCAs with control adult male searchers and (C) adult male vs. adult hermaphrodite targets, (D) male vs. hermaphrodite targets at L3, L4, and adult stages, and (E) L4 vs. adult hermaphrodite targets. (F) MCA with control adult male and hermaphrodite searchers and adult male vs. hermaphrodite targets. In this and all following figures, schematic diagrams show the nature of the cues potentially produced by each target class, with red indicating hermaphrodite and blue indicating male. Each data point represents an individual MCI derived from a MCAs using ~10 searchers; the vertical gray bar indicates the mean MCI. For each tester-target condition, we carried out a Wilcoxon signed-rank test to ask whether the MCI was consistent with the null hypothesis that males had no preference for either targets class (e.g., MCI = 0). Black squares to the right of each row indicate the p values associated with these tests: ■, p ≤ 0.05; ■■, p ≤ 0.005; ■■■, p ≤ 001. To compare MCI values between different classes of searchers, we used a Mann-Whitney test (to compare two genotypes) or a Kruskal-Wallis test with Dunn’s correction (to compare more than two genotypes). Asterisks indicate p values associated with these tests: *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.001. We only carried out tests necessary to evaluate specific pre-established hypotheses. Comparisons made whose outcomes were not statistically significant are shown in gray dotted lines and are labeled “n.s.”.

Figure 2.. Ascaroside pheromones, whose sexual specificity is determined by the intestine, have central roles in sex and stage discrimination.

(A, B) MCAs with control male searchers and daf-22 targets that lack short-chain ascaroside production. (A) Between-sex MCAs with daf-22 adults and L4 larvae. (B) Within-sex MCAs with daf-22 vs. control adult hermaphrodites and males. The break in the vertical axis here and in subsequent panels below indicates that these experiments were not done in parallel, so we chose not to carry out a direct statistical comparison between them. (C, E) Epifluorescence images of mab-3::GFP in control and int^masc hermaphrodites (C) and in control and int^fem males (E). Dashed white lines outline the body; yellow arrows indicate the intestine. (D, F) Within-sex MCAs with control male searchers and control vs. sex-reversed intestine targets with or without short-chain ascarosides. (D) int^masc vs. control hermaphrodites in a wild-type and daf-22 mutant background. (F) int^fem vs. control males in a wild-type and daf-22 mutant background.

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

(A, B) MCAs with control male searchers and (A) Vul lin-39 vs. control hermaphrodites and (B) Vul lin-39 vs. control hermaphrodites in the absence of short-chain ascarosides. (C-E) MCAs with control male searchers and (C) within-sex comparisons of bus-2 vs. control hermaphrodites and males; (D) between-sex comparisons using control targets, bus-2 targets, daf-22; bus-2 targets, and daf-22 targets; and (E) within-sex comparisons of daf-22; bus-2 vs. daf-22 and daf-22; bus-2 vs. bus-2 targets. (F) 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.

Figure 4.. Volatile sex pheromones (VSPs) that signal fertility status are powerful mate choice signals.

(A-D) MCAs with wild-type and srd-1 mutant male searchers. (A) Between-sex MCAs with daf-22; bus-2 targets. (B) Within-sex MCAs with glp-1 vs. control targets. (C) Within-sex MCAs with daf-22; glp-1 vs. daf-22 targets. (D) Within-sex MCAs with daf-22; glp-1 vs. glp-1 targets.

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

(A,B) MCAs with control male searchers and food-deprived hermaphrodite targets. (A) 4-hr and 28-hr nutrient-deprived vs. control hermaphrodite targets. (B) 4-hr and 28-hr nutrient-deprived vs. control hermaphrodite targets, with all targets carrying a daf-22 mutation. (C-I) MCAs with male searchers and virgin or previously mated hermaphrodite targets. (C) Control male searchers with previously mated vs. virgin young adult hermaphrodites. (D) Control and srd-1 mutant male searchers with mated vs. virgin hermaphrodites. (E) Control male searchers with mated vs. virgin hermaphrodites, either in a wild-type or daf-22 background. (F) Control male searchers with mated vs. virgin bus-2 hermaphrodites. (G) Control male searchers with Vul lin-39 targets previously exposed to males (“mated”) vs. control targets exposed to unc-54 males than cannot mate (“virgin”). (H) Control male searchers with mated vs. virgin targets that had not (control) or had been briefly washed before testing. (I) Control male searchers with washed vs. control targets, either virgin or mated.

Figure 6.. The male state of shared neural circuits is necessary and sufficient for aspects of male mate-choice behavior.

(A-C) MCAs with control male, neuro^fem male, control hermaphrodite, and neuro^masc hermaphrodite searchers. (A) Between-sex MCAs with control male and hermaphrodite targets. (B) Within-sex MCAs with daf-22 and control hermaphrodite targets. (C) Between-sex MCAs with daf-22 male and hermaphrodite targets. (D) MCA with control and neuro^fem male searchers and daf-22; bus-2 vs. daf-22 hermaphrodite targets.

Figure 7.. The sex-shared ADF and ASH neurons have key roles in ascaroside-mediated mate-choice decisions.

(A-E) MCAs with daf-22 vs. control hermaphrodite targets. (A) MCAs with control and sens^fem male searchers. (B) MCAs with control and ASK-ablated male searchers. Strains used in this panel contained him-8(e1489) rather than him-5(e1490). (C) MCAs with control and ADL-ablated male searchers. (D) MCAs with control searchers and those with ablation of AWB; AWE and AWC; or ASI. (E) MCAs with control, ADF-ablated, and ASH-ablated male searchers. (F) Quadrant chemotaxis assay for ascr#3 attraction using control or ASH-ablated males.

Figure 8.. C. elegans males to assess multiple features of potential mates using a hierarchical behavioral strategy that incorporates multimodal sensory cues.

(A) The hierarchy of mate-choice behavior is dictated by the nature of these diverse signals and the mechanisms by which they are detected. In this scenario, a hermaphrodite has been migrating to the right and has recently transitioned from roaming to dwelling behavior. A trail of ascaroside-containing deposits is indicated. As volatile cues,^, VSPs likely attract males from a distance; this is probably most relevant for older, sperm-depleted hermaphrodites “advertising” for males. Less-diffusible ascaroside cues are thought to be deposited as frass and are likely non-uniform in distribution. These may allow males to identify regions in which hermaphrodites have recently been present and could trigger a transition to a local-search-like state. Detection and evaluation of 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 of these three phases of mate-detection carries allows the male to assess specific subsets of the features of potential mates. (B) A table summarizing various categories of potential mates, ordered according to male preference. For each category, the role of different classes of mate-choice signals is indicated. “—” indicates that this signal is absent or does not play a detectable role in mate-choice. “?” indicates that the status of this signal is unknown.