Fig 1.
Male scent, likely mediated by ascaroside pheromones, facilitates recovery of fecundity after heat stress.
(A) The protocol for heat stress and recovery experiments is summarized. At 48 hours post L1 arrest (young adulthood) hermaphrodites were singled onto either control or scented plates. Unless otherwise indicated, worms were exposed to scent during both stress and recovery. (B) The fraction of hermaphrodites that recover fecundity after 24 hours of heat stress at 29°C. A schematic is depicted above each condition. **P < 0.01, ***P < 0.001. P-values were calculated using binomial test and Bonferroni corrected for multiple comparisons. Error bars are ±SD among separate trials. See S1 Table for raw experimental data including numbers of independent trials and worms tested in each trial.
Fig 2.
Different ratios of two ascarosides have distinct effects on reproductive recovery from stress.
The effect of single ascarosides and cocktails of two ascarosides on the recovery of fecundity after heat stress. *P < 0.05, ***P < 0.001. P-values were calculated using binomial test and Bonferroni corrected for multiple comparisons. Error bars are ±SD among separate trials. See S1 Table for raw experimental data including numbers of independent trials and worms tested in each trial. The dashed line marks the average recovery fraction with male scent (the value shown in Fig 1B). Ethanol on the control plates made recovery somewhat higher than in Fig 1B.
Fig 3.
The male pheromone signal is conserved and affects brood size of gonochoristic Caenorhabditis nematodes.
(A) Fraction of C. elegans hermaphrodites that recover self-fecundity after heat stress on plates with male scent of three related species. See S1 Table for raw experimental data including numbers of independent trials and worms tested in each trial. Recovery on control and C. elegans male-scented plates are given for comparison (shown as white bars); these data are the same as shown in Fig 1B. Error bars are ±SD among separate trials. (B) Brood sizes of C. remanei females exposed to male scent for 16 hours were significantly higher than naïve females after 10-minute matings (at 20°C) (P = 9.5 x 10−3, Kolmogorov-Smirnov test). Red lines mark the median values. Means are: control = 160.1 and male-scented = 205. See S2 Table for numbers of independent trials and worms tested in each trial.
Fig 4.
Effects of male scent on brood sizes of selfing hermaphrodites at 20°C and after recovery from heat stress.
(A) Self-brood sizes of C. elegans hermaphrodites raised at 20°C with or without C. elegans male scent. Broods from 24 hermaphrodites were determined for each condition. Red lines mark the median values. Means are: control = 285.3 and male-scented = 277.3. They are not significantly different (P = 0.99, Kolmogorov-Smirnov test). (B) Brood sizes for hermaphrodites recovering from heat stress alone (i.e. recovery is due to self-fertilization) on control or male-scented plates. The animals whose brood sizes were counted were the same as whose recovery is shown in Fig 1B (see S1 Table for raw experimental data). Only worms with offspring were considered. Red lines mark the median values. Means are: control = 2.3 and male-scented = 2.6. They are not significantly different (P = 0.27, Kolmogorov-Smirnov test). See S2 Table for numbers of independent trials and worms tested in each trial.
Fig 5.
Male scent enhances sperm guidance following stress.
Schematic drawing of the gonad of a hermaphrodite stressed at 29°C (A) and unstressed control (B). Representative images of worm gonads (with mCherry-labeled sperm) on control plates after heat stress (C) or at 20°C (D) and on male-scented plates after heat stress (E) or at 20°C (F). For all photographs, anterior is to the left and ventral is down. Quantification of sperm distribution in heat stressed animals grown on control vs. male-scented plates (G) and in unstressed animals (20°C) grown on control vs. male-scented plates (H). Red lines mark the median values. The ratios are significantly different in G (P = 1.3 x 10−4, Kolmogorov-Smirnov test), but not in H (P = 0.79, Kolmogorov-Smirnov test). Red lines mark the median values. Means in G are: control = 0.4 and male-scented = 2.1. Means in H are: control = 2.3 and male-scented = 2.4. See S2 Table for numbers of independent trials and worms tested in each trial.
Fig 6.
ascr#10 and ascr#3 have different effects on the C. elegans reproductive system.
(A) Sperm guidance in heat-stressed hermaphrodites on plates with individual ascarosides and ascaroside cocktails. Sperm guidance on plates with male cocktail (P = 3.1 x 10−3, Kolmogorov-Smirnov test Bonferonni corrected for four comparisons) and ascr#10 (P = 4.3 x 10−3, Kolmogorov-Smirnov test Bonferonni corrected for four comparisons) were significantly different from control. Red lines mark the median values. Means are: control = 1.1, male cocktail = 3.2, hermaphrodite cocktail = 1.2, ascr#10 = 2.8, and ascr#3 = 1.3. (B) Representative photographs of heat-stressed hermaphrodites during recovery. Hermaphrodites were monitored during recovery for the clearance of large concretions formed in the uterus during heat stress. Worms were examined at 48, 72, and 96 hours of recovery–that is, the time corresponding to when most of recovery occurs (See S8 Fig). Worms on plates with ascr#10 were significantly worse at clearing large concretions from the uterus (P = 8.7 x 10−3, binomial test Bonferroni corrected for two comparisons). In both (A) and (B) individual ascarosides were at 10 fmol. Male and hermaphrodite ascaroside cocktails were as in Fig 2 (1.92 fmol ascr#3 + 7.2 fmol ascr#10 and 6.0 fmol ascr#3 + 1.68 fmol ascr#10, respectively). See S2 Table for numbers of independent trials and number of worms tested in each trial.
Fig 7.
DAF-7 function in ASI neurons is necessary and sufficient for improved reproductive recovery caused by the male scent.
The fraction of hermaphrodites that recover fecundity after 24 hours of heat stress at 29°C. P = 4.2 x 10−8, binomial test. Error bars are ±SD among separate trials. See S1 Table for raw experimental data including numbers of independent trials and worms tested in each trial.