Figure 3.

Vital Dye Method Demonstrates Adult-Specific Density Effect on Mouth Form

(A–F) (A) The wild isolate RSC017 grown in standard conditions (5 young adults passed to fresh plates, progeny phenotyped 4 days later) are highly stenostomatous (<10%, n = 102). Mouth form ratios of neutral red-stained J2s (B–D) and dauers (E and F), with increasing number of CellTracker Green-stained competitors (total number of animals n > 100 per experiment, with 3–5 independent biological replicates for J2 and adult crowding, and 2 for J3/J4s). Overall significance between strain and age was determined by a binomial linear regression (see Transparent Methods), and pairwise comparisons were assessed by Fisher's exact test on summed Eu counts (^∗∗∗p < 0.001, **p < 0.01, *p < 0.05). Mouth forms were phenotyped at 40–100× on a Zeiss Axio Imager 2 light microscope.

(G and H) (G) Percent reaching adulthood and percent Eu of those that reached adulthood (H) after increasing numbers of J2s were added to standard 6-cm Nematode Growth Media (NGM) agar plates with 300 μL OP50 E. coli bacteria (n = 2 biological replicates, with total n > 200 for percent reaching adulthood, and total n > 100 for mouth form. Significance was determined by a binomial regression; Error bars represent standard deviation of the two biological replicates).