Supplemental Material for Carstensen et al., 2021
SM Figure 1. Ppa-hsd-2 transcript is absent in csu88. (A) RT-PCR using primers targeting Ppa-hsd-2transcript did not amplify from Ppa-hsd-2(csu88) mixed-stage cDNA, but did amplify from PS312 cDNA. The expected band size was 832 bp using RHL927 and RHL930. For no RT controls, RNA was processed without reverse transcriptase. (B) The Ppa-odr-3 reaction was used as a positive control for the same csu88 cDNA sample. The expected band size was 303 bp using RHL1097 and RHL1100.
SM Figure 2. Olfactory responses of Daf-c dauer larvae. (A) Dauer larvae of Daf-c mutants do not respond to insect pheromones known to be attractive to adults (1% E-11-tetradecenyl acetate (E-TDA), 10% hexadecenal, 10% 2-(E)-nonenol) and the plant defense volatile 1% ß-caryophyllene. Sample sizes: dfc-1ETDA = 523, csu60 ETDA = 193, dfc-1 hexadecenal = 175, csu60 hexadecenal = 147, dfc-1 2-(E)-nonenol = 204, csu60 2-(E)-nonenol = 992, dfc-1 ß-caryophyllene = 656, csu60 ß-caryophyllene = 278. For each category, 11-24 assays were performed over at least 2 trials. (B) Dauer larvae of Daf-c mutants also do not respond to small molecular weight compounds that are strong attractants for C. elegans: 1% 2,3-butanedione (diacetyl), 1% 2,3-pentanedione, and 1% isoamyl alcohol. *P<0.05, two-way ANOVA with Sidak’s multiple comparisons post-test between tu391 and csu60. Sample sizes: dfc-1 butanedione = 313, csu60 butanedione = 1658, dfc-1 pentanedione = 261, csu60 pentanedione = 176, dfc-1 isoamyl alcohol = 243, csu60 isoamyl alcohol = 252. For each category, 11-16 assays were performed over at least two trials. Violin plots show medians with quartiles.
SM Figure 3. Amphid neuron marker expressions for two reporters are wildtype in Ppa-hsd-2(csu60). Representative overlay images of adult hermaphrodites expressing the Ppa-odr-7p::rfp reporter in AM9(ADF) and AM7(AWC) neurons of (A) wild-type PS312 and (B) hsd-2(csu60) mutant. Overlay images of adult hermaphrodites expressing the Ppa-che-1p::rfp reporter in AM5(ASE) and AM6(ASG) neurons of (C) wild-type PS312 and (D) hsd-2(csu60) mutant. Anterior is left and dorsal is up. Scale bars represent each reporter strain: 20 µm.
SM Figure 4. RT-qPCR results of Ppa-odr-3 expression. Over-expression of Ppa-odr-3 in hsd-2(csu60) is ~10-fold more abundant than wild-type PS312 (arbitrarily set as one). Data represents 12 technical replicates from two biological RNA extraction replicates. Error bars denote s.e.m.
SM Figure 5. The effect of temperature on dauer formation in Ppa-hsd-2(csu60) and Ppa-hsd-2(csu88).Synchronized eggs from 20°C were transferred to the designated temperatures and scored for pre-dauer J2d, dauer, or J3 larvae. Census was taken on both day 6 and day 11 at 15°C due to the slow development. Wild-type PS312 do not form any dauer larvae under any of these temperatures in the presence of food. Error bars represent s.e.m. See SI Table 1 for details.
SM Figure 6. J2d are committed to the dauer larvae fate. Ppa-hsd-2(csu60)(N = 6, n = 109) and Ppa-hsd-2(csu88) (N = 5, n = 127) J2d larvae with dark intestine lacking radial constriction and buccal cap were picked onto new plates with food and scored 24 and 48 hours later. Error bars represent s.e.m.