Supplemental Material for Carstensen et al., 2021
Creators: name: Carstensen, Heather R.
name: Reinard M. Villalon
name: Navonil Banerjee
name: Hallem, Elissa A.
name: Hong, Ray L.
Publisher: name: GSA Journals
Year: 2021
Subjects: Developmental Biology

description: <b>SM Figure 1. <i>Ppa-hsd-2</i> transcript is absent in <i>csu88</i>.</b> <b>(A)</b> RT-PCR using primers targeting <i>Ppa-hsd-2</i>transcript did not amplify from <i>Ppa-hsd-2</i>(<i>csu88) </i>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>(B)</b> The <i>Ppa-odr-3</i> reaction was used as a positive control for the same <i>csu88 </i>cDNA sample. The expected band size was 303 bp using RHL1097 and RHL1100.<b> </b><b>SM Figure 2. Olfactory responses of Daf-c dauer larvae<i>.</i> (A) </b>Dauer larvae of Daf-c mutants do not respond to insect pheromones known to be attractive to adults (1% <i>E</i>-11-tetradecenyl acetate (E-TDA), 10% hexadecenal, 10% 2-(<i>E</i>)-nonenol) and the plant defense volatile 1% ß-caryophyllene. Sample sizes: <i>dfc-1</i>ETDA = 523, <i>csu60 </i>ETDA = 193, <i>dfc-1</i> hexadecenal = 175, <i>csu60</i> hexadecenal = 147, <i>dfc-1</i> 2-(<i>E</i>)-nonenol = 204, <i>csu60</i> 2-(<i>E</i>)-nonenol = 992, <i>dfc-1</i> ß-caryophyllene = 656, <i>csu60</i> ß-caryophyllene = 278. For each category, 11-24 assays were performed over at least 2 trials. <b>(B)</b> Dauer larvae of Daf-c mutants also do not respond to small molecular weight compounds that are strong attractants for <i>C. elegans</i>: 1% 2,3-butanedione (diacetyl), 1% 2,3-pentanedione, and 1% isoamyl alcohol. *<i>P</i>&lt;0.05, two-way ANOVA with Sidak’s multiple comparisons post-test between <i>tu391</i> and <i>csu60</i>. Sample sizes: <i>dfc-1</i> butanedione = 313, <i>csu60 </i>butanedione = 1658, <i>dfc-1</i> pentanedione = 261, <i>csu60 </i>pentanedione = 176, <i>dfc-1</i> isoamyl alcohol = 243, <i>csu60 </i>isoamyl alcohol = 252. For each category, 11-16 assays were performed over at least two trials. Violin plots show medians with quartiles. <b>SM Figure 3. Amphid neuron marker expressions for two reporters are wildtype in <i>Ppa-hsd-2(csu60)</i></b>. Representative overlay images of adult hermaphrodites expressing the <i>Ppa-odr-7p::rfp</i> reporter in AM9(ADF) and AM7(AWC) neurons of <b>(A) </b>wild-type PS312 and<b> (B) </b><i>hsd-2(csu60)</i> mutant<i>.</i> Overlay images of adult hermaphrodites expressing the <i>Ppa-che-1p::rfp</i> reporter in AM5(ASE) and AM6(ASG) neurons of <b>(C) </b>wild-type PS312 and<b> (D) </b><i>hsd-2(csu60)</i> mutant<i>.</i> Anterior is left and dorsal is up. Scale bars represent each reporter strain: 20 µm. <b> </b><b>SM Figure 4. RT-qPCR results of <i>Ppa-odr-3</i> expression</b>. Over-expression of <i>Ppa-odr-3</i> in <i>hsd-2(csu60)</i> 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.<b> </b><b>SM Figure 5. The effect of temperature on dauer formation in <i>Ppa-hsd-2(csu60)</i> and <i>Ppa-hsd-2(csu88).</i></b><i></i>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 <b>Table 1</b> for details.<b> </b><b>SM Figure 6. J2d are committed to the dauer larvae fate.</b> <i>Ppa-hsd-2(csu60)</i>(N = 6, n = 109) and <i>Ppa-hsd-2(csu88)</i> (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.<b></b>