Ethosuximide enhanced <i>daf-2(e1370)</i> L1 arrest.

∞<p>This value includes arrested L1 larvae and eggs that failed to hatch, as described by Gems et al. (1998) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000230#pgen.1000230-Gems1" target="_blank">[55]</a>. Unhatched eggs were only a small proportion of this total. Statistical comparisons are to the same genotype with no drug treatment. Numbers with no asterisks are not significant (P>0.05); *, P<0.05; **, P<0.005; ***, P<0.0001. P values determined by Student T-test. The 47.5 value in line four is significantly larger than the 2.3 value in line eight (P<0.0001).</p

<i>osm-3</i> mutants were long-lived and failed to respond to lifespan extension by ethosuximide.

<p>Wild-type (WT) or <i>osm-3(p802)</i> hermaphrodites were cultured with 0 mg/ml or with 4 mg/ml ethosuximide (+ETH 4) from conception until death. Day 0 represents the L4 stage.</p

Ethosuximide treated animals and <i>che-3(am178)</i> mutants cultured at 20°C did not display dye-filling defects.

<p>Animals were incubated in the lipophilic dye DiO and observed using fluorescence microscopy at 200× magnification. Each panel illustrates the anterior tip of the animal (top) to the base of the bi-lobed pharynx (bottom). DiO stains amphid neuron processes (arrows) and cell bodies (arrowheads). Wild-type (WT) animals displayed robust staining that was not affected by treatment with 4 mg/ml ethosuximide (WT+ETH4). <i>che-3(am178)</i> mutants cultured at 20°C displayed robust staining, whereas <i>osm-3(p802)</i> mutants displayed no detectable staining (the Dyf phenotype).</p

Ethosuximide disrupted chemotaxis toward the volatile attractants isoamyl alcohol and diacetyl.

<p>Wild-type animals grown from conception to adulthood on NGM plates containing 2 mg/ml ethosuximide or no drug were transferred to the center of a 10 cM chemotaxis plate containing 4 mg/ml of ethosuximide or no drug, respectively. A chemotaxis index (CI) to isoamyl alcohol or diacetyl was determined. (A) Chemotaxis to isoamyl alcohol. The mean CI±SD for untreated and ethosuximide treated animals was 0.8±0.2 and 0.3±0.2, respectively (p<0.0001, n = 8) (Student T-test). (B) Chemotaxis to diacetyl. The mean CI±SD for untreated and ethosuximide treated animals was 0.8±0.14 and 0.2±0.2, respectively (p = 0.0009, n = 6) (Student T-test). Error bars represent standard error of mean.</p

The <i>che-3(am178)</i> mutation caused temperature-sensitive defects in DiO staining of amphid neurons.

∞<p>For lines 1–4, hermaphrodites were cultured from conception to L4 at the indicated temperature. For lines 5–8, hermaphrodites were cultured from conception to L4 at 20°C and then shifted to the indicated temperature for approximately 24 hours.</p

Positioning the <i>am178</i> mutation.

<p>The horizontal line represents a portion of Chromosome I. Genes that can be mutated to cause visible phenotypes and SNP markers are shown above; map units are shown below. From <i>dpy-5 am178</i>/CB4856 hermaphrodites, we selected 56 ethosuximide resistant, non-Dpy self-progeny. From <i>am178 unc-75</i>/CB4856 hermaphrodites, we selected 72 ethosuximide resistant, non-Unc self-progeny. An analysis of SNP markers in these strains positioned the recombination events in the intervals shown below. The double-headed arrow indicates a 250 kBp interval between the SNP markers <i>uCE-952</i> and <i>snp_T01G9</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000230#pgen.1000230-Collins1" target="_blank">[<i>1</i>]</a> that contains <i>am178</i>.</p

Ethosuximide treatment extended lifespan and stimulated egg laying in <i>cca-1</i> null-mutants.

<p>(A) Ethosuximide extended the lifespan of <i>cca-1(gk30)</i> mutants. Wild-type or <i>cca-1(gk30)</i> hermaphrodites were cultured with 0 mg/ml or 4 mg/ml ethosuximide (+ETH 4) from conception until death. The mean lifespans in days±SD for WT, WT+ETH 4, <i>cca-1</i>, and <i>cca-1</i>+ETH 4 were 16.1±3.3 (n = 110), 19.5±4.8 (n = 120), 15.2±4.9 (n = 140), and 20.8±6.6 (n = 110), respectively. The differences in mean lifespan of WT compared to WT+ETH 4 and <i>cca-1</i> compared to <i>cca-1</i>+ETH 4 were statistically significant, p<0.0001 (Student T-test). (B) <i>cca-1</i> mutants responded to ethosuximide stimulated egg-laying. The mean number of eggs laid±SD by wild-type animals (blue circles) was 0.0±0.0, 0.7±2.2, 2.0±2.5, and 6.8±5.2 for ethosuximide concentrations of 0, 4, 8, and 12 mg/ml, respectively. The mean number of eggs laid±SD by <i>cca-1(ad1650)</i> mutant animals (red squares) was 0.0±0.0, 0.25±1.0, 2.7±3.1 and 8.0±5.6 for ethosuximide concentrations of 0, 4, 8, and 12 mg/ml, respectively. n>23 for all trials. Differences in egg-laying between WT and <i>cca-1</i> at ethosuximide concentrations of 0, 4, 8 and 12 mg/ml were not statistically significant, p>0.05 (Student T-test).</p

Ethosuximide stimulated dauer formation in a low-density, well-fed population at 27°C.

***<p>p<0.0001, comparisons are to the same genotype with no drug treatment. p values determined by Student T-test.</p

Mutants with defects in cilia structure were resistant to ethosuximide toxicity.

∞<p>Animals cultured at 20°C were classified as normal (+) or defective (Dyf) for amphid neuron dye filling (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000230#s4" target="_blank">Materials and Methods</a>).</p>#<p>Experiments were done with 12 mg/ml ethosuximide. The differences in percent resistance between <i>che-3</i> alleles were not statistically significant except that <i>che-3(am162)</i> was significantly different from <i>che-3(am178)</i>, <i>che-3(am165)</i> and <i>che-3(e1124)</i> (p<0.0001). The differences in percent resistance between <i>osm-3</i> alleles were not statistically significant except that <i>osm-3(p802)</i> was significantly different from <i>osm-3(am161)</i> and <i>osm-3(am177) (p<0.0001)</i>.</p>†<p>The dye-filling phenotype of these mutants was described previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000230#pgen.1000230-Perkins1" target="_blank">[28]</a>.</p>*<p>These values may underestimate the fraction of animals that were resistant to ethosuximide, since we observed multiple animals that were mature, indicating they were ethosuximide resistant, but they were desiccated on the side of the dish and not included in the data. We have observed that mutants with severe chemotaxis defects have a propensity to leave the agar surface.</p

<i>am177</i> is a missense mutation of <i>osm-3</i>.

<p>(A) A schematic of the OSM-3 protein showing amino acid numbers (below) and four functional regions: the motor (left diagonal lines), neck (shaded), rod (open) and tail (right diagonal lines) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000230#pgen.1000230-Snow1" target="_blank">[47]</a>. The position of <i>am177</i> and six previously characterized mutations are shown above with the mutation name and molecular change <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000230#pgen.1000230-Snow1" target="_blank">[47]</a>. (B) The predicted amino acid sequence of OSM-3 (M02B7.1B). The <i>am177</i> mutation changes codon 329 from AAC (N) to CAC (H), affecting the neck region of the protein.</p