Overall effect of different rearing temperatures on the masculinization rate in Experiment A (pool of four different <i>mal</i>-carrying progeny).

<p>Panel A: Individual masculinization rates (each individual is considered masculinized when at least one gonad is masculinized) different temperature treatments (8, 12 and 18°C) (in percentage ± Confidence Interval at p = 0.05; χ²; *** p<0.001). Panel B: Masculinization rates of left versus right gonads following different temperature treatments (8, 12 and 18°C) (in percentage ± Confidence Interval at p = 0.05; χ²; *** p<0.001). Numbers of animals analyzed at 8°C n = 357, 12°C n = 386 and 18°C n = 375.</p

Rates of masculinization of the different <i>mal-carrying</i> progeny exposed to different temperatures in experiment A.

<p>Panels A to D: Individual masculinization rates (each individual is considered masculinized when at least one gonad is masculinized) following different temperature treatments (8, 12 and 18°C). Panels E to H: Masculinization rates of left versus right gonads following different temperature treatments (8, 12 and 18°C) (in percentage ± Confidence Interval at p = 0.05%+CI; χ²; * p<0.05, ** p<0.01, *** p<0.001). Numbers of animals analyzed at 8, 12 and 18°C, respectively: mal1  = 99, 108 and 99; mal2  = 92, 101 and 101; mal3 = 91, 87 and 85; mal4 = 75, 90 and 90.</p

Experimental conditions for the common garden experiment A.

<p>Eggs from four <i>mal</i>-carrying progeny were incubated at 10°C until hatching and were then shifted to the 8, 12 or 18°C temperature treatment. At first feeding, the total number of fish per progeny was reduced to 110 and all four progeny were mixed for each temperature condition. At the end of the temperature treatment, the fish were transferred and maintained at 12°C (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113355#s2" target="_blank">Materials and Methods</a> section for more details).</p

Overall mean survival rates and assignment success according to temperature treatments in experiment A.

<p>* : mortality significantly higher at 8°C (<i>P<0.05</i>).</p><p>Overall mean survival rates and assignment success according to temperature treatments in experiment A.</p

Overall mean frequency of the different gonadal phenotypes according to temperature treatments in experiment A.

1<p>: Glimmix analysis performed with for each phenotype the complete set of data (3 temperatures, 4 families) on the logit scale assigning binary gonadal phenotypes (1 =  individual with the target phenotype, 0 =  other individuals) and temperature and family effects modeled as fixed and random effects respectively. Within line: different subscripts (a, b) indicate significant differences among temperatures for the proportion of the gonadal phenotype (paired comparisons of temperature using Glimmix analysis, <i>P<0.05</i>).</p><p>Overall mean frequency of the different gonadal phenotypes according to temperature treatments in experiment A.</p

Effect of temperature treatment on the masculinization rate in Experiment B.

<p>Intersex, male: number of each phenotype in the family; n_T: total number of fish in the family;</p>1<p>χ² test (df = 1) for effect of temperature on total masculinization rate within each family;</p>2<p>results of Glimmix analysis performed with the whole set of data (2 temperatures, 3 families) on the logit scale assigning binary gonadal phenotypes (1 =  intersex or males, 0 =  females) and temperature and family modeled as fixed and random effects respectively.</p><p>Effect of temperature treatment on the masculinization rate in Experiment B.</p

Comparative description of mammalian cases of SLC25A46 mutations.

<p>The age of onset refers to the onset of neurological symptoms. ND, non-detected; NA, non-analyzed.</p

MS/MS results (protein extracts from brain tissues).

<p>The MS/MS data were analyzed for three WT and four Tg^-/- mice. Downregulated and upregulated proteins were selected based on the adjusted p-value (threshold = 0. 2). Total MS/MS data are provided in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006597#pgen.1006597.s010" target="_blank">S5 Table</a></b>.</p

Biochemical analysis of plasma from WT and homozygous Tg^-/- mice (n≥7 for each genotype).

<p>For glucose and bile acids, measures were repeated on another group of mice.</p

Construction of two mouse lines with disruption of <i>Slc25a46</i>.

<p><b>(A)</b> Schematic diagram of the <i>Slc25a46</i> gene in mice, located on chromosome 18, with positions of mutations in Tg18 and Tg26 lines (arrows). <b>(B)</b> Total proteins were extracted from brain, muscle and liver of WT, Tg18 homozygous and Tg26 homozygous mice. Samples were analyzed by immunoblotting, with antibodies against mitochondrial proteins Slc25a46 and Cox2 (internal loading control). <b>(C)</b> Proteins were extracted with a Mitochondria Isolation kit from brains of WT and Tg18 mice. Samples were analyzed by immunoblotting with antibody against the mitochondrial proteins Slc25a46 and Cox2. WT, Wild-Type; Aff, affected. Note: entire Western Blots with SLC25A46 antibody are shown in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006597#pgen.1006597.s005" target="_blank">S5 Fig</a></b>.</p