DAF-16 dependence of metabolites. A. PCA of metabolic profiles of wild-type, single mutant daf-2(e1370) and daf-16(m26) and double-mutant daf-2(e1370);daf-16(m26) samples. PCA and cluster analysis shows that daf-2(e1370) have the most divergent metabolism of the four genotypes, implying that some of the distinctive features of the daf-2 metabolome is daf-16 dependent. The first three PCs explain, respectively, 31, 21 and 13% of the variance; most of the separation between daf-2 and other genotypes is along PC2. In this experiment mutant and wildype worms were initially raised at 15°C, transferred to 22.5°C at L4, and assayed at 144 hours post bleaching. B. Relative concentrations of 11 metabolites in worms sampled at 22.5°C and 144 hours post hatch. Four metabolites - isoleucine, valine, leucine and phosphocholine - show classical DAF-16 dependence, the rest show more complex patterns of epistasis or none.