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. 2015 Jun 25;2:172–178. doi: 10.1016/j.bbrep.2015.06.007

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© 2015 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 1 — Effect of fat-5 and/or cyp-35A2 mutation on life history traits and Nile Red positive compartments. (A and B) Developmental changes were followed from 24 h post-hatch until 144 h. Length (A) and body surface area (B) were measured every 24 h, in wild-type, fat-5(tm420), cyp-35A2-(gk317) and fat-5(tm420);cyp-35A2(gk317) nematodes. The overall length was significantly reduced upon double deletion of fat-5 and cyp-35A2 whereas the overall length of fat-5(tm420) and cyp-35A2(gk317) was not statistically different from wild-type nematodes. The surface area was significantly (*p<0.05) affected in fat-5(tm420) mutants or cyp-35A2(gk317) mutants and resulted in a considerable reduction. The overall decrease in volumetric growth of fat-5(tm420);cyp-35A2 was highly significant (***p<0.001) compared to wild-type and single mutants. Each data represents the average surface area of 20 individual nematodes taken from two biological repeats±the standard error of mean (SEM). (C) Survival rate of wild-type, fat-5(tm420), cyp-35A2(gk317) and the double mutant were compared. Median lifespan and the maximum survival of each strain were determined, and graphs were plotted by the Kaplan–Meier method followed by a Log-Rank (Mantel–Cox) test with 95% significance (GraphPad Prism v5). The median lifespan (50% alive) and maximum lifespan in the double knockout nematodes was significantly extended in comparison to the wild-type as well as the corresponding single knockout strains. Each experimental group of the nematodes was started with a population of 400 age-matched worms and if the nematode was lost/died before day eight of adulthood, the individual nematode was censored from further analysis. (D) The Nile Red staining profile was analyzed in all four age-matched strains at 72 h post-hatch. Quantitative analyses using ImageJ software revealed that the red fluorescent intensity was lower in fat-5(tm420) in comparison to the wild-type, yet this reduction was insignificant. In cyp-35A2(gk317) the relative fluorescent intensity was decreased with a statistical significance (*p<0.05). The fat-5(tm420);cyp-35A2(gk317) double knockout exhibited an analogous, statistically even more significant (***p<0.001) reduction in the relative fluorescent intensity. Each bar graph represents the mean Nile Red intensity measured from 10 worms±the standard error of the mean (SEM). A one-way ANOVA was applied to define the statistical significance.