Figure 2. Methionine supplementation reverses the negative effect of calorie restriction (CR) on small body size of the progenies. (A) Representative image of progenies from worms raised under Ad libido (AL) and CR conditions. AL and CR were achieved by plating 1X1011/mL and 1X108/mL OP-50 bacteria, respectively, on solid nematode growth (NG) medium containing carbenicillin and kanamycin. Parental worms were raised on NG medium supplemented with indicated concentration of methionine (Met) from hatching to day-1 adulthood. Worms were allowed to lay eggs for 2 hours to obtained synchronized embryos. Progenies were raised on NG medium with growing OP-50 to day-1 adulthood and imaged with Leica microscope. (B) CR reduces body size of progenies, which can be mitigated by methionine. Parental worms raised under Ad libido (AL) and CR conditions were treated with 5mM methionine (Met) as in A and progenies were collected and imaged as in A. Data were collected from 2 independent experiments. Body length of 20 worms were measured by using ImageJ and plotted by using Graphpad Prism software. P values were obtained by t-test: ***, P<0.0001. (C) The negative effect of CR on body size of progenies is transient. The first (F1) and second (F2) generations of progenies from parental worms raised under Ad libido (AL) and CR conditions were raised and imaged as in A and measured as in B. Data were collected from 2 independent experiments. P values were obtained by t-test: ns, not significant; ***, P<0.0001.