Fig 1.
Outline of experimental design.
Fresh feces are collected from mice. MCB homogenates are produced by immersing the feces in M9 or PBS buffers, and quantified by bacterial DNA analysis. Bacterial viability is then evaluated quantitatively by resazurin assay (Alamar blue) and qualitatively by the presence of spores (Schaeffer-Fulton staining). Nutritional value of the MCB is evaluated by quantifying energy metabolites (glucose, glycogen, triglycerides, and glycerol). Subsequently, half of MCB extract is inactivated with PFA and the other half is kept alive to allow symbiosis. Alive and inactivated MCB homogenates are then used to characterize phenotypes and biological traits in the nematode C. elegans.
Fig 2.
Bacterial viability of fecal MCB homogenates.
(a) Quantitative evaluation of bacterial viability in MCB homogenates obtained after immersion of fresh feces of male mice in either M9 or PBS over 28 days. Bacterial viability at day 0 (after feces collection and homogenization) is considered as 100%. Mean ± SEM values. Each data point represents the average of three resazurin assays in technical duplicates. Statistical comparisons by ANOVA repeated measures. (b) Representative images of Schaeffer-Fulton staining (100x) at days 0, 1, 7, 14, 21 and 28. This staining allows the qualitative evaluation of bacterial viability by coloring spores, which are an indicator of viability. Spores are green and bacterial vegetative cells are red—pink. Spores are indicated by an arrow on each image.
Fig 3.
Bacterial food quality of MCB and C. elegans food intake and preferences.
(a) Concentrations of triglycerides, glycerol, glucose and glycogen in E. coli OP50 and in MCB homogenates described in Fig 2, as determined based on similar DNA content. Each bar represents the average of three measurements performed in technical duplicates. (b) Schematic illustration of food preference assay between heat-inactivated versus PFA-inactivated microbiota. (c) Percentage of adult worms on and off the bacterial lawn. n = 40 worms/replicate for a total of 120 worms. (d) Schematic illustration of food choice assay between two different bacteria. (e) Percentage of adult worms on and off of the bacterial lawns. n = 40/replicate for a total of 240 worms. (f) Schematic choice assay between alive versus inactivated MCB. (g) Percentage of adult worms on and off the only one bacterial lawn. n = 40 /replicate for a total of 240 worms. (h) Pharyngeal pumping per minute of worms fed with E. coli OP50 or MCB either alive or inactivated. Statistical comparisons by 2 x 2 ANOVA. ns: Not significant.
Fig 4.
Comparative effects of MCB on reproduction and healthspan of C. elegans.
(a) Body length based on relative axial length (Time of flight, TOF) of worms fed E. coli OP50 versus MCB either alive or inactivated for the indicated time points. Each point represents an individual worm. (b) Body size of worms fed E. coli OP50 versus MCB either alive or inactivated. Each point represents an individual worm. (c) Brood size of worms fed E. coli OP50 versus MCB either alive or inactivated. 30 worms were tested individually per group. (d) Percentage of worms showing a Bag phenotype over the reproductive period in worms fed with E. coli OP50 or MCB either alive or inactivated. Data represent the mean ± SEM values on 35 worms/replicate for a total of 105 worms. (e) Percentage of worms showing an Avid phenotype over the reproductive period in worms fed with E. coli OP50 or MCB either alive or inactivated. Data represent the mean ± SEM values on 35 worms/replicate for a total of 105 worms. Statistical comparisons by 2 x 2 ANOVA. ns, no significant. (g) Survival curves of worms fed on E. coli OP50 versus MCB either alive or inactivated. n = 150 per group, see also S4 Table in S1 File).
Fig 5.
Effects of OP50 and MCB on fat accumulation.
(a) Nile red fluorescence relative to length (TOF) in worms fed E. coli OP50 or MCB either alive or inactivated for up to 15 days. Each point represents an individual worm. Statistical comparisons by 2 x 2 ANOVA followed by Bonferroni’s multiple comparison test. ns, no significant. (b) Representative images of Nile red staining at days 3, 9 and 15 days in worms described in (a) Scale bar: 1000 μm. (c) Representative images of Oil red O staining at days 3, 9 and 15 in worms described in (a). Magnification 25x.