Bacterial diets differentially alter lifespan and healthspan trajectories in C. elegans - PubMed (original) (raw)
Bacterial diets differentially alter lifespan and healthspan trajectories in C. elegans
Nicole L Stuhr et al. Commun Biol. 2020.
Abstract
Diet is one of the more variable aspects in life due to the variety of options that organisms are exposed to in their natural habitats. In the laboratory, C. elegans are raised on bacterial monocultures, traditionally the E. coli B strain OP50, and spontaneously occurring microbial contaminants are removed to limit experimental variability because diet-including the presence of contaminants-can exert a potent influence over animal physiology. In order to diversify the menu available to culture C. elegans in the lab, we have isolated and cultured three such microbes: Methylobacterium, Xanthomonas, and Sphingomonas. The nutritional composition of these bacterial foods is unique, and when fed to C. elegans, can differentially alter multiple life history traits including development, reproduction, and metabolism. In light of the influence each food source has on specific physiological attributes, we comprehensively assessed the impact of these bacteria on animal health and devised a blueprint for utilizing different food combinations over the lifespan, in order to promote longevity. The expansion of the bacterial food options to use in the laboratory will provide a critical tool to better understand the complexities of bacterial diets and subsequent changes in physiology and gene expression.
Conflict of interest statement
The authors declare no competing interests.
Figures
Fig. 1. Characterization of bacterial diets.
a Bacterial streak plate with the six bacterial diets fed to C. elegans. b Growth curves of the bacteria with antibiotics, with optical density measurements every hour for 12 h and another measurement taken at 24 h. c Principal component analysis (PCA) of metabolite concentrations in the different bacterial diets. Bacteria were collected during the log phase of growth for bomb calorimetry (water content) and metabolite kits.
Fig. 2. Gene expression analysis of L4 C. elegans on each bacterial food source after 30 generations.
a Volcano plot of all differentially expressed genes in each food relative to OP50-reared worms. All genes considered to be significant have a p value < 0.01. b–f Volcano plots of each individual food with all significant genes that are differentially expressed. g Venn diagram of all significant genes. Shows the number of genes shared between two, three, four, and five of the bacterial diets, along with the number of genes unique to each bacterium. h Heat map contains genes that are shared between all five bacterial diets. Gene Ontology (GO) terms for uniquely expressed genes are noted below and split between genes that were upregulated and downregulated. More information for the GO term enrichment analysis of RNAseq data in larval stage 4 animals can be found in Supplementary Data 1 and 2.
Fig. 3. Developmental timing of C. elegans is dependent upon bacterial diet.
a The molting reporter strain _mgls49_[_mlt-10p::gfp-pest ttx-3p::gfp_]IV allows for the visualization of molting between larval stages of development into adulthood. The time between peaks represents the period at each larval stage, while time between troughs represent time spent molting. Analysis was carried out after this strain was raised on each bacterial food for 20 generations. b–f Developmental timing of worms raised on each food relative to OP50. All diets showed accelerated development into adulthood when compared to worms raised on OP50. g Volcano plot showing how many of the differentially expressed genes are related to development. The number of genes on each bacterial food are labeled in the legend. All genes have a p value < 0.01. h Heat map of development genes that are significant with a p value < 0.01 in at least one of the five bacterial diets, relative to the OP50 bacterial diet.
Fig. 4. Fat content and distribution vary depending on the food C. elegans are exposed to.
a–f Nile Red staining of L4 worms, scale bar 50 μm. HT115 (b) and Orange (e) have significantly higher fat content, while HB101 (c), Red (d), and Yellow (f) have lower fat contents. g Quantification of Nile Red staining with comparisons made to the OP50 control. GFP fluorescence is measured and then normalized to area before being normalized to OP50. Statistical comparisons by Tukey’s multiple comparison test. ***p < 0.001; ****p < 0.0001. h–m Oil Red O lipid staining in day 3 adult C. elegans. OP50, HT115, HB101, Red, and Orange show similar lipid distribution of fat throughout the animal (representative image, scale bar 50 μm). m Yellow-reared worms display age-dependent somatic depletion of fat (Asdf) with loss of fat in the intestine, while fat is retained in the germline (representative image). n Quantification of lipid distribution across tissues. o Pumping in day 1 adult worms is not significantly different between any of the bacterial foods and OP50. p Food intake of C. elegans raised on each bacterial food in liquid nematode growth media. q Volcano plot of differentially expressed metabolism genes in all bacterial diets compared to OP50. All genes have a p value < 0.01. r Heat map of metabolism genes that are significant with a p value < 0.01 in at least one of the five bacterial diets, relative to the OP50 bacterial diet. All studies were performed in biological triplicate.
Fig. 5. C. elegans raised on Red and Yellow have decreased reproductive output.
a Hermaphrodites have reduced brood size when fed Red or Yellow bacteria. b Reproductive timing is also altered, revealing differences between all bacterial diets and OP50 in total output each day of their reproductive span. HB101 and Red halt output of viable progeny before OP50, HT115, Orange, and Yellow. c OP50 and Red hermaphrodites mated with OP50 or Red males yield similar number of progeny. d Yellow has similar number of unfertilized oocytes compared to OP50. HT115, HB101, Red, and Orange have significantly fewer unfertilized oocytes. Statistical comparisons by Tukey’s multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. e Volcano plot of differentially expressed reproduction genes on all bacterial foods compared to OP50. The number of significant genes have a p value of <0.01. f Heat map of reproduction genes that are significant with a p value < 0.01 in at least one of the five bacterial diets, relative to the OP50 bacterial diet.
Fig. 6. Lifespan of C. elegans on each bacterial diet.
a–e Lifespan comparisons of OP50 versus each bacterial diet. HB101 (a) had a very small significant difference in lifespan compared to OP50. HT115 (b), Red (c), and Yellow (d) worms had increased lifespans, and Orange (e) worms had a shorter lifespan. Lifespan comparisons between OP50-reared worms and the other diets made with log-rank test. For lifespan quartile comparisons, refer to Supplementary Data 2. OP50 n = 155, HT115 n = 51, HB101 n = 53, Red n = 113, Orange n = 122, and Yellow n = 105. f Volcano plot of differentially expressed genes related to lifespan and stress on all bacterial foods compared to OP50. The number of significant genes have a p value of <0.01. g Heat map of lifespan and stress-related genes that are significant with a p value < 0.01 in at least one of the five bacterial diets. h Volcano plot of differentially expressed genes related to dietary restriction on all bacterial foods compared to OP50. The number of significant genes have a p value of <0.01. i Heat map of dietary restriction-related genes that are significant with a p value < 0.01 in at least one of the five bacterial diets, relative to the OP50 bacterial diet.
Fig. 7. C. elegans have a food-dependent decline in thrashing with age.
a–e The rate of thrashing in worms raised on each bacterial diet at different developmental stages. All comparisons were made to OP50-reared worms. a All foods except for Orange showed a significantly higher thrashing rate at the L4 stage. b All foods except for Orange showed a significantly higher thrashing rate at the day 1 adult stage. c HT115 and Orange thrashed significantly less than OP50, while the other bacterial diets were similar in thrashing rate at the day 3 adult stage. d Red and Orange had significantly lower thrashing rates compared to OP50, and all other bacterial foods at the day 8 adult stage. e Red, Orange, and Yellow had lower thrashing rates at day 11 adult when compared to OP50. Statistical comparisons by Tukey’s multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. f Average rate of thrashing over time on each bacterial diet. All studies performed in biological triplicate; refer to Supplementary Data 3 for n for each comparison. g Volcano plot of differentially expressed locomotion genes on all bacterial foods compared to OP50. The number of significant genes have a p value of <0.01. h Heat map of locomotion genes that are significant with a p value < 0.01 in at least one of the five bacterial diets, relative to the OP50 bacterial diet.
Fig. 8. C. elegans food choice.
a Schematic of the food choice assay. b Food choice of C. elegans raised on OP50 when presented with all six bacterial foods. Worms were found less often to be dwelling on the Red bacteria compared to the other bacterial diets. Each data point represents one plate of 50 worms (nine plates total; n = 450 worms assessed). Each box represents 95% confidence intervals with the median value defined. c Proportion of worms on and off of the bacterial lawn at the L4 stage of development. n = 200/replicate for a total of five replicates. d Proportion of worms on and off of the bacterial lawn at day 1 of adulthood. n = 200/replicate for a total of five replicates. e Volcano plot of differentially expressed immune-related genes on all bacterial foods compared to OP50. The number of significant genes have a p value of <0.01. f Heat map of immune-related genes that are significant with a p value < 0.01 in at least one of the five bacterial diets, relative to the OP50 bacterial diet.
Fig. 9. The introduction of the Red bacteria at the post-reproductive stage in C. elegans extends lifespan.
a Schematic of the bacterial diet as a nutraceutical experiment. Food 0 was OP50 for all worms. After synchronization and allowing L1s to hatch overnight, L1s were dropped on bacterial food 1 and moved at L4 to bacterial food 2 before being moved to bacterial food 3 at day 3 of adulthood. Lifespans were then measured. b–g Lifespan curves of C. elegans on each of the different bacterial diet combinations. Lifespan comparisons between the bacterial diet combinations and Red-only, and Orange-only were made with log-rank test; refer to Supplementary Data 4. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
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