Arch Med Sci. A comparative perspective on lipid storage in animals. A lipid droplet-associated GFP reporter-based screen identifies new fat storage regulators in C. J Genet Genomics. Intermediary metabolism in the dauer larva of the nematode Caenorhabditis elegans — 1. Glycolysis, gluconeogenesis, oxidative phosphorylation and the tricarboxylic acid cycle.
Rothstein M, Mayoh H. Nematode biochemistry: IV. On isocitrate lyase in Caenorhabditis briggsae. Nematode biochemistry—VII. Presence of isocitrate lyase in Panagrellus redivivus , turbatrix aceti , and Rhabditis anomala. Nematode biochemistry—VIII malate synthetase. Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. Alcohol Clin Exp Res. Caenorhabditis elegans battling starvation stress: low levels of ethanol prolong lifespan in L1 larvae.
Intermediary metabolism. WormBook; Cytochemical localization of malate synthase in amphibian fat body adipocytes: possible glyoxylate cycle in a vertebrate. J Histochem Cytochem. Identification of glyoxylate cycle enzymes in chick liver—the effect of vitamin D3: Cytochemistry and biochemistry.
Evidence of the glyoxylate cycle in the liver of newborn rats. Med Sci Monit. Evolution of glyoxylate cycle enzymes in metazoa: evidence of multiple horizontal transfer events and pseudogene formation.
Biol Direct. The demonstration of sterols as requirements for the growth, development and reproduction of the DD- nematode. A sterol requirement in Turbatrix aceti and Panagrellus redivivus.
Sterol composition of the nematodes Ditylenchus triformis and Ditylenchus dipsaci , and host tissues. Nematode biochemistry—IX.
Lack of sterol biosynthesis in free-living nematodes. Recent developments in nematode steroid biochemistry. Kurzchalia TV, Ward S. Why do worms need cholesterol? Nat Cell Biol. Chitwood DJ. Biochemistry and function of nematode steroids. Crit Rev Biochem Mol Biol. Regulation of sterol synthesis in eukaryotes. Annu Rev Genet. Metabolism of sterols of varying ring unsaturation and methylation by Caenorhabditis elegans.
Distribution and transport of cholesterol in Caenorhabditis elegans. Mol Biol Cell. Identification of ligands for DAF that govern dauer formation and reproduction in C. Influence of steroid hormone signaling on life span control by Caenorhabditis elegans insulin-like signaling.
The role of dafachronic acid signaling in development and longevity in Caenorhabditis elegans : digging deeper using cutting-edge analytical chemistry.
Front Endocrinol. DAF-9, a cytochrome P regulating C. Sharma V, Hiller M. Loss of enzymes in the bile acid synthesis pathway explains differences in bile composition among mammals.
Genome Biol Evol. Rothstein M, Coppens M. Nutritional factors and conditions for the axenic culture of free-living nematodes. Lack of heme synthesis in a free-living eukaryote. Biosynthesis of heme in mammals. Haem homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins.
Severance S, Hamza I. Trafficking of heme and porphyrins in metazoa. Chem Rev. An intercellular heme-trafficking protein delivers maternal heme to the embryo during development in C. Topologically conserved residues direct heme transport in HRGrelated proteins. Heme utilization in the Caenorhabditis elegans hypodermal cells is facilitated by heme-responsive gene Control of metazoan heme homeostasis by a conserved multidrug resistance protein.
Dougherty EC, Hansen E. He folic acid requirement and its antagonism by aminopterin in the nematode Caenorhabditis briggsae Rhabditidae. Vanfleteren JR, Avau H. Selective inhibition of reproduction in aminopterin-treated nematodes. Accumulation of Formimino-l-glutamic acid in the free-living nematode Caenorhabditis briggsae as related to folic acid deficiency. One-carbon metabolism in health and disease. Biomarkers of nutrition for development-folate review.
J Nutr. Human intestinal folate transport: cloning, expression, and distribution of complementary RNA. Cloning and functional characterization of a folate transporter from the nematode Caenorhabditis elegans. Am J Phys Cell Physiol. The B-vitamins required by Caenorhabditis briggsae Rhabditidae. Nicholas WL, Jantunen R. A biotin requirement for Caenorhabditis Briggsae Rhabditidae. Effect of vitamin B12 and folate on biosynthesis of methionine from homocysteine in the nematode Caenorhabditis briggsae.
Vitamin B12 deficiency in Caenorhabditis elegans results in loss of fertility, extended life cycle, and reduced lifespan. Vitamin B 12 deficiency results in severe oxidative stress, leading to memory retention impairment in Caenorhabditis elegans.
Redox Biol. Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes. Worms, bacteria, and micronutrients: an elegant model of our diet. Trends Genet. The effects of dietary vitamin B12 deficiency on sperm maturation in developing and growing male rats. Congenit Anom.
Prevalence of low serum cobalamin in infertile couples. Systems genetics of mineral metabolism. Wada O. What are trace elements? Their deficiency and excess states. Japan Med Assoc J. A study of mineral requirements in Caenorhabditis elegans. Zinc deficiency reduces fertility in C. Hidiroglou M, Knipfel JE. Zinc in mammalian sperm: a review. J Dairy Sci. Zinc availability regulates exit from meiosis in maturing mammalian oocytes.
Nat Chem Biol. Zinc levels modulate lifespan through multiple longevity pathways in Caenorhabditis elegans. Download references. You can also search for this author in PubMed Google Scholar. Correspondence to Bart P. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions. The nutritional requirements of Caenorhabditis elegans. Genes Nutr 14, 15 Download citation.
Received : 04 March Accepted : 10 April Published : 06 May Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Download PDF. Abstract Animals require sufficient intake of a variety of nutrients to support their development, somatic maintenance and reproduction.
Caenorhabditis elegans ecology and diet in nature Habitat Caenorhabditis elegans is a free-living nematode with cosmopolitan distribution [ 1 ]. Food In nature, C.
Feeding and food-related behaviour Food ingestion in C. Dietary choices in the lab Commonly used bacterial strains While information about the C. Biomass composition of E. Nutritional requirements of C. Caloric compounds and building blocks Proteins and peptides Early studies of nutritional requirements of free-living nematodes underlined the absolute requirement for supplementation of axenic media with a heat-labile proteinaceous factor to achieve continuous growth and reproduction [ 41 , 44 , 54 ].
Amino acids In , Abderhalden classified amino acids as nutritionally essential and nutritionally non-essential [ 77 ]. Carbohydrates An early study of carbohydrate requirements of free-living nematodes reported that fecundity of C. Small organic compounds The ability of free-living nematodes to utilise two-carbon compounds e.
Vitamins Vitamins are defined as a group of organic compounds essential in small amounts for organismal function that cannot be synthesised by the body and thus must be provided through the diet or via the biosynthetic activity of intestinal bacteria. Sterols Early studies into the dietary requirements of free-living nematodes and attempts to formulate a chemically defined medium that would support growth and reproduction gradually resulted in a defined medium CbMM that required supplementation with crude substances for sustainable cultures [ 54 , 56 , 57 ].
Heme Supplementation of chemically defined axenic media with organic substances could possibly provide nematodes with yet another essential growth factor which these media lacked: heme [ 54 , 56 , 57 ]. Other vitamins First insights into the vitamin requirements of nematodes emerged in the s in a study in which was shown that continuous growth of C.
Minerals Dietary minerals represent a class of inorganic nutrients that are essential for many metabolic and physiological processes in the body, usually required in small amounts naturally found in different types of food. Concluding remarks For decades, C. Known common and specific essential nutrients of C. Full size image. References 1. Google Scholar Article Google Scholar CAS Google Scholar Availability of data and materials Not applicable.
View author publications. Ethics declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. About this article.
Copy to clipboard. Contact us Submission enquiries: Access here and click Contact Us General enquiries: info biomedcentral. B Protein levels normalized per cell in four strains of E. C Fatty acid levels normalized per cell in four strains of E.
D Average dry weight of bacterial lawns normalized per cell in four strains of E. Total sugars in hydrolyzed E. G Growing bacteria on high glucose media increases carbohydrate levels in bacteria but does not cause decreased triacylglycerol stores in C. To determine if dietary carbohydrates regulate fat storage in C. Addition of glucose resulted in increased concentration of cellular carbohydrates in both bacterial strains, from These experiments indicate that it is unlikely that carbohydrate content of dietary E.
We then examined the fatty acid composition of the four bacterial strains and the fatty acid composition of the total lipids, as well as the TAG and phospholipid fractions in the worms. We found significant differences in the fatty acid composition of HB compared to the other three strains Figure 4A. The cyclopropane fatty acids are produced by bacteria during stationary culture and, theoretically, the differences among the strains may be accounted for by the activity of one gene, cyclopropane synthase, which converts monounsaturated fatty acids into cyclopropane fatty acids [19].
The worm lipids reflect their dietary lipids, with higher monounsaturated fatty acids levels accumulating in worms feeding on HB and higher cyclopropane levels accumulating in worms feeding on the other strains Figure 4B.
However, these fatty acid composition changes do not correlate with fat stores, because nematodes growing on HT show similar fatty acid composition to nematodes growing on OP50, yet their levels of fat storage differ significantly.
Pelleted bacteria were derivatized to produce fatty acid methyl esters FAMEs for gas chromatography analysis. We then examined the fatty acid composition of phospholipid and TAG fractions to determine whether the relative fatty acid levels of any of the other worm fatty acids corresponded to TAG levels. We found that in the TAG fraction, the composition of one monomethyl branched-chain fatty acid, methylpalmitic acid C17iso , corresponds inversely to TAG levels in worms Figure 5A and 5B.
None of the other C. It is important to note that C17iso is not a dietary nutrient, because the four E. This fatty acid is synthesized de novo by C. Although fat storage in many strains is greater or less than wild type, fat stores in most strains were reduced when grown on E.
Only pept-1 mutants, defective in intestinal peptide transport, show no significant difference in fat stores when grown on OP50 and HB F Brood size is reduced in pept-1 animals growing on HB compared to OP50, while brood size does not depend on dietary E.
Food seeking behaviors that mediate dietary choice are regulated by the AIY neurons [10]. The ttx-3 gene encodes a LIM homeodomain transcription factor required for the differentiation of AIY interneurons [23].
We found that, like wild type, ttx-3 mutants stored less TAG when feeding on HB than on OP50, indicating that AIY interneurons, critical for mediating thermotaxis and food-seeking behaviors, are not required for mediating differential fat storage Figure 5C. We also examined two other mutants reported to contain high fat stores.
One strain carries a mutation in tub-1 , which is homologous to one of the few single-gene mutations that cause obesity in mice, Tub [24]. Another gene, egl-4 , shows a bright Nile Red phenotype in gain-of-function mutants [25]. Even though our live staining experiments verified previous reports of bright Nile Red staining of live worms is increased in tub-1 and egl-4 gf mutants [26] — [29] , we found that measurements of lipids consistently showed wild-type levels of TAG accumulation in both mutants feeding on OP50 and HB Two independently isolated tub-1 mutant strains nr and nr both showed wild-type TAG accumulation.
Finally, we examined several eating-defective mutants and found that both eat-2 and eat-5 mutants stored lower fat on both bacteria Figure 5C. Taken together, these studies show that sensory and feeding pathways necessary for the regulation of fat storage on OP50 are also necessary for wild-type levels of fat stored on HB, yet these pathways are not necessary for distinguishing the difference between the two strains. The only strain examined in this study that showed no difference in levels of fat storage when growing on OP50 and HB was a mutant pept-1 , previously called opt-2 and pep-2 , which carries a deletion in a gene encoding an intestinal peptide transporter [30].
This gene was previously identified as a low-fat gene by live Nile Red staining [26]. Our measurements of TAG levels, however, revealed that this strain stores very high levels of fat, even when growing on HB Our lipid analysis also revealed fatty acid composition differences in pept-1 mutants compared to wild type, with decreased amounts of monomethyl branched-chain fatty acids C15iso and C17iso as well as decreased levels of polyunsaturated fatty acids compared to wild type Table 2.
The fatty acid composition of pept-1 mutants is similar to that reported by others [31] , [32]. We found a significant inverse correlation of C17iso vs. Finally, we asked whether the differences in fat stores in worms grown on OP50 or HB affected reproductive success by counting the number of live progeny produced from individuals of various genotypes raised on either HB or OP50 E.
We found that for wild type, as well as for daf-2 and eat-2 mutants, similar numbers of offspring were produced regardless of the food source.
For pept-1 , however, there was a significant reduction of progeny production in worms growing on HB compared to OP50 Figure 5F. These results suggest that the range of TAG storage levels in wild type and daf-2 on either food source are adequate to ensure efficient progeny production, but the feeding defects in eat-2 and the peptide transport defects of pept-1 may prevent adequate assimilation of nutrients and, consequently, reduced progeny production.
Obesity is a disorder in energy homeostasis that develops when energy intake exceeds energy expenditure. In order to prevent and treat obesity, it is important to develop a deeper understanding of the effects of dietary macronutrients on energy regulation pathways. We demonstrate that fat storage in C. In the wild, C. Some bacterial species are pathogenic [33] , [34] , and worms can learn to avoid pathogenic food sources [13].
Given a choice, C. Worms tend to leave undesirable bacteria food by engaging in increased roaming behavior, and this behavior depends on AIY interneurons [10]. Food choice studies have shown that compared to HB, DA is considered to be a less desirable food, with cells that tend to clump together and may be difficult to ingest [10]. We suspected that differences in macronutrients of bacterial strains may be responsible for the range of fat stores observed in C. We found differences in fatty acid composition, as well as differences in carbohydrate content among the four E.
Our analysis showed that fatty acid composition differences in dietary E. Higher carbohydrate content of HB and HT correlates inversely with fat content, however, increasing carbohydrate content in HB and OP50 did not lead to a reduction in fat content, indicating that carbohydrate levels in bacteria per se do not dictate fat storage levels.
Analysis of TAG stores in a range of mutants indicated that sensory pathways are not necessary to store fat differentially on OP50 and HB food. Furthermore, the ttx-3 mutants, which are incapable of differentiating AIY interneurons critical for multiple sensory pathways, also accumulate less TAG when feeding on HB than on OP Only one mutant, pept-1 , showed equally high fat stores when grown on both OP50 and HB This mutant is deficient in a peptide transporter expressed in the intestine [30].
Recent work demonstrates that even though endogenous fat synthesis is reduced in pept-1 mutants, these worms accumulate high levels of fat due to accelerated uptake of dietary fatty acids [32]. This uptake is presumed to occur by way of a flip-flop mechanism that is dependent on intracellular and extracellular pH differences that are exacerbated in pept-1 mutants. Feeding behavior may also contribute to differential fat stores. A recent study showed that worms feeding on HB, considered a high-quality food that is easy to ingest, entered into periods of quiescence, characterized by cessation of movement and pharyngeal pumping [36].
High-fat mutants such as daf-2 and daf-7 show reduced quiescence on HB [36]. Thus, wild-type nematodes growing on OP50, as well as daf-2 and daf-7 mutants growing on HB, spend less time in quiescent states than wild-type worms growing on HB The reduced quiescence means more time is spent actively feeding, which correlates with higher fat stores. C — D Exogenous serotonin treatment selectively increases feeding rates of wild type worms on novel food to the level of the worms on familiar food.
The average values of the feeding rates presented in C are The average values of the feeding rates presented in D are E — F ser-7 tm is defective in increasing feeding response to familiar food compared to novel food.
Familiarity of food does not alter the feeding rates in ser-4 ok ; mod-1 ok ; ser-7 tm The average values of the feeding rates presented in E are The average values of the feeding rates presented in F are The feeding rate of the ser-4; mod-1; ser-7 triple null mutant is not altered by serotonin treatment. These assays were conducted on 3- to 5-hr-old L1 larvae, which pumped much more slowly than the adults used in other measurements.
The average values of the feeding rates presented in G are H Serotonin signaling via SER-7 that activates the feeding response is more active on familiar food than novel food. The y axis indicates the difference in the feeding rates between wild-type and ser-7 tm animals.
Each value corresponds to the difference in the feeding rates between wild-type and the ser-7 null mutant presented in Figure 7A and B. Feeding rates of tph-1 mg on HB A and JU54 B after a 7- to 8-hr interval from training the animals on one or the other bacterium.
Among five serotonin receptor null mutants, only ser-7 tm failed to activate feeding in response to serotonin. A null mutation in ser- 4 also decreased the feeding rate in presence of serotonin but the effect was relatively small. The average values of the feeding rates presented in this figure are Wild-type worms feed more actively on familiar food than novel food.
On novel food the feeding rate of wild-type is slightly higher than that of the ser-7 null mutant. The difference may be due to constitutive activity of SER-7; that is, SER-7 is active to some extent even in absence of its ligand, serotonin Hobson et al.
C — D Feeding rates of ser-4 ok ; mod-1 ok ; ser-7 tm and ser-7 tm on HB C and DA D after a 7- to 8-hr interval from training the animals on one or the other bacterium.
Familiarity of food does not alter feeding rates in ser-4 ok ; mod-1 ok ; ser-7 tm Like the positive SERmediated signal, the inhibitory SER and MODmediated serotonin signaling is more active on familiar food than novel food, but it decreases the feeding rate.
E A simple linear model explaining how different serotonin receptors might contribute to the regulation of pumping on familiar food and on novel food. While it is presented as an aid to thinking about the results, none of the results presented in the paper depend on this model.
A change in this number suggests the action of serotonin via SER We use this as the measure of serotonin action via SER-7 because it is model-independent and robust. The y axis indicates the difference in the feeding rates between ser-4 ok ; mod-1 ok ; ser-7 tm and ser-7 tm animals. Each value corresponds to the difference in the feeding rates between the triple null mutant and the ser-7 null mutant presented in C and D.
The rescue effect is suppressed by loss of ser-7 , but not by loss of mod No difference was found in feeding rates between the tph-1 single null mutant, the ser-7 single null mutant and the tph-1; ser-7 double null mutant.
The average values of the feeding rates presented in A are E Expression of ser-7 cDNA driven either by the flp-2 promoter or by the flp promoter MC, M4, and other neurons but not by the ser-7b promoter M4 only fully restored the feeding rate in the ser-7 null mutant in response to serotonin. The rescue effect was suppressed by blocking cholinergic transmission from MC to the pharyngeal muscles.
The difference suggests that acetylcholine marginally activates pumping in an EATindependent manner and that there is residual acetylcholine release in absence of SER-7 in response to serotonin. No difference in feeding rates was found between the eat-2; ser-7 mutant expressing pflpgfp and the mutant expressing pflpser-7 cDNA. F Expression of ser-7 cDNA driven either by the flp-2 promoter or by the flp promoter fully restored the feeding rate in the ser-7 null mutant in response to familiar food.
Expression of ser-7 cDNA in M4 and occasionally in M2 driven by the ser-7b promoter also increased the feeding rate, but the effect was relatively small. Feeding rates of ser-4 ok ; mod-1 ok on HB A and DA B after a 7- to 8-hr interval from training the animals on one or the other bacterium. Like wild type worms, ser-4 ok ; mod-1 ok show increased feeding response on familiar food compared to novel food. To test if serotonin feeding signaling via SER-7 indeed gets activated by recognition of familiar food, we compared the differences between feeding rates of wild-type and the ser-7 null mutant animals the SER-7 effect on familiar food with the differences on novel food.
Any feeding rate difference between wild-type and the ser-7 mutant animals indicates active serotonin signaling via SER-7 because ser-7 specifically affects serotonergic signaling, with some contribution from basal activity of SER-7 in the absence of serotonin Hobson et al.
If serotonin signaling is equally active on familiar food and novel food, we expect the SER-7 effect to be similar on familiar food and novel food. We concluded that recognition of familiar food increases the feeding response mainly by activating serotonin signaling via SER To gain insight into how serotonin signals familiar bacteria, we asked which serotonergic neurons regulated the feeding response. Serotonin is detected in five types of neurons in C.
HSN is also unlikely to be necessary for the behavioral plasticity because feeding rates of males, which do not have HSN, were also greater on familiar food than the rates on novel food Figure The NSM neurons are a pair of secretory neurons located in the pharynx, whereas the ADF neurons are a pair of chemosensory neurons located outside the pharynx Sze et al.
We asked if serotonin either in ADF or in NSM regulates the feeding response by expressing tph-1 cDNA in the tph-1 null mutant using either the srh promoter or the ceh-2 promoter. The srh promoter drives expression specifically in ADF and the ceh-2 promoter drives expression in NSM and three additional neurons Liang et al.
A — B Feeding rates of wild-type male worms on HB A and DA B after a 7- to 8-hr interval from training the animals on one or the other bacterium. This suggests that serotonin synthesized by ADF might act in either of two possible ways: it could activate SER-7 directly, or it could be taken up and subsequently released by other serotonergic neurons.
MOD-5 is a serotonin transporter required to take up extracellular serotonin into some serotonergic neurons Ranganathan et al. A Schematic of experimental design for anti-serotonin staining. Filled arrowheads and open arrowheads indicate ADFs and serotonin-uptaking cells, respectively. The serotonin signals not marked by arrowheads are neuronal processes. The baseline was 2. A ser-7 null mutation suppressed the rescue effect of restoring serotonin in ADF in the tph-1 null mutant Figure 8A.
To understand how serotonin increases feeding at a neural circuit level, we asked where SER-7 acts. SER-7 is expressed mostly in pharyngeal neurons Hobson et al. Among the pharyngeal neurons, MC is particularly interesting because it is essential for normal fast feeding on bacteria Avery and Horvitz, , and SER-7 was suggested to activate MC Hobson et al. The flp and the flp-2 promoters drive expression in several neurons, and the expression patterns of the two promoters overlap only in MC and M4 Kim and Li, We found that both pflp SER-7 and pflp SER-7 fully rescued the feeding rate in the ser-7 mutant in response to familiar food as well as serotonin Figure 8E,F ; Part of the data in Figure 8E,F were reported previously in Song and Avery, and were re-analyzed and presented here.
In contrast, expression of SER-7 in M4 and occasionally in M2 using the ser-7b promoter failed to alter the pumping rate and had only a small effect on pumping in the ser-7 null mutant in response to serotonin and familiar food, respectively Figure 8E,F , suggesting that SER-7 in MC activates pharyngeal pumping. The failure of rescue is unlikely to be due to insufficient expression because expression of the rescue construct significantly activated isthmus peristalsis, the other feeding motion in C.
To test whether SER-7 indeed acts through MC, we used an eat-2 null mutation to test if blocking neurotransmission from MC suppresses the rescue effect of pflp ::SER-7 in the ser-7 mutant. Thus, an eat-2 null mutation selectively blocks cholinergic transmission from MC to the pharyngeal muscles. In summary, we conclude that serotonin released from extrapharyngeal ADF increased feeding in response to familiar bacteria mainly by activating SER-7 in MC directly, which in turn activates cholinergic transmission from MC to the pharyngeal muscles.
We next asked why ADF increases feeding response on familiar but not novel bacteria. A simple explanation is that only familiar bacteria can activate ADF, and this activation causes increased serotonin release. We tested the hypothesis first by asking if ADF is more active on familiar bacteria than novel bacteria by directly measuring the response of the ADF neurons to novel and familiar bacteria using ratiometric calcium imaging. In contrast, only marginal changes in the activity were observed in response to novel food Figure 12A,B.
Consistent with our hypothesis, the increases in ADF activity were greater in response to familiar food than novel food Figure 12C. The activities in response to novel food were not different from baseline data not shown. These data indicate that familiar food, but not novel food, activates ADF neurons. Traces represent the average percentage change from baseline over time of the fluorescence emission ratio of the ratiometric calcium sensor Cameleon YC3.
The number of individual recordings is indicated in parenthesis next to each group. C Average response to familiar or novel food. We then asked if ADF releases more serotonin in response to familiar bacteria than novel bacteria. Since direct measurement of serotonin release from ADF in response to food is challenging, we developed a method to detect released serotonin indirectly by its uptake into other serotonergic neurons.
No serotonin signal was detected in the tph-1 mg ;Is[ptphgfp] A and in the mod-5 n ;tph-1 mg ;Is[ptphgfp] B mutant animals. Filled arrowheads indicate ADFs.
There are several other possible explanations for the greater increase on familiar food than novel food that we cannot exclude, such as increased efficiency of serotonin uptake in serotonin-uptaking cells or increased serotonin release from NSM on familiar food compared to novel food.
However, considering that an increase in the efficiency of serotonin uptake is likely to result in a decrease in the level of extracellular serotonin that can activate SER-7 in MC and that restoring serotonin in NSM in the tph-1 null mutant or killing NSM in wild type did not affect the feeding response on familiar food, it is more likely that the greater increase is due to increased serotonin release from ADF.
In conclusion, only familiar bacteria activate ADFs, which increases serotonin release from the neurons and subsequently activates the feeding response. Worms may recognize familiar bacteria by taste, smell or texture. To get insight into the mechanism, we tested if worms recognize familiar bacteria by their taste or smell. For this, we examined feeding rates of worms on bacteria mixed with LB broth or with medium conditioned by one of the bacteria Figure 14A.
The conditioned media do not contain any bacterial particles, thus, if the media alter the feeding responses to familiar food or novel food, it suggests that gustatory or olfactory cues in the media were sensed by worms and affected discrimination of familiar food from novel food. We found that the media from bacteria that are familiar to the tested worms did not alter the feeding responses to the novel bacteria Figure 14B,C. Each condition is coded by three symbols. The first and the second letter above the bar represent training and test food in order.
The third symbol, below the bar, represents the conditioned media that was mixed with the test food. B — C Conditioned media from novel bacteria override the stimulatory effect of familiar bacteria on feeding. We used both Orange bacteria, which decreases lifespan, and Red bacteria, which increases lifespan, as the basis of our model. Remarkably, after assessing eight combinations of bacterial diet switching, we discovered that the bacteria fed during development food 1 and reproductive period food 2 contributed little to overall lifespan, and the last bacteria exposed food 3 exerted the most impact.
In brief, when animals were exposed to the Red bacteria after experiencing the Orange bacteria, the normally shortened lifespan resulting from ingestion of Orange is suppressed Fig. Conversely, exposure to Orange suppresses the normally extended lifespan linked to whole-life feeding of the Red bacteria Fig.
Intriguingly, when compared to animals raised on OP50, ingestion of Red bacteria post-reproductively, regardless of ingestion of Orange bacteria at any other life stage, results in a relatively normal lifespan, not shortened Supplementary Fig. Moreover, animals that eat the Red bacteria post-reproductively have an extended lifespan, which is further enhanced if the food is introduced after the development.
Taken together, our studies reveal that expanding the available foods for C. Future studies to integrate genetic analyses to define new gene—diet pairs 6 , 59 and gene—environment interactions in general, will be of significant interest. 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. Lifespan comparisons between the bacterial diet combinations and Red-only, and Orange-only were made with log-rank test; refer to Supplementary Data 4.
Here, we augment that model by introducing a comprehensive phenotypic analysis of C. Interestingly, these microbes have been found in the normal C.
Previous works have identified how C. Because of this, we decided to investigate the effects of bacterial diet on both physiological attributes and transcriptional signatures of C. Recent studies have shown that bacterial diet can alter transcriptional responses in C. Our work supports this observation with three new menu options for C. Although each of these foods evokes a unique transcriptional signature Fig. Our RNAseq analysis was limited to gene expression changes observed as animals enter adulthood, prior to reproduction.
Given the extent of changes in reproductive capacity, health movement and fat , and aging, future work to examine how gene expression is altered on each food over the lifespan will be of great interest. Moreover, a fine-tuned analysis of transcription at each development stage will be informative based on our observation that different foods can accelerate the transition of animals across specific developmental stages.
Nevertheless, it is clear that the standard laboratory E. Knowing that even different strains of E. Bacteria serve as a live food source for C. Prior studies have shown animals pumping at similar rates in the presence of bacteria they can and cannot eat 12 , The rates of pharyngeal pumping were not significantly different on any of the bacteria in L4 stage and day 1 adult animals Fig.
Because pharyngeal pumping may not be the best way to measure the overall consumption of food, we employed a food intake assay to measure the quantity of food ingested by worms fed each bacterial diet.
These data demonstrated that worms were eating at similar rates on the bacterial diets between the L4 larva and day 4 adult stages Fig. We also found that when examining the bacterial load in day 1 adults, it appears that all bacterial diets are able to be broken down and digested by the worms due to the lack of colony growth on LB plates after the lysis of individual worms Supplementary Fig. In support of this data, we also observed faster developmental timing Fig.
Nevertheless, based on the similarities of animals eating Red and animals undergoing dietary restriction, it remains possible that this bacterium allows ad libitum ingestion with the physiological benefits of reduced eating, which is further supported by the Red bacteria causing worms to have the highest proportion of dietary restriction-related genes deregulated in comparison to the other bacterial diets Fig.
Clearly, changing food sources can potently impact multiple phenotypic attributes and several of these aging-relevant phenotypes are interrelated. For example, reproduction, fat, and stress resistance are intrinsically tied to overall life expectancy 9. In support of this model, the Red and Yellow bacteria, which reduce the overall reproduction Fig. However, our study reveals that this relationship is more complex as these two foods have different effects on lipid storage and Yellow evokes an Asdf response Fig.
It may be that C. The Yellow bacteria also does not produce a lawn avoidance phenotype at L4 or day 1 of adulthood Fig. In addition, these data are supported by the RNAseq data analysis of immune system-related genes, which shows that the Yellow bacteria induces a proportionally smaller number of gene expression changes compared to the other bacterial diets used in this study Fig.
Lastly, genetic and environmental mechanisms that delay developmental timing have been tied to increased longevity in adulthood 40 , 67 , Each of the bacterial diets we tested results in the faster development into a reproductive adult Fig.
Taken together our study reveals that each bacterial food can exert a specific life history changing response, but also questions previously established models of aging. Many aspects of the bacterial communities, besides that of their nutritional composition, have been shown to influence multiple attributes in C. We decided to explore the food preference aspect by carrying out a food choice assay that contained all six of our bacterial diets.
We hypothesized that bacterial diets shown to extend lifespan, like Red and Yellow Fig. Interestingly, in the food choice assay, we saw that worms are less often found on the Red bacteria and more often found on the other bacteria.
Finding worms more often on both Yellow and Orange was somewhat surprising due to the Asdf phenotype at day 3 of adulthood in Yellow-raised worms Fig. Both of these phenotypes are consistent with phenotypes when worms are exposed to pathogens. However, the pathogenic effect of specific bacteria is often associated with intestinal colonization 71 , 72 , 73 , which we did not observe Supplementary Fig. Moreover, the increased lifespan of animals fed the Yellow bacteria disagrees with the idea that the Yellow bacteria is pathogenic.
Nevertheless, these results motivate future experimentation to understand why worms are found less frequently on a longevity-promoting bacterium and more frequently on a bacterium that decreases lifespan. Inspired by previously described dietary interventions that are capable of extending lifespan in multiple species 47 , 74 , 75 , we wanted to investigate whether, instead of keeping animals on one bacterial diet their entire life, altering food exposure at critical life stages could affect the overall lifespan.
We asked whether these foods could alter lifespan without any previous generational exposure. Strikingly, acute exposure to these bacterial foods was capable of altering lifespan Fig. Furthermore, our study revealed that the bacteria fed during the post-reproductive period exerted the strongest influence over the lifespan of the cohort. This result, however, is potentially confounded by the amount of time longitudinally that is spent on each bacteria. Regardless, our study reveals that any potential early-life development and reproductive span exposure to foods that normally shorten lifespan can be mitigated by eating a lifespan-promoting food option.
This result is reminiscent of previous studies showing the mortality rate of DR-treated animals is accelerated when switched to an ad libitum diet, while mortality rate is reduced when ad libitum-fed animals are switched to a DR diet In our case, calories are perhaps not different on Red or Orange, but rather the overall nutritional composition of the bacteria is different. Given that in our model animals are chronically exposed to as much food as they can eat throughout their life, we posit that if a similar human diet were discovered that this treatment protocol would be more accessible as food restriction is difficult, socially, and psychologically 77 , 78 , Ultimately, this study reveals the impact that diet can have on both physiology and the transcriptomics of animals that thrive on them.
These alterations caused by differential bacterial diet exposure can not only be seen at the surface level in the diverse phenotypes that present themselves, but also at the genetic level, causing fluctuations in gene expression important for multiple physiological processes, including development, fat content, reproduction, healthspan, and lifespan Table 1. These discoveries in the worm can aid in understanding how dietary exposure influences different phenotypes, and continuing work in the effects of bacterial diet on overall health and aging will unequivocally contribute to more personalized diets to promote healthier and longer lives in individuals.
Taken together, our study expands the menu of bacterial diets available to researchers in the laboratory, identifies bacteria with the ability to drive unique physiological outcomes, and provides a food quality approach to better understand the complexities of gene—diet interactions for health over the lifespan.
For experiments, NGM plates without streptomycin were seeded with each bacterium at the optical density of 0. Red, Yellow, and Orange bacteria were isolated from stock plates in the laboratory and selected for with antibiotics before inoculating. Optical density curves were made by averaging the readings together after performing the experiment in biological triplicate. The antibiotic versus no antibiotic growth curves were conducted in a similar manner.
Bacteria was grown overnight in LB liquid culture with corresponding antibiotics. Red bacteria samples were seeded onto LB plates and then allowed to grow overnight before scraping off and collecting. Bacteria were homogenized based on metabolite kit instructions and corresponding metabolites were measured via Bio Vison kit instructions.
Bacteria was grown overnight in liquid culture of LB with corresponding antibiotics. The next day, bacteria were collected at the log phase, seeded onto LB plates at 0. Bacteria was then collected with autoclaved water from the seeded plates and spun down to remove excess water. Water content measurements were also obtained after measuring wet weight and dry weight of the samples. Wild-type worms grown on each food for at least 30 generations were egg prepped and eggs were allowed to hatch overnight for a synchronous L1 population.
The next day, L1s were dropped on NGM plates seeded with 0. The RNA samples were sequenced and read counts were reported by Novogene. Statistically significant genes were chosen based on the adjust p values that were calculated with the DESeq2 package.
Worm areas were measured in ImageJ using the polygon tool. GR worms grown on each bacterial food for at least 20 generations were egg prepped and eggs were allowed to hatch overnight for a synchronous L1 population. Each hour worms were scored as green molting or nongreen not molting.
Wells without worms, wells with two worms, and worms that crawled to the side of the plate were censored.
Worm strains on each bacterial diet were egg prepped to generate a synchronous L1 population and dropped on the corresponding food.
Every diet was moved at the same time. Worms were scored daily for survival by gentle prodding with a platinum wire. Animals that burst or crawled to the side of the plate were censored and discarded from this assay. Wild-type worms grown on each bacteria for at least 30 generations were egg prepped and eggs were allowed to hatch overnight for a synchronous L1 population.
Progeny were removed from the plate during counting in order to ensure accuracy. Wild-type males raised on Red and OP50 were egg prepped at the same time, as wild-type hermaphrodite populations raised on Red and OP50, and allowed to hatch overnight in M9 to create synchronous L1 populations.
Hermaphrodites were moved every day to a new plate until egg laying ceases. Progeny were removed from the plate during counting, in order to ensure accuracy. ORO-stained worms were placed on glass slides and a coverslip was placed over the sample. Fat levels of worms were placed into three categories: non-Asdf, intermediate, and Asdf.
Non-Asdf worms display no loss of fat and are stained a dark red throughout most of the body somatic and germ cells. Asdf worms have had most, if not all, observable somatic fat deposits depleted germ cells only. Wild-type worms grown on each bacterial diet for at least 30 generations were egg prepped and eggs were allowed to hatch overnight for a synchronous L1 population. Food intake experiments were adapted from Gomez-Amaro et al. Plates contained 10—40 worms per well.
The next day, bacteria were collected at the log phase, seeded onto NGM plates at 0. Bacteria was then collected with autoclaved water from the seeded plates, and spun down to wash and resuspended in water. Plates were sealed with Parafilm to prevent evaporation and rocked continuously to prevent drowning of the nematodes. The fraction of animals alive was scored microscopically at day 4 of adulthood.
Food intake per worms was calculated as bacterial clearance divided by worm number in well. Measurements were then normalized to the L4 to day 1 adult clearance rate for each bacterial diet. Worms were grown on each bacterial diet until the L4, day 1 adult, day 3 adult, day 8 adult, or day 11 adult stage.
Worms were then moved to an unseeded NGM plate to remove bacteria from the cuticle of the worms. Once food choice assay plates were seeded and allowed to grow overnight, OPreared worms were egg prepped, and eggs were allowed to hatch overnight for a synchronous L1 population.
The next day, L1s were dropped into the center of the NGM plate and counted. If worms were found on the bacterial lawn, then those worms were counted as on that food. Worms found outside bacterial lawns were counted as not on food.
The proportion of worms found on each food or off of food was then calculated and graphed. Each assay was done in biological triplicate with technical triplicates for a total of nine plates.
Worms reared on each bacterial food were egg prepped and eggs were allowed to hatch overnight for a synchronous L1 population. The next day, L1s were dropped onto NGM plates with each bacterial diet. Worms were then washed with water and then dropped on an unseeded NGM plate. Worms were allowed to crawl for an hour before washing again and dropping onto a new unseeded NGM plate.
This was done in duplicate for a total of 48 individual worms per bacterial diet. The next day, L1s were dropped onto NGM plates with each bacterial diet and counted.
The proportion of worms was calculated and graphed using Graphpad Prism 8. Comparisons and significance were analyzed in Graphpad Prism 8. Lifespan comparisons were done with log-rank test.
Sample size and replicate number for each experiment can be found in figures and corresponding figure legends.
This information is also in the experimental methods. Exact values for graphs found in the main figures can be found in Supplementary Data 5. All other relevant data is available upon request from the corresponding author. Fontana, L. Extending healthy life span—from yeast to humans. Science , — Fitzpatrick, M. Food fads. Lancet ,
0コメント