Phenotypic and metabonomics studies of FMOs in C. elegans and their roles in lifespan extension

Phenotypic and metabonomics studies of FMOs in C. elegans and their roles in lifespan extension | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Phenotypic and metabonomics studies of FMOs in C. elegans and their roles in lifespan extension Mohamed Said, Rafael Freire, Filipe Cabreiro, Jose Ivan Serrano-Contreras, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7122670/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Nov, 2025 Read the published version in Metabolomics → Version 1 posted 9 You are reading this latest preprint version Abstract Introduction: Flavin-Containing Monooxygenases (FMO) are widely conserved, xenobiotic-detoxifying enzymes whose additional endogenous functions have been revealed in recent studies. Those roles include the regulation of longevity in the model nematode Caenorhabtidis elegans. Objectives: The purpose of this study was to compare aspects of the phenotypes of C. elegans worms with mutations in all fmo genes, particularly focusing on the metabolome and its relationship with lifespan-extension and the worm life cycle. Methods: NMR Spectroscopic analysis of the extracts of metabolites from C. elegans worms of different ages and fmo genotypes was used to compare metabolite profiles of C. elegans worms and determine how these changed with genotype and ageing. Results: Loss of both fmo-4 and fmo-3 and over-expression of fmo- 2, resulted in increased levels of tryptophan in the metabolome, which correlated with an extended lifespan in these mutants. Loss of fmo-4 also led to decreased embryo hatching, along with increased sensitivity to bleach during sterilisation protocols. In contrast, in the extended lifespan fmo-1 knockout worm, the metabolome did not reveal any significant metabolite changes and therefore lifespan effects may occur through another mechanism, or hidden metabolic changes. Conclusion: Genetic interventions coupled with metabolome profiling in C. elegans can provide insights into biological mechanisms in ageing that might lead to strategies for healthy lifespan extension in human old age. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Ageing is a progressive and intrinsic biological degenerative process in the body, with accumulated molecular deterioration in many tissues and cellular pathways. This physical decline over time leads to cellular damage and dysfunction and, finally, to death (DiLoreto and Murphy, 2015 ; Bratic and Larsson, 2013 ; Leiser et al., 2015 , López-Otín et al, 2023 ). One of the key drivers in 21st century healthcare research is the extension of healthy lifespan in an ageing population (Mount et al., 2016 ). As part of these efforts to enhance quality of life and healthy ageing, it is desirable to reduce the risk, or delay the onset, of chronic diseases associated with ageing, such as Alzheimer’s and Parkinson’s diseases (Hoffman et al., 2017 ; DiLoreto and Murphy, 2015 ). C. elegans is an ideal model for the study of ageing as it has a relatively short lifespan of around 2–3 weeks under normal conditions (Corsi, Wightman and Chalfie, 2015 ; Altun and Hall, 2009 ).Importantly, C. elegans lifespan has also been shown to be extended by single mutations in specific genes, such as components of insulin/IGF-1 like signalling pathways (van Heemst, 2010 ). The discovery of systems that act in ageing and their mechanisms of action in the model organism Caenorhabditis elegans may be applicable to the improvement and extension of heathy human lifespans. Metabonomics, defined as “the quantitative measurement of the multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification” (Lindon et al., 2000 ; Everett et al., 2019 ), is a useful tool for investigating metabolic changes during ageing and age-related disease (Balashova et al., 2022 ). Among the factors that influence metabolism are diet, environment, disease, genetics and microbiome variation, resulting in a metabolic profile that reflects the health status of an individual (Everett et al., 2019 ). Metabonomics can also be used to investigate changes to the metabolism in model organisms such as C. elegans , and reveal relationships between genotype and phenotype that integrate across genomic and environmental factors, including the microbiome (Everett et al., 2019 ; Raamsdonk et al., 2001 ). The measurement of metabolites in biofluids or in tissue extracts of organisms such as C. elegans can be performed using NMR spectroscopy (Everett et al., 2019 ; Lindon et al., 2000 ), as in this study, or mass spectrometry (MS) (Scalbert et al., 2009 ; Watson, 2013 ). Flavin-containing monooxygenases (FMOs) are NADPH-dependent enzymes, located in the membrane of the endoplasmic reticulum (ER), which catalyse the oxygenation of a wide variety of medicines and dietary-derived compounds (Krueger and Williams, 2005 ; Phillips and Shephard, 2020 ), detoxifying nitrogen- and sulphur-containing drugs and xenobiotics (Phillips and Shephard, 2017 ). Beyond their roles as xenobiotic-metabolising enzymes, however, FMOs are now known to be involved in various important endogenous functions in mammals. Human FMO1 was recently found to catalyse the conversion of hypotaurine to taurine, an amino acid critical for human health, utilising either NADPH or NADH as co-factor (Veeravalli et al., 2020 ). Also, host hepatic FMO3 is the primary FMO responsible for trimethylamine N -oxide (TMAO) production from trimethylamine. Genetic mutations reducing FMO3 activity result in trimethylaminuria (or fish-odour syndrome) in humans, caused by build-up of trimethylamine (Shephard, Treacy and Phillips, 2012 ; Yamazaki and Shimizu, 2007 ; Dolphin et al., 1997 ; Phillips and Shephard, 2020 ). Another recent study of the endogenous functions of FMOs in human cells revealed that the five mammalian FMOs share a common and redundant function in stress resistance: the overexpression of the five FMOs increased stress resistance, regardless of whether they were normally expressed in that cell-type (Huang et al., 2021 ). FMOs are conserved across the eukaryotes and, notably, can be induced by multiple lifespan-extending interventions in mice: this poses the question over whether these enzymes might play a critical role in promoting health and longevity across phyla (Leiser et al., 2015 ). Fmo5 knockout (KO) mice exhibited an age-related phenotype with lower body fat and weight, despite higher food intake, and lower blood glucose and cholesterol (Malagon et al., 2015 ). Changes in metabolism due to disruption of Fmo5 indicated that metabolic ageing was slowed through pleiotropic effects (Malagon et al., 2015 ; Varshavi et al., 2018 ) and recently, a specific compound, 2,3-butanediol, was shown to be a microbiome-derived biomarker for Fmo5 KO in mice. Moreover, 2,3-butanediol treatment prompted lower cholesterol and epididymal body fat in wild-type (WT) mice, recreating aspects of the phenotype of the Fmo5 KO (Veeravalli et al., 2022 ). Given the formation of 2,3-butanediol by the host microflora, it is interesting that mammalian FMO5 was recently shown to act as a sensor of gut bacteria (Scott et al., 2017 ). Like mammals, the nematode C. elegans encodes five FMOs, again named FMO1-5 (Petalcorin et al., 2005b ). In the case of Fmo-2 , overexpression (OE) in the C. elegans intestine was reported to increase worm lifespan through activation of hypoxia inducible factor (HIF)-1 (Uno and Nishida, 2016a ; Leiser et al., 2015 ). fmo-2 OE in C. elegans also enhanced resistance to proteotoxic stress within the ER and increased proteostasis in worms undergoing hypoxic responses (Leiser et al., 2015 ). Intestinal fmo-2 transcription was increased by dietary restriction (DR) and is necessary for DR-mediated lifespan extension (Uno and Nishida, 2016b ). Further, intestinal fmo-2 , which is regulated in C. elegans by serotonergic signalling originating in neurons, subsequently activates the transcription factor HLH-30 (another factor also activated by DR) in the intestine. FMO-2 is thus an enzyme both necessary and sufficient for a majority of the beneficial effects of either of these longevity pathways (Leiser et al., 2015 ). Correspondingly, fmo-2 and also fmo-4 transcription was upregulated by hypoxia (Shen et al., 2005 ). In more recent studies, oxidative stress (Goh et al., 2018 ) and infection with either Pseudomonas aeruginosa (PA14) (Dasgupta et al., 2020 ) or Staphylococcus aureus was found to induce fmo-2 , with FMO-2 required for pathogen resistance (Wani et al., 2021 ). Recent work also supported a role for fmo-2 in C. elegans innate immunity, as it’s transcription was strongly induced via NHR-49 and HLH-30 (Wani et al., 2021 ) in a pathogen-specific manner to impact infection survival. C. elegans FMO-4 was expressed prominently in hypodermis, duct and pore cells but was absent from excretory cells (Petalcorin et al., 2005a ). FMO-4 was hypothesised to possess an osmoregulatory role, promoting clearance of excess water that enters during periods of hypotonicity, potentially by synthesising an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells (Hirani et al., 2016 ). Sequence alignments of C. elegans FMO-2 and mammalian FMO5 showed high conservation of catalytic residues (Choi et al., 2023 ). Although C. elegans fmo-4 was thought to be orthologous to human FMO4, as they had similar predicted protein structures (Hirani et al., 2016 ), the human FMO4 was not able to rescue an hypoosmotic stress sensitivity phenotype in the fmo-4 KO worm strain (Hirani et al., 2016 ). Previous observations of the role of mammalian FMO5 in ageing raised the possibility that modulation of all or any C. elegans FMOs may be a conserved mechanism for enhancing protein homeostasis and extending lifespan. The appropriate modulation of all FMOs might equally promote healthy ageing, improving health span, in mammals and people (Leiser et al., 2015 ). We recently demonstrated that knockout of fmo-1, fmo- 3 and fmo- 4 statistically significantly extended C. elegans lifespan relative to wild type (Said et al., 2024 ). Therefore, in this study, metabolic profiles were obtained and more detailed phenotypic analyses (i.e., development, behaviour, ageing and egg-laying and hatching) were analysed for all C. elegans fmo KO lines, to further delineate or identify conserved roles for all fmo’s in development and ageing. Results fmo-2 KO , fmo-3 KO and fmo-4 KO delayed C. elegans development Phenotypic differences were identified between WT and fmo mutant C. elegans worms in terms of development. The length of KO mutants of fmo-2 (0.70 mm +/- 0.02; p < 0.003), fmo-3 (0.58 mm +/- 0.015; p < 0.0001) and fmo-4 (0.67 mm +/- 0.02; p 0.05, not significant (ns)) and fmo-5 KO worms (0.76 mm +/- 0.02; p > 0.05, ns) (Figure S1 A Error! Reference source not found. ). In addition, at the beginning of day 3, the plates of WT, fmo-1 KO fmo-2- OE and fmo-5 KO C. elegans contained the expected mix of eggs and mother worms, whereas plates of fmo-2 KO, fmo-3 KO and fmo-4 KO contained mainly mother worms and were delayed in their development by approx. 5–6 h (Figure S1 B). At the end of day 3, the worms of fmo-2 KO, fmo-3 KO and fmo-4 KO mutants began to lay eggs, whereas on WT and fmo-1 KO and fmo-5 KO mutant plates, many eggs were visible (Figure S2). Loss of fmo-4 increased C. elegans chemical/osmotic sensitivity A difference was observed in resistance to a strong oxidising agent in fmo- 4 KO. During C. elegans egg preparation, the fmo-4 KO strain resistance of its cuticle to bleach treatment (see Methods) was decreased compared with WT worms: the time required for bleach to disrupt ca. 95% of worms in fmo-4 KO was 2.12 +/- 0.13 min; p < 0.0001), whereas the time required for WT C. elegans was 5.19 +/- 0.12 min (Figure S3A-C) Error! Reference source not found. . Loss of fmo-4 in C. elegans was deleterious to embryo hatching At day 3 post-hatching, a difference could be seen clearly in the number of adult C. elegans worms between WT and fmo-4 KO. A mean (+/- SEM) of only 57 +/- 2% of fmo-4 KO eggs hatched, compared with 95 +/- 2% of eggs hatching in WT (p < 0.0002, Figure S3D-G). Interestingly, fmo-4 KO was the only C. elegans mutant that showed a large decrease in the number of adult worms in comparison to WT at day 3 post-hatching (data not shown). fmo-2 KO C. elegans swarming phenotype fmo-2 KO worms feeding behaviour occurred in aggregates, with more coherent groups of worms swarming across a bacterial lawn (Ding et al., 2019 ) than seen for WT or the other four fmo knockout lines, fmo-1,3,4 and − 5 . This phenotype was evident from the first day of hatching, with fmo-2 KO worms tending to move in groups from the centre to the edges of the bacterial lawn until they had consumed the bacteria (Fig. 1 ). Metabolic profiles of extracts of fmo mutants at day 3 post-hatching At day 3 post-hatching, unsupervised and unbiased principal components analysis (PCA) of all mutants showed no sample overlaps of fmo-4 KO worm metabolite profiles on PC1 with those of WT (Figure 2 A) and similarly for fmo-4 KO alone vs WT (Figure 2 B, Figure S4). Moreover, fmo-3 KO alone (Figure S5) had no sample overlaps with WT on PC1. PCA of fmo-2 KO vs WT (Figure S6) showed a complete overlapping on PC1 and a partial overlap with WT on PC2, and the fmo-2 OE extracts (Figure S7) versus WT showed no sample overlap on PC2. fmo-1 KO (Figure S8) and fmo-5 KO (Figure S9) were the only strains that had no PCA separation from WT in pairwise comparisons. Interestingly, at the earlier, embryo, life stage, PCA of NMR-based metabonomics of fmo-4 KO and WT metabolite extracts showed no group separation (Figure S10). When the composition of the metabolome of each worm mutant was compared at day 3 post-hatching, using PCA and ANOVA, branched chain amino acids (BCAA; isoleucine, leucine and valine) were present at significantly higher levels in fmo-2 KO, fmo-4 KO and fmo-2 OE strains than in WT (Table S1 ). The levels of agmatine (a metabolite of arginine), phosphorylcholine and choline were decreased in fmo-2 KO and fmo-2 OE strains, whereas the levels of 5'-adenosine monophosphate (AMP), 5'-adenosine triphosphate (ATP) and 5'-uridine monophosphate (UMP) were decreased in fmo-2 KO, fmo-4 KO and fmo-2 OE. In contrast, fmo-4 KO showed increased threonine, phenylalanine and tyrosine. Finally, fmo-3 KO showed an increased level of cystathionine, agmatine, lactate and threonine (Table S1 Error! Reference source not found. ). At this day 3, post-hatching, timepoint, the metabolites discriminating fmo-2 KO, fmo-4 KO and fmo-2 OE strains from WT shared similar pathways and included valine, leucine and isoleucine degradation (Figure S11, S12). Interestingly, fmo-2 KO and fmo-2 OE shared common pathway changes including phosphatidylcholine and phospholipid biosynthesis (Figure S11, S12) but differences between WT and the fmo-2 OE line were observed in other pathways, such as mitochondrial beta-oxidation of short, medium and long chain fatty acids, ethanol metabolism, riboflavin metabolism and urea cycle, that did not occur in fmo-2 KO extracts. For fmo-4 KO, the discriminating metabolites were also involved in phenylalanine and tyrosine metabolism, purine metabolism, glutamate metabolism and phenylacetate metabolism (Figure S12). Metabolic profiles of extracts from fmo mutants at day 6 post-hatching At day 6 post-hatching, PCA of NMR spectra of the extracts of WT and all fmo mutants combined showed no sample overlaps between the WT and fmo-2 KO, fmo-2 OE and fmo-3 KO on PC1 (see Fig. 3 A). fmo-1 KO, in contrast, showed a degree of separation but with some overlap with WT. Conducting a PCA of WT separately versus each fmo mutant no sample overlaps with fmo-3 KO (Fig. 3 B; Figure S13), fmo-2 KO (Figure S14) and fmo-2 OE (Figure S15) on PC1. PCA of fmo-4 KO (Figure S16) versus WT showed complete overlap on PC1 and some overlap on PC2, and the PCA of fmo-1 KO (Figure S17) versus WT also showed some overlaps on PC1 and PC2. The metabolome of each mutant at day 6 post-hatching was once again evaluated for discriminating metabolites using PCA loadings and ANOVA (Table S2). The discriminating metabolites of each strain allowed enriched pathways to be constructed for each strain (Figures S18-19). In the two strains with increased lifespan ( fmo-3 KO and fmo-2 OE) the discriminating metabolites were from pathways including the urea cycle and phenylacetate metabolism (Figure S19 Error! Reference source not found. ). fmo-2 OE also had discriminating metabolites involved in pyrimidine metabolism and nicotinate and nicotinamide metabolism (Figure S19 Error! Reference source not found. ) and those for fmo-2 KO included betaine metabolism, and transfer of acyl groups into mitochondria (Figure S18). Metabolic profiles of extracts of fmo mutants at day 9 post-hatching The metabonomics study extended to an additional timepoint for fmo-4 KO vs WT, at day 9 post-hatching, following its significant metabolic differences vs WT at day 3, and because fmo-4 KO had a significant lifespan extension over WT (Said et al., 2024 ). At day 9 post-hatching, PCA once again showed that fmo-4 KO and WT had distinct metabolic profiles (Fig. 4 Error! Reference source not found. , Figure S20) with increased levels of tryptophan, choline and phosphorylcholine, but decreased glutamine, asparagine, cystathionine, aspartate, agmatine, 5′-UMP, trehalose and 5'-guanosine monophosphate (GMP) in fmo-4 KO (Table S3 Error! Reference source not found. ). The discriminating metabolites of fmo-4 KO at day 9 post-hatching were involved in several pathways including aspartate metabolism, valine, leucine and isoleucine metabolism, phosphatidylcholine metabolism and betaine metabolism ( S21 Error! Reference source not found. ). Metabolic profile changes as a result of ageing in WT and fmo mutant worms Having characterised WT and fmo-4 KO worms at three different stages (day 3, 6 and 9 post-hatching), PCA of NMR-based metabolite extract profiles determined the metabolic trajectories of both strains over time. Both strains followed a similar metabolic trajectory from day 3 to day 6 post-hatching, with both groups moving to lower PC1 values and becoming more diffuse and overlapped (Figure S22). Notably, from day 6 to day 9, the WT spectral profiles did not move whereas the fmo-4 KO worm metabolic profiles moved to lower PC1 and PC2 values and became distinct again from the WT (Figure S22 Error! Reference source not found. ). The metabolic changes that occurred with ageing were identified by comparing 1 H NMR metabolic profiles of WT and mutant worms at day 3 post-hatching relative to those at day 6 and day 9 post-hatching stages (Table S4-S11). The levels of alanine, lactate, asparagine, agmatine, succinate, cystathionine, histidine, 5′-AMP, 5′-ATP and 5′-UMP were decreased in day 6 post hatching WT worms whereas D-glucose levels were increased at days 6 and 9 compared with day 3 post-hatching (Table S12). Most of the fmo mutants exhibited patterns of ageing progression that largely or partially overlapped with that of the WT worms, with changes particularly in for glucose, alanine, lactate, succinate and agmatine levels as the worms aged from day 3 to day 6 post-hatching (Table S12 Error! Reference source not found. ). The most notable differences in the long-lived lines were the increased glutamine, asparagine, aspartate and glutamate levels in fmo-2 OE and increased tryptophan in fmo-4 KO, fmo-3 KO and fmo-2 OE strains. Differences were seen between fmo-4 KO and WT lines at day 9 post-hatching (see Figure S20-21), with glutamine, glutamate, leucine, citrate and 5′-GMP decreased, and the levels of formate, tryptophan, choline, phosphoryl choline, ethanol and methanol, increased in the day-9, fmo-4 KO strain (Table S12 Error! Reference source not found. ). Discussion Previous work already showed that the absence of Fmo5 in mouse had a significant impact upon ageing, fat metabolism and other metabolic processes.(Malagon et al., 2015 ) This raised the question as to whether the inactivation of fmo genes in C. elegans would alter the metabolism associated with ageing. The systematic study of phenotypic and metabolic impacts of the fmo s was particularly of interest with reference to C. elegans , whose five fmo sequences all appear to be related to human (and mouse) FMO5 (Huang et al., 2021 ). Metabonomics was applied to investigate the metabolic profiles of a series of fmo mutant worms after our observation of lifespan extension in fmo-1 KO, fmo-3 KO, fmo-2 OE and fmo-4 KO mutants (Said et al., 2024 ). We investigated whether these long-lived mutants had common changes to their metabolism, or if they were mutant-specific. Phenotypic changes due to genetic mutation In a previous study, the loss of fmo-1 , fmo-3 and fmo-4 and the overexpression of fmo-2 increased longevity compared with WT C. elegans worms (Said et al., 2024 ). In this study, we showed that fmo-4 KO, fmo-2 KO and fmo-3 KO worms exhibited a delayed developmental phenotype, reaching the adult stage about 4–5 h later than WT, timed from the beginning of egg production. Fmo-1 KO and fmo-2 OE mutants did not show the same developmental delay. (Table 1 ; Figure S1 ). These results indicated that FMO enzymes have a role in C. elegans development and in extending C. elegans lifespan. Although another recent study reported that fmo-4 KO had no effect on C. elegans lifespan (Tuckowski et al., 2024), feeding levels were higher in the latter study, a difference in experimental conditions that could affect this model, which is highly sensitive to environment and diet (Ezcurra et al 2011). A role for fmo-1 and fmo-4 in development is suggested by their transcriptional upregulation in C. elegans at larvae stage versus adult (Said et al., 2024 ). However, phenotype was not correlated with fmo transcriptional regulation at larval versus embryo and adult stages (Said et al., 2024 ), suggesting that individual fmo genes play distinct and nonredundant roles that are not all correlated with worm development. The fmo-2 KO was the only C. elegans mutant studied here that showed a distinctive behaviour phenotype, of swarming or aggregation (Table 1 ; Fig. 1 ). Swarming is one of the most complex social behaviours exhibited by C. elegans (Avery et al., 2021 ). It was reported that starvation in larvae (L1) induced swarming (Artyukhin et al., 2015 ). C. elegans was reported to exhibit a strong behavioural preference for 5–12% oxygen, avoiding lower or higher levels of oxygen, and a link with both swarming and starvation is found in the observation that social feeding occurred only when oxygen exceeded the preferred level (Gray et al., 2004 ). Mutation in the npr-1 gene, which encodes a predicted G protein-coupled receptor similar to neuropeptide Y receptors, causes a solitary strain to take on social behaviour (de Bono and Bargmann, 1998 ), suggesting the inclusion of Npr1 in further experiments to explain swarming behaviour upon loss of fmo-2 . Phenotypic analysis supports an important role in C. elegans for fmo-4 as its loss both increased sensitivity to bleach treatment (Figure S3) and increased embryo lethality relative to WT. The decreased rate of egg hatching was not a result of decreased egg formation rate (Table 1 ; Figure S3). The increased sensitivity to bleach treatment agrees with a reported osmoregulatory role for fmo-4 , loss of which affects clearance of excess water during hypotonicity (Hirani et al., 2016 ). A role for fmo-4 is plausible in the synthesis of an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells (Hirani et al., 2016 ). Alternatively, loss of fmo-4 could affect composition of the cuticle of the worm, which provides the first line of defence against chemical and microbial stressors (Page and Johnstone, 2007 ). As disruption of cuticle collagen activates osmolyte- and antimicrobial- response genes (Dodd et al., 2018 ), this aspect of inactivation of fmo-4 requires further investigation for multiple aspects of the phenotype. There may be other, redundant functions for fmo-4 that, at least partially, overlap with fmo-2 , since inactivation of fmo-4 led to up-regulation of fmo-2 by ca. 15- to 30-fold (Said et al., 2024 ). Shared fmo-2 functions would therefore be interesting regarding the fmo-4 KO’s differences in egg hatching and worm development (Table 1 ). It was reported that mutation of C. elegans drp-1 (dynamin related protein 1; mitochondrial pro-fission gene) is associated with defects in mitochondria segregation in gonads, which could be associated with increased embryonic lethality (Labrousse et al., 1999 ) but also with ageing, as mitochondrial size may be related with ageing in C. elegans and other organisms. Therefore, a future investigation of the effect of fmo-4 KO on the transcription level of drp-1 gene is also suggested. This study uncovered several new potential endogenous roles of C. elegans fmo s. Table 1 shows the different phenotypes associated with different fmo mutant strains. Table 1 Summary of phenotypes of fmo mutant C. elegans worms Strain Phenotypes Lifespan (Said et al., 2024 ) Development Egg hatching Distinctive social behaviour fmo-1 KO Extended lifespan No effect No difference observed No effect fmo - 2 KO No effect Delayed development No difference observed aggregation and grouped feeding behaviour fmo-3 KO Extended lifespan Delayed development No difference observed No effect fmo-4 KO Extended lifespan Delayed development Decreased hatching rate No effect fmo-5 KO No effect No effect No difference observed No effect fmo-2 OE Extend lifespan No effect No difference observed No effect Changes in metabolite profiles of adult worms resulting from genetic mutation The metabolome of each strain was analysed at day 3 post-hatching to compare the effect of each fmo mutation on the adult stage worm composition. Multivariate analysis (PCA) revealed that all fmo mutant strains possessed a distinct metabolic profile compared with WT, except for fmo-1 KO, which had a similar although more diffuse metabolome (Figure S8). Higher levels of BCAAs were observed in fmo-2 KO, fmo-4 KO and fmo-2 OE strains compared to WT (day 3; by PCA and ANOVA, Table S1 ). Like other animals, C. elegans cannot synthesise BCAAs (Payne and Loomis, 2006 ), and so any difference in their relative concentrations must be due to a change in either protein turnover or BCAA catabolism (Brosnan and Brosnan, 2006 ). Down-regulation of the branched-chain α-ketoacid dehydrogenase complex was hypothesised to be responsible for increased BCAA pool sizes in a mutant in a developmental arrest gene, daf-2 (Fuchs et al., 2010 ) which was linked with longevity, and affects fertility and embryonic development. The increased day-3 BCAAs levels in the fmo-4 KO, which had effects in all these aspects of the C elegans life cycle, may be caused by the same mechanism. fmo-4 KO also showed increased levels of threonine, phenylalanine and tyrosine at day 3 post-hatching compared with WT. These changes of amino acid level in the fmo mutants could also result from a role in development for fmo-4 because these amino acids are the building blocks of proteins formed during growth and development of an organism (Edwards et al., 2015 ). Interestingly, the levels of agmatine, phosphorylcholine (PCho) and choline were decreased in both KO and OE strains of fmo-2 . PCho is produced from choline phosphorylation by choline kinase. Meanwhile, endoplasmic reticulum (ER) stress activated choline kinase (CKB-2) expression, which was linked with ageing, and low phosphocholine (PCho) was correlated with high life expectancy (Pontoizeau et al., 2014 ). The decreased level of PCho in these fmo-2 mutant worms tallies with this and could therefore be related to effects on or from CKB-2. Changes of metabolite levels due to ageing If the metabolome is causally linked to lifespan extension, then the metabolomes of fmo-1 KO, fmo-3 KO, fmo-4 KO and fmo-2 OE mutants should be different from that of wild-type at some, or even all, stages of the C. elegans life cycle. Multivariate analysis by PCA showed a distinct metabolic profile compared with WT for the fmo-4 KO C. elegans mutant at days 3 and 9 but not at day 6 post-hatching. The fmo-1 KO was the only lifespan-extended strain that did not show any metabolic separation at day 3 or 6 post-hatching (Figures S8 and S17). The lack of effect of removing fmo-1 on 1 H NMR-detectable C. elegans metabolite profiles suggests that lifespan extension may not always be correlated with patent metabolite profiles changes. The number of significantly different metabolites between fmo-2 KO, fmo-2 OE and fmo-3 KO relative to WT, however, increased with ageing (Table S1 &S2). There were no significant differences in the BCAA levels at day 6 post-hatching between WT and any fmo mutants (Table S2) but BCAA levels were down-regulated in fmo-4 KO at day 9 post-hatching (Table S3). The same was also seen in two other long-lived strains, namely eat-2 ( ad465 ) and slcf-1 ( tm2258 ), both of which had decreased leucine vs WT in 7-day-old adult worms (Pontoizeau et al., 2014 ). Although the metabolomes of both glp-1 KO and the long-lived daf-2 mutant, mentioned above, showed elevated levels of BCAAs at 10 days’ of age (Fuchs et al., 2010 ); (Wan et al., 2017 ). The abundance of PCho and choline pathway activation with age (Pontoizeau et al., 2014 ) means that the increased level of PCho in 9-day, fmo-4 KO worms could be due to CKB-2 activation, perhaps to compensate for stress from ageing. This contrasts, however, with a previous study of other long-lived mutants which concluded that low PCho levels correlated with high life expectancy in C. elegans (Pontoizeau et al., 2014 ) (Fuchs et al., 2010 ). The results for fmo-4 KO at day 9 post-hatching, with increased levels of PCho, choline and trimethylglycine, are consistent with reports of the WT dauer metabolome which shows elevated levels of phosphoserine, hydroxyproline and choline compounds (Fuchs et al., 2010 ). These changes were also correlated for long lived mutants in this study and also for dauer stage nematodes, grown in the same conditions (Fuchs et al., 2010 ). The long-lived mutants, fmo-3 KO and fmo-2 OE at day 6 and fmo-4 KO at day 9 post-hatching, showed higher levels of tryptophan relative to WT, seen previously in dauer stage and long-lived mutants daf-2 and ife-2 (Fuchs et al., 2010 ). In C. elegans , blocking tryptophan catabolism may extend lifespan via regulation of proteotoxicity (accumulation of damaged or misfolded proteins) (van der Goot et al., 2012 ) whereas in human serum, tryptophan levels decreased with ageing (Yu et al., 2012 ) and toxic tryptophan catabolites increased (Ramos-Chávez et al., 2018 ). An increase in tryptophan catabolism with ageing may result from increased levels of the enzyme indoleamine-2, 3-dioxygenase (IDO) whereas depletion of tryptophan 2, 3-dioxygenase ( tdo-2 ) increased tryptophan levels (Ramos-Chávez et al., 2018 ). Literature reports (Edwards et al., 2008 ; − Bennett and Kaeberlein, 2014 ; Bennett et al., 2014 ), support a role for the increased tryptophan levels seen in fmo-3 KO, fmo-4 KO and fmo-2 OE mutants in extending lifespan in these strains. Although there were some differences between the results of the present work and previous ageing studies, metabolomes are sensitive to precise experimental conditions and genetic changes, the exact life-stage targeted, and to extraction and analysis methods. Furthermore, using FUdR resulted in genotype-specific effects (in daf-2 mutant vs WT nematodes) on levels of eight specific metabolites (Davies, Leroi and Bundy, 2012 ), but collecting sufficient biomass for metabolite analysis would not be possible without the use of FUdR, or a similar intervention, to synchronise cultures (Davies, Leroi and Bundy, 2012 ).Therefore, we developed a gravity worm filtering protocol (Said et al. , manuscript in preparation) removing the need for use of FUdR. It is likely therefore that there would be some variation between the results in this study compared with those studies that made use of chemically synchronised samples. A proposed mechanism of lifespan extension for fmo mutants One of the major functions of HLH-30 is to regulate autophagy and promote longevity in C. elegans (Lapierre et al., 2013 ; O'Rourke and Ruvkun, 2013 ). fmo-2 is a target of HLH-30, and was induced both by hypoxia and starvation (complete bacterial food source removal) and induction by starvation was dependent on HLH-30 (Leiser et al., 2015 ). Interestingly, reduction in tald-1 activity by RNAi knockdown also resulted in high lifespan worms that lacked the hypoxic response transcription factor HIF-1 (Bennett et al., 2017 ). The life-extending effects of hypoxia in C. elegans begin in neurons with HIF-1-upregulated transcription and increased serotonergic signalling (Leiser et al., 2015 ). These effects increased production of FMO-2 in the intestine, and increased longevity, whereas knocking out fmo-2 did not affect C. elegans lifespan (Leiser et al., 2015 ). The latter findings (Leiser et al., 2015 ) were similar to those in the present study. A new endogenous metabolic pathway of FMOs that relates to ageing processes was found to involve oxygenation of tryptophan, forming N -formyl-kynurenine, which was then converted to kynurenine by formamidase (Choi et al., 2023 ). Formate is produced as a byproduct when kynurenine is synthesised from N -formyl-kynurenine by formamidase (Brosnan and Brosnan, 2016 ). A hypothesis whereby increased levels of formate confer stress resistance and lifespan extension under metabolically stressful conditions, such as hypoxia or DR (Choi et al., 2023 ), could explain increased tryptophan and formate in fmo-4 KO at day 9 post-hatching, and could link C. elegans lifespan extension in fmo-4 KO with that in the fmo-2 OE and fmo-3 KO mutants (Choi et al., 2023 ). Interestingly, fmo-1 KO was a long-lived mutant that did not show elevated tryptophan with ageing. Thus, we propose that fmo-1 KO extends worm lifespan via a different mechanism. This is also supported by the previous study with the same lines, in which fmo-2 transcription was upregulated 15- to 30-fold compared with WT upon loss of fmo-4 , whereas loss of fmo-1 up-regulated fmo-2 by only approx. threefold (Said et al., 2024 ). In contrast, long-lived tald-1 mutant C. elegans had 30-40-fold increased fmo-2 transcription relative to WT (Bennett et al., 2017 ). Thus, the up-regulation of fmo-2 upon loss of fmo-4 could be the cause of lifespan extension in the fmo-4 KO strain. As the overexpression of fmo-2 was reported to extend C. elegans lifespan in four different conditions including hypoxia, DR (Leiser et al., 2015 ), MAPK and HLH-30 activation, (Bennett et al., 2017 ), an important future experiment would be to determine which environmental condition or transcription factor is responsible for fmo-2 up-regulation in fmo-4 KO. The proposed, linked mechanisms of action in fmo-4 KO resulting in extended C. elegans lifespan are shown in Fig. 5 Error! Reference source not found. . Conclusion This study indicated that there are interlinked but non-redundant roles for C. elegans fmo s, and revealed phenotypic differences between C. elegans fmo mutants. Loss of fmo-4 significantly affected on egg hatching and sensitivity to bleach, but, for example, the fmo-2 KO strain showed distinct swarming and aggregation behaviour. The metabolome of the long-lived fmo-3 KO, fmo-4 KO and fmo-2 OE lines displayed higher tryptophan levels during ageing. fmo-3 KO and fmo-4 KO, but not fmo-1 KO, may therefore extend the C. elegans lifespan via the same mechanism as fmo-2 OE. Because fmo-4 KO showed significant upregulation of fmo-2 over WT levels, it was hypothesised that overexpression of fmo-2 in fmo-4 KO was responsible for fmo-4 lifespan extension. Further experiments are needed, including identifying transcription factor involvement, to confirm whether fmo-4 KO and fmo-2 OE do indeed act by the same mechanism. Material and methods C. elegans strains and maintenance Animals were cultures at 20°C and maintained on OP50 seeded NGM plates. C. elegans strains were purchased from the Caenorhabditis Genetic Center (CGC, Minnesota, USA). C. elegans strains used in this study: Bristol (N2) strain as the wild-type strain, fmo-1 KO (RB671 [ fmo-1 ( ok405 ) IV ]), fmo-2 KO (VC1668 [ fmo-2 ( ok2147 ) IV ]), fmo-3 KO (RB686[ fmo-3 (ok354) III]), fmo-4 KO (RB562 [ fmo-4 ( ok294 ) V ]), fmo-5 KO (tm2438) and fmo-2 OE (KAE10 [ seaSi40 I; unc-119(ed3) III ]). Worm length measurement Synchronised cultures of WT and fmo mutants ( fmo-1 KO, fmo-2 KO, fmo-3 KO and fmo-4 KO) were obtained using our developed egg preparation protocol. Egg of each strains were transferred to a new seeded NGM plate and left for 3 days until they reached the adult stage. 15–20 worms of each strain were immobilised using 40 µl of 1mM tetramisole hydrochloride (levamisole; Sigma). Images were taken using an M80 stereomicroscope (Leica, Wetzlar, Germany). Worm length was measured using Image J (Schindelin et al., 2012 ). The analysis was done using One-way ANOVA. Worm length of WT and fmo-5 KO was also measured using an AI software ( C. elegans length measurement; Rapid Biolabs). Bleach time test (cuticle sensitivity test) 0.2 ml worm pellet of each strain were re-suspended in 7 ml M9 buffer, and 2 ml of 1M NaOH and 3 ml of 7.5% sodium hypochlorite (bleach) were added to each sample. The time for 95–100% of worms to be disrupted of each strain was counted. Each experiment consisted of three biological replicates and the analysis was done using student t-test. Egg hatching rate 100–150 eggs were transferred to each of three plates for each strain. Eggs were obtained using egg preparation protocol or by picking from the original plates. All plates were incubated at 20°C for three days, then adult hatched worms were counted on each plate. The percentage of the hatched worms on each plate was calculated. The mean and SEM of the three repeats for each strain were calculated and plotted using Graphpad Prism 6.0 software (La Jolla, California, USA). The data were analysed using student t-test. Images of each plate was taken to double check the number of adult worms and to determine the visual differences between strains. Egg laying rate Normally this assay was done by letting number of L4/adult worms to lay eggs for 12 hrs and repetitive transfer to new plates every 12 h and let eggs laid on each plate to hatch, two days later, number of worms per each plate was counted. This normal assay is not appropriate to the strains with reduced egg hatching rate as the count of hatched eggs will not be indicative for the total number of eggs laid. Modified egg laying rate was developed to overcome this problem, this assay aimed to count eggs instead of counting worms post hatching. 3–5 adult (1st day of adulthood) worms were transferred to 35mm NGM plates seeded with OP50. All plates were left for 12 h. Number of eggs and hatched larvae were counted and recorded. The advantages of the modified assay was being suitable for the stains with reduced egg hatching rate, it was a combination of egg hatching and egg laying rate as results could be divided into number of laid eggs in 12 h and the rate of eggs hatched could be calculated from the ratio of hatched worms to the total number of laid eggs. Maintaining and growing C. elegans to the required stage C. elegans WT and different fmo mutants ( fmo-1 KO, fmo-2 KO, fmo-3 KO, fmo-4 KO, fmo-5 KO and fmo-2 OE) were tested metabolically at day 3 and day 6 post-hatching, in addition WT and fmo-4 KO were also tested at day 9 post-hatching. Different strains were maintained and the synchronised cultures were obtained by using the developed egg preparation protocol. 100 µl egg pellet was transferred to each OP50 seeded NGM plate, hatched larvae were checked under the microscope daily for growth. For each strain, 5 different biological repeats were obtained at each tested stage. Worms were combined from three individual 90 mm NGM plates to make a single replicate. For day 3 post-hatching stage, once they reach the adulthood stage (defined as the point where there were eggs seen on the plates but no new-generation worms had hatched), they were ready for metabolite extraction. The adult worms of each strain were collected in 15 ml Falcon tubes using M9 buffer, all tubes were spun at 1,300 g for 1 min. The supernatant was discarded, worm pellets were washed three times with 10 ml M9 buffer to get rid of the E. coli . Samples were snap frozen and stored at -80°C until metabolite extraction. For the day 6 and 9 post-hatching stages, worms of each strain after they reached the adult stage (day 3 post-hatching) were washed and filtered from the larvae worms (new progeny) daily using our developed gravity worm filter protocol (Said et al. , manuscript in preparation). Mother worms were transferred to new seeded plates to supply the worms with OP50. At each stage, worms were collected and harvested using the same method as day 3 stage. Metabolite extraction of C. elegans is critical, and required sufficient worms to give a 0.3–0.5 ml pellet of synchronised adult worms, so these steps were repeated several times to obtain the ideal mass of the required worms. Metabolite extraction and sample preparation for NMR analysis Metabolite extraction from different C. elegans strains was performed using methanol and worms were disrupted using zirconium beads (Sigma, Dorset, UK) for 5 min in TissueLyser II (Qiagen) at 30 Hz. The metabolite extracts of all C. elegans strains were dried overnight in a RapidVap Vertex Evaporator (LABCONCO, UK) before NMR analysis. Dried extracts were vortexed and resuspended with 240 µl of phosphate buffer (pH. 7.4; 0.93 g NaH 2 PO 4 (Fisher), 1.04 g K 2 HPO 4 (Fisher), 0.86 mg TSP (Sigma) and 5.85 mg sodium azide (Fisher), all dissolved in 10 ml D 2 O (Sigma)). Eppendorf tubes were centrifuged for 10 min at 12,000 g at 4°C. 200 µl of each extract was transferred to new 3mm diameter NMR tubes (SampleJet Tube 3.0x103.5mm) by using eVol XR electronic syringe (SGE-Analytical Science, UK). Nuclear Magnetic Resonance Spectroscopy. All sample were analysed at 300.0 K by 600 MHz 1 H NMR (Bruker Spectrospin) using a cooled Bruker Sample Jet to store the samples prior to pre-heating and then insertion in the magnet. 1D 1 H NMR spectra were acquired with the Bruker 1D NOESY water suppression pulse sequence noesygppr1d. 128 scans were accumulated into 32K data points with a sweep widthe of ca 20 ppm (12,019 Hz) and a relaxation delay of 4.0 s. The 1 H NMR free induction decays were zero-filled to 128 K data points and apodised with a line broadening of 0.3 Hz, prior to Fourier transformation, manual phasing and baseline correction where necessary using MNova version 12.0.1.20560 (Mestrelab, 2018). Various 2D 1 H NMR experiments were acquired as previously described (Veeravalli et al, 2022 ) Metabolite identification Metabolites were identified by three complementary methods: (I) comparison with reference spectra from the human metabolome database (HMDB; http://www.hmdb.ca/ ; Wishart et al. , 2007) and the biological magnetic resonance data bank (BMRB; http://www.bmrb.wisc.edu/metabolomics/ ); (II) analysis of previously published data; and (III) the interpretation of a series of two-dimensional (2D) spectra such as 1 H COSY, JRES and HSQC, using published methods (Dona et al., 2016 ). Multivariate Statistical Analyses. Regions of the 1 H NMR spectra without signals (upfield of 0.5 ppm and downfield of 9.5 ppm) and the region containing residual water signals at ca 4.8 ppm were removed. The spectra were normalised to a total signal area of 100 and thensuperimposed on one another by stacking in MNova (Mestrelab, 2018). The spectra were saved as a csv file (transposed comma separated variable) and then imported into Matlab 2018b (Mathworks, UK) for processing as a cellular array. The data were carefully aligned using the isoshifft algorithm (Tomasi et al, 2011 ), the relevant sample metadata (sample name, sample genotype, sample age) were imported and the spectra were bucketed into segments typically of 0.04 ppm spectral width. Further statistical analysis was performed in PLS-Toolbox 9.3 (Eigenvector USA) using standard methods such as principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA). Declarations Additional Information Supplementary information accompanies this paper at … Competing Interests Dr M. Said is a founder and CSO of Rapid Biolabs, an AI company involved in C. elegans and other imaging. The other authors declare no conflicts of interest. Funding We thank the University of Greenwich for support for the PhD studentship of MS. 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(2016a) 'Lifespan-regulating genes in C. elegans', Npj Aging and Mechanisms of Disease, 2. Uno, M. and Nishida, E. (2016b) 'Lifespan-regulating genes in C. elegans', Npj Aging and Mechanisms of Disease, 2, pp. 8. van der Goot, A. T., Zhu, W., Vázquez-Manrique, R. P., Seinstra, R. I., Dettmer, K., Michels, H., Farina, F., Krijnen, J., Melki, R., Buijsman, R. C., Ruiz Silva, M., Thijssen, K. L., Kema, I. P., Neri, C., Oefner, P. J. and Nollen, E. A. A. (2012) 'Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation', Proceedings of the National Academy of Sciences, 109(37), pp. 14912. van Heemst, D. (2010) 'Insulin, IGF-1 and longevity', Aging Dis, 1(2), pp. 147-57. Varshavi, D., Scott, F. H., Veeravalli, S., Phillips, I. R., Veselkov, K., Strittmatter, N., Takats, Z., Shephard, E. A. and Everett, J. R. (2018) 'Metabolic Biomarkers of Ageing in C57BL/6J Wild-Type and Flavin-Containing Monooxygenase 5 (FMO5)-Knockout Mice', Front Mol Biosci, 5, pp. 28. Veeravalli, S., Phillips, I. R., Freire, R. T., Varshavi, D., Everett, J. R. and Shephard, E. A. (2020) 'Flavin-Containing Monooxygenase 1 Catalyzes the Production of Taurine from Hypotaurine', Drug Metab Dispos, 48(5), pp. 378-385. Veeravalli, S., Varshavi, D., Scott, F. H., Pullen, F. S., Veselkov, K., Phillips, I. R., Everett, J. R. and Shephard, E. A. (2022) 'Treatment of wild-type mice with 2,3-butanediol, a urinary biomarker of Fmo5 (-/-) mice, decreases plasma cholesterol and epididymal fat deposition', Front Physiol, 13, pp. 859681. Wan, Q. L., Shi, X., Liu, J., Ding, A. J., Pu, Y. Z., Li, Z., Wu, G. S. and Luo, H. R. (2017) 'Metabolomic signature associated with reproduction-regulated aging in Caenorhabditis elegans', Aging (Albany NY), 9(2), pp. 447-474. Wani, K. A., Goswamy, D., Taubert, S., Ratnappan, R., Ghazi, A. and Irazoqui, J. E. (2021) 'NHR-49/PPAR-α and HLH-30/TFEB cooperate for C. elegans host defense via a flavin-containing monooxygenase', eLife, 10, pp. e62775. Watson, D. G. (2013) 'A rough guide to metabolite identification using high resolution liquid chromatography mass spectrometry in metabolomic profiling in metazoans', Computational and structural biotechnology journal, 4(5), pp. 1-10. Yamazaki, H. and Shimizu, M. (2007) 'Genetic polymorphism of the flavin-containing monooxygenase 3 (FMO3) associated with trimethylaminuria (fish odor syndrome): observations from Japanese patients', Curr Drug Metab, 8(5), pp. 487-91. Yu, Z., Zhai, G., Singmann, P., He, Y., Xu, T., Prehn, C., Römisch-Margl, W., Lattka, E., Gieger, C., Soranzo, N., Heinrich, J., Standl, M., Thiering, E., Mittelstraß, K., Wichmann, H.-E., Peters, A., Suhre, K., Li, Y., Adamski, J., Spector, T. D., Illig, T. and Wang-Sattler, R. (2012) 'Human serum metabolic profiles are age dependent', Aging Cell, 11(6), pp. 960-967. Yurekten, O., Payne, T., Tejere, N., Amaladoss, F.X., Martin, C., Williams, M. and O'Donovan, C. (2023) MetaboLights: open data repository for metabolomics, Nucleic Acids Research , 52 (D1) D640-D646; doi.org/10.1093/nar/gkad1045 Additional Declarations Competing interest reported. Dr M. Said is a founder and CSO of Rapid Biolabs, an AI company involved in C. elegans and other imaging. The other authors declare no conflicts of interest. Supplementary Files SUPPINFO10072025.pdf Cite Share Download PDF Status: Published Journal Publication published 15 Nov, 2025 Read the published version in Metabolomics → Version 1 posted Editorial decision: Revision requested 27 Sep, 2025 Reviews received at journal 26 Sep, 2025 Reviewers agreed at journal 29 Aug, 2025 Reviews received at journal 28 Aug, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviewers invited by journal 15 Jul, 2025 Editor assigned by journal 15 Jul, 2025 Submission checks completed at journal 15 Jul, 2025 First submitted to journal 14 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7122670","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486179725,"identity":"852daa72-3522-4315-a0eb-0d97b464dd22","order_by":0,"name":"Mohamed Said","email":"","orcid":"","institution":"University of Greenwich","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"Said","suffix":""},{"id":486179728,"identity":"e20d3b4f-7abf-414a-b54e-830a42c9b75d","order_by":1,"name":"Rafael Freire","email":"","orcid":"","institution":"University of Greenwich","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"Freire","suffix":""},{"id":486179729,"identity":"6e2994a5-510b-48de-9a41-b4e5dc6f23c0","order_by":2,"name":"Filipe Cabreiro","email":"","orcid":"","institution":"University of Cologne","correspondingAuthor":false,"prefix":"","firstName":"Filipe","middleName":"","lastName":"Cabreiro","suffix":""},{"id":486179731,"identity":"98d357ac-6514-428a-9004-aa72adfa3f98","order_by":3,"name":"Jose Ivan Serrano-Contreras","email":"","orcid":"","institution":"Imperial College","correspondingAuthor":false,"prefix":"","firstName":"Jose","middleName":"Ivan","lastName":"Serrano-Contreras","suffix":""},{"id":486179733,"identity":"a2c7c6ec-6afc-490e-8c3d-b6a6539b53e8","order_by":4,"name":"Elinor P Thompson","email":"","orcid":"","institution":"University of Greenwich","correspondingAuthor":false,"prefix":"","firstName":"Elinor","middleName":"P","lastName":"Thompson","suffix":""},{"id":486179735,"identity":"429a1ce0-dba0-4fec-8e4d-76d96832795b","order_by":5,"name":"Jeremy R Everett","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYBACxgYk9gOYGDOEwUZACxszswFRWhCAjZlNAsbGq4W5gfcBw8c2m3x++f5jlV/32MiZSyQ3fi5gsJNnkEhLwO4wdgPGmW1pljPbmNluyzxLM7ackdgsPYMh2bBBIu0Adi1sDMw8Zw4bGBwDapE4cDhxw+3ENmYeBuYEBon0Bpxa/pz5b2AP1FIsceB/PVRLPX4tDBUHDAyA3mf8cOBAggFEy2GgFhwOa2ZjONhTkWwgcSzZWJrhQLLhhvsPm6V5DI4btvE8w+p9w/Y2xgc/DOwM+JsPPvz444CdvMGZ4w8/81RUy/Ozpxlg1dLMwAC3HugeGDDAHZHyKK78gUPVKBgFo2AUjGwAAG4oVcb9PobOAAAAAElFTkSuQmCC","orcid":"","institution":"University of Greenwich","correspondingAuthor":true,"prefix":"","firstName":"Jeremy","middleName":"R","lastName":"Everett","suffix":""}],"badges":[],"createdAt":"2025-07-14 15:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7122670/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7122670/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11306-025-02367-4","type":"published","date":"2025-11-15T15:58:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86967193,"identity":"7d83377c-e496-4555-864c-459d55423968","added_by":"auto","created_at":"2025-07-17 17:50:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":520753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003efmo-2\u003c/em\u003e KO \u003cem\u003eC. elegans \u003c/em\u003eswarming phenotype. Groups of worms on the bacterial lawn (black arrows) were observed on \u003cem\u003efmo\u003c/em\u003e-2 KO plates versus WT worms spread across the plate.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7122670/v1/e69b8821a50202271b016da2.png"},{"id":86967190,"identity":"606c2b14-1787-405e-b53e-044db838c5b3","added_by":"auto","created_at":"2025-07-17 17:50:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA,\u003c/strong\u003e PCA of data from 600 MHz \u003csup\u003e1\u003c/sup\u003eH NMR spectra of metabolic extracts of WT and \u003cem\u003efmo\u003c/em\u003e mutant \u003cem\u003eC. elegans\u003c/em\u003e worms at day 3 post-hatching. In the two-component model, PC1 explained 46% of the total variance and PC2 explained 29%. N = 5. \u003cstrong\u003eB,\u003c/strong\u003e PCA of data from 600 MHz \u003csup\u003e1\u003c/sup\u003eH NMR of metabolic extract of WT and \u003cem\u003efmo-4\u003c/em\u003e KO at day 3 post-hatching. In the two-component model, PC1 explained 33% of the total variance and PC2 explained 21%. N = 5.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7122670/v1/51d27b5afa4d6c6ef34ef9ae.png"},{"id":86967818,"identity":"8a032d2c-ada3-45a5-b285-a363c18e54b6","added_by":"auto","created_at":"2025-07-17 17:58:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":86536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA,\u003c/strong\u003e PCA of data from 600 MHz \u003csup\u003e1\u003c/sup\u003eH NMR of metabolic extracts of WT and \u003cem\u003efmo\u003c/em\u003e mutants at day 6 post-hatching. In the two-component model, PC1 explained 23% of the total variance and PC2 explained 11%. \u003cstrong\u003eB,\u003c/strong\u003e PCA of data from 600 MHz \u003csup\u003e1\u003c/sup\u003eH NMR of metabolic extract of WT and \u003cem\u003efmo-3\u003c/em\u003e KO at day 6 post-hatching. In the two-component model, PC1 explained 28% of the total variance and PC2 explained 18%.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7122670/v1/3cb90a6733bbf1b4ded1ca49.png"},{"id":86967191,"identity":"e976f69d-c97b-4b3f-8d7c-dd93f4a1154c","added_by":"auto","created_at":"2025-07-17 17:50:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39996,"visible":true,"origin":"","legend":"\u003cp\u003ePCA of data from 600 MHz \u003csup\u003e1\u003c/sup\u003eH NMR of metabolic extract of \u003cem\u003eC. elegans\u003c/em\u003e WT and \u003cem\u003efmo-4\u003c/em\u003e KO at day 9 post-hatching. In the two-component model, PC1 explained 34% of the total variance and PC2 explained 15%.\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7122670/v1/f5621a63b2d13c0589b0b59a.png"},{"id":86967819,"identity":"ba043e1d-ea8a-4f09-8bed-3cdbe54e0ed8","added_by":"auto","created_at":"2025-07-17 17:58:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":163251,"visible":true,"origin":"","legend":"\u003cp\u003eA model for \u003cem\u003efmo-4\u003c/em\u003eKO-mediated longevity.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7122670/v1/2d003b9c59544f4916612c45.png"},{"id":96105109,"identity":"93ad00d8-9b46-4aec-8d43-96cea2fc69a9","added_by":"auto","created_at":"2025-11-17 16:08:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2350430,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7122670/v1/74e4af4d-9185-46f2-aef3-b7b387d0b5dd.pdf"},{"id":86967824,"identity":"3ca2fe07-c196-417b-aab6-c5824ba5acde","added_by":"auto","created_at":"2025-07-17 17:58:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":8577623,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPINFO10072025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7122670/v1/0599837a26eb4c2f8c4cc277.pdf"}],"financialInterests":"Competing interest reported. Dr M. Said is a founder and CSO of Rapid Biolabs, an AI company involved in C. elegans and other imaging. The other authors declare no conflicts of interest.","formattedTitle":"Phenotypic and metabonomics studies of FMOs in C. elegans and their roles in lifespan extension","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAgeing is a progressive and intrinsic biological degenerative process in the body, with accumulated molecular deterioration in many tissues and cellular pathways. This physical decline over time leads to cellular damage and dysfunction and, finally, to death (DiLoreto and Murphy, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bratic and Larsson, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, L\u0026oacute;pez-Ot\u0026iacute;n et al, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). One of the key drivers in 21st century healthcare research is the extension of healthy lifespan in an ageing population (Mount et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As part of these efforts to enhance quality of life and healthy ageing, it is desirable to reduce the risk, or delay the onset, of chronic diseases associated with ageing, such as Alzheimer\u0026rsquo;s and Parkinson\u0026rsquo;s diseases (Hoffman et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; DiLoreto and Murphy, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eC. elegans\u003c/em\u003e is an ideal model for the study of ageing as it has a relatively short lifespan of around 2\u0026ndash;3 weeks under normal conditions (Corsi, Wightman and Chalfie, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Altun and Hall, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).Importantly, \u003cem\u003eC. elegans\u003c/em\u003e lifespan has also been shown to be extended by single mutations in specific genes, such as components of insulin/IGF-1 like signalling pathways (van Heemst, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The discovery of systems that act in ageing and their mechanisms of action in the model organism \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e may be applicable to the improvement and extension of heathy human lifespans.\u003c/p\u003e\u003cp\u003eMetabonomics, defined as \u0026ldquo;the quantitative measurement of the multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification\u0026rdquo; (Lindon et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Everett et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), is a useful tool for investigating metabolic changes during ageing and age-related disease (Balashova et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among the factors that influence metabolism are diet, environment, disease, genetics and microbiome variation, resulting in a metabolic profile that reflects the health status of an individual (Everett et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Metabonomics can also be used to investigate changes to the metabolism in model organisms such as \u003cem\u003eC. elegans\u003c/em\u003e, and reveal relationships between genotype and phenotype that integrate across genomic and environmental factors, including the microbiome (Everett et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Raamsdonk et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The measurement of metabolites in biofluids or in tissue extracts of organisms such as \u003cem\u003eC. elegans\u003c/em\u003e can be performed using NMR spectroscopy (Everett et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lindon et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), as in this study, or mass spectrometry (MS) (Scalbert et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Watson, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFlavin-containing monooxygenases (FMOs) are NADPH-dependent enzymes, located in the membrane of the endoplasmic reticulum (ER), which catalyse the oxygenation of a wide variety of medicines and dietary-derived compounds (Krueger and Williams, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Phillips and Shephard, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), detoxifying nitrogen- and sulphur-containing drugs and xenobiotics (Phillips and Shephard, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Beyond their roles as xenobiotic-metabolising enzymes, however, FMOs are now known to be involved in various important endogenous functions in mammals. Human FMO1 was recently found to catalyse the conversion of hypotaurine to taurine, an amino acid critical for human health, utilising either NADPH or NADH as co-factor (Veeravalli et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Also, host hepatic FMO3 is the primary FMO responsible for trimethylamine \u003cem\u003eN\u003c/em\u003e-oxide (TMAO) production from trimethylamine. Genetic mutations reducing FMO3 activity result in trimethylaminuria (or fish-odour syndrome) in humans, caused by build-up of trimethylamine (Shephard, Treacy and Phillips, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yamazaki and Shimizu, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Dolphin et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Phillips and Shephard, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Another recent study of the endogenous functions of FMOs in human cells revealed that the five mammalian FMOs share a common and redundant function in stress resistance: the overexpression of the five FMOs increased stress resistance, regardless of whether they were normally expressed in that cell-type (Huang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). FMOs are conserved across the eukaryotes and, notably, can be induced by multiple lifespan-extending interventions in mice: this poses the question over whether these enzymes might play a critical role in promoting health and longevity across phyla (Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eFmo5\u003c/em\u003e knockout (KO) mice exhibited an age-related phenotype with lower body fat and weight, despite higher food intake, and lower blood glucose and cholesterol (Malagon et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Changes in metabolism due to disruption of \u003cem\u003eFmo5\u003c/em\u003e indicated that metabolic ageing was slowed through pleiotropic effects (Malagon et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Varshavi et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and recently, a specific compound, 2,3-butanediol, was shown to be a microbiome-derived biomarker for \u003cem\u003eFmo5\u003c/em\u003e KO in mice. Moreover, 2,3-butanediol treatment prompted lower cholesterol and epididymal body fat in wild-type (WT) mice, recreating aspects of the phenotype of the \u003cem\u003eFmo5\u003c/em\u003e KO (Veeravalli et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given the formation of 2,3-butanediol by the host microflora, it is interesting that mammalian FMO5 was recently shown to act as a sensor of gut bacteria (Scott et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLike mammals, the nematode \u003cem\u003eC. elegans\u003c/em\u003e encodes five FMOs, again named FMO1-5 (Petalcorin et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2005b\u003c/span\u003e). In the case of \u003cem\u003eFmo-2\u003c/em\u003e, overexpression (OE) in the \u003cem\u003eC. elegans\u003c/em\u003e intestine was reported to increase worm lifespan through activation of hypoxia inducible factor (HIF)-1 (Uno and Nishida, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003efmo-2\u003c/em\u003e OE in \u003cem\u003eC. elegans\u003c/em\u003e also enhanced resistance to proteotoxic stress within the ER and increased proteostasis in worms undergoing hypoxic responses (Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Intestinal \u003cem\u003efmo-2\u003c/em\u003e transcription was increased by dietary restriction (DR) and is necessary for DR-mediated lifespan extension (Uno and Nishida, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e). Further, intestinal \u003cem\u003efmo-2\u003c/em\u003e, which is regulated in \u003cem\u003eC. elegans\u003c/em\u003e by serotonergic signalling originating in neurons, subsequently activates the transcription factor HLH-30 (another factor also activated by DR) in the intestine. FMO-2 is thus an enzyme both necessary and sufficient for a majority of the beneficial effects of either of these longevity pathways (Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCorrespondingly, \u003cem\u003efmo-2\u003c/em\u003e and also \u003cem\u003efmo-4\u003c/em\u003e transcription was upregulated by hypoxia (Shen et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In more recent studies, oxidative stress (Goh et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and infection with either \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (PA14) (Dasgupta et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) or \u003cem\u003eStaphylococcus aureus\u003c/em\u003e was found to induce \u003cem\u003efmo-2\u003c/em\u003e, with FMO-2 required for pathogen resistance (Wani et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recent work also supported a role for \u003cem\u003efmo-2\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e innate immunity, as it\u0026rsquo;s transcription was strongly induced via NHR-49 and HLH-30 (Wani et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) in a pathogen-specific manner to impact infection survival. \u003cem\u003eC. elegans\u003c/em\u003e FMO-4 was expressed prominently in hypodermis, duct and pore cells but was absent from excretory cells (Petalcorin et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2005a\u003c/span\u003e). FMO-4 was hypothesised to possess an osmoregulatory role, promoting clearance of excess water that enters during periods of hypotonicity, potentially by synthesising an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells (Hirani et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSequence alignments of \u003cem\u003eC. elegans\u003c/em\u003e FMO-2 and mammalian FMO5 showed high conservation of catalytic residues (Choi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although \u003cem\u003eC. elegans fmo-4\u003c/em\u003e was thought to be orthologous to human FMO4, as they had similar predicted protein structures (Hirani et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the human FMO4 was not able to rescue an hypoosmotic stress sensitivity phenotype in the fmo-4 KO worm strain (Hirani et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious observations of the role of mammalian FMO5 in ageing raised the possibility that modulation of all or any \u003cem\u003eC. elegans\u003c/em\u003e FMOs may be a conserved mechanism for enhancing protein homeostasis and extending lifespan. The appropriate modulation of all FMOs might equally promote healthy ageing, improving health span, in mammals and people (Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We recently demonstrated that knockout of \u003cem\u003efmo-1, fmo-\u003c/em\u003e3 and \u003cem\u003efmo-\u003c/em\u003e4 statistically significantly extended \u003cem\u003eC. elegans\u003c/em\u003e lifespan relative to wild type (Said et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, in this study, metabolic profiles were obtained and more detailed phenotypic analyses (i.e., development, behaviour, ageing and egg-laying and hatching) were analysed for all \u003cem\u003eC. elegans fmo\u003c/em\u003e KO lines, to further delineate or identify conserved roles for all \u003cem\u003efmo\u0026rsquo;s\u003c/em\u003e in development and ageing.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003efmo-2\u003c/b\u003e \u003cb\u003eKO\u003c/b\u003e, \u003cb\u003efmo-3\u003c/b\u003e \u003cb\u003eKO and\u003c/b\u003e \u003cb\u003efmo-4\u003c/b\u003e \u003cb\u003eKO delayed\u003c/b\u003e \u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003edevelopment\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePhenotypic differences were identified between WT and \u003cem\u003efmo\u003c/em\u003e mutant \u003cem\u003eC. elegans\u003c/em\u003e worms in terms of development. The length of KO mutants of \u003cem\u003efmo-2\u003c/em\u003e (0.70 mm +/- 0.02; p\u0026thinsp;\u0026lt;\u0026thinsp;0.003), \u003cem\u003efmo-3\u003c/em\u003e (0.58 mm +/- 0.015; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and \u003cem\u003efmo-4\u003c/em\u003e (0.67 mm +/- 0.02; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) was significantly shorter at the beginning of day 3 post-hatching relative to WT (0.81 mm +/- 0.01), \u003cem\u003efmo-1\u003c/em\u003e KO (0.76 mm +/- 0.02; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, not significant (ns)) and \u003cem\u003efmo-5\u003c/em\u003e KO worms (0.76 mm +/- 0.02; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, ns) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003cb\u003eError! Reference source not found.\u003c/b\u003e). In addition, at the beginning of day 3, the plates of WT, \u003cem\u003efmo-1\u003c/em\u003e KO \u003cem\u003efmo-2-\u003c/em\u003eOE and \u003cem\u003efmo-5\u003c/em\u003e KO \u003cem\u003eC. elegans\u003c/em\u003e contained the expected mix of eggs and mother worms, whereas plates of \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-3\u003c/em\u003e KO and \u003cem\u003efmo-4\u003c/em\u003e KO contained mainly mother worms and were delayed in their development by approx. 5\u0026ndash;6 h (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). At the end of day 3, the worms of \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-3\u003c/em\u003e KO and \u003cem\u003efmo-4\u003c/em\u003e KO mutants began to lay eggs, whereas on WT and \u003cem\u003efmo-1\u003c/em\u003e KO and \u003cem\u003efmo-5\u003c/em\u003e KO mutant plates, many eggs were visible (Figure S2).\u003c/p\u003e\u003cp\u003e\u003cb\u003eLoss of\u003c/b\u003e \u003cb\u003efmo-4\u003c/b\u003e \u003cb\u003eincreased\u003c/b\u003e \u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003echemical/osmotic sensitivity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA difference was observed in resistance to a strong oxidising agent in \u003cem\u003efmo-\u003c/em\u003e4 KO. During \u003cem\u003eC. elegans\u003c/em\u003e egg preparation, the \u003cem\u003efmo-4\u003c/em\u003e KO strain resistance of its cuticle to bleach treatment (see Methods) was decreased compared with WT worms: the time required for bleach to disrupt ca. 95% of worms in \u003cem\u003efmo-4\u003c/em\u003e KO was 2.12 +/- 0.13 min; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas the time required for WT \u003cem\u003eC. elegans\u003c/em\u003e was 5.19 +/- 0.12 min (Figure S3A-C)\u003cb\u003eError! Reference source not found.\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLoss of\u003c/b\u003e \u003cb\u003efmo-4\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003ewas deleterious to embryo hatching\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt day 3 post-hatching, a difference could be seen clearly in the number of adult \u003cem\u003eC. elegans\u003c/em\u003e worms between WT and \u003cem\u003efmo-4\u003c/em\u003e KO. A mean (+/- SEM) of only 57 +/- 2% of \u003cem\u003efmo-4\u003c/em\u003e KO eggs hatched, compared with 95 +/- 2% of eggs hatching in WT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0002, Figure S3D-G). Interestingly, \u003cem\u003efmo-4\u003c/em\u003e KO was the only \u003cem\u003eC. elegans\u003c/em\u003e mutant that showed a large decrease in the number of adult worms in comparison to WT at day 3 post-hatching (data not shown).\u003c/p\u003e\u003cp\u003e\u003cb\u003efmo-2\u003c/b\u003e \u003cb\u003eKO\u003c/b\u003e \u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003eswarming phenotype\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003efmo-2\u003c/em\u003e KO worms feeding behaviour occurred in aggregates, with more coherent groups of worms swarming across a bacterial lawn (Ding et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) than seen for WT or the other four \u003cem\u003efmo\u003c/em\u003e knockout lines, \u003cem\u003efmo-1,3,4\u003c/em\u003e and \u003cem\u003e\u0026minus;\u0026thinsp;5\u003c/em\u003e. This phenotype was evident from the first day of hatching, with \u003cem\u003efmo-2\u003c/em\u003e KO worms tending to move in groups from the centre to the edges of the bacterial lawn until they had consumed the bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolic profiles of extracts of\u003c/b\u003e \u003cb\u003efmo\u003c/b\u003e \u003cb\u003emutants at day 3 post-hatching\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt day 3 post-hatching, unsupervised and unbiased principal components analysis (PCA) of all mutants showed no sample overlaps of \u003cem\u003efmo-4\u003c/em\u003e KO worm metabolite profiles on PC1 with those of WT (Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and similarly for \u003cem\u003efmo-4\u003c/em\u003e KO alone vs WT (Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Figure S4). Moreover, \u003cem\u003efmo-3\u003c/em\u003e KO alone (Figure S5) had no sample overlaps with WT on PC1. PCA of \u003cem\u003efmo-2\u003c/em\u003e KO vs WT (Figure S6) showed a complete overlapping on PC1 and a partial overlap with WT on PC2, and the \u003cem\u003efmo-2\u003c/em\u003e OE extracts (Figure S7) versus WT showed no sample overlap on PC2. \u003cem\u003efmo-1\u003c/em\u003e KO (Figure S8) and \u003cem\u003efmo-5\u003c/em\u003e KO (Figure S9) were the only strains that had no PCA separation from WT in pairwise comparisons. Interestingly, at the earlier, embryo, life stage, PCA of NMR-based metabonomics of \u003cem\u003efmo-4\u003c/em\u003e KO and WT metabolite extracts showed no group separation (Figure S10).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen the composition of the metabolome of each worm mutant was compared at day 3 post-hatching, using PCA and ANOVA, branched chain amino acids (BCAA; isoleucine, leucine and valine) were present at significantly higher levels in \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-4\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE strains than in WT (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The levels of agmatine (a metabolite of arginine), phosphorylcholine and choline were decreased in \u003cem\u003efmo-2\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE strains, whereas the levels of 5'-adenosine monophosphate (AMP), 5'-adenosine triphosphate (ATP) and 5'-uridine monophosphate (UMP) were decreased in \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-4\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE. In contrast, \u003cem\u003efmo-4\u003c/em\u003e KO showed increased threonine, phenylalanine and tyrosine. Finally, \u003cem\u003efmo-3\u003c/em\u003e KO showed an increased level of cystathionine, agmatine, lactate and threonine (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003cb\u003eError! Reference source not found.\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eAt this day 3, post-hatching, timepoint, the metabolites discriminating \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-4\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE strains from WT shared similar pathways and included valine, leucine and isoleucine degradation (Figure S11, S12). Interestingly, \u003cem\u003efmo-2\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE shared common pathway changes including phosphatidylcholine and phospholipid biosynthesis (Figure S11, S12) but differences between WT and the \u003cem\u003efmo-2\u003c/em\u003e OE line were observed in other pathways, such as mitochondrial beta-oxidation of short, medium and long chain fatty acids, ethanol metabolism, riboflavin metabolism and urea cycle, that did not occur in \u003cem\u003efmo-2\u003c/em\u003e KO extracts.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003efmo-4\u003c/em\u003e KO, the discriminating metabolites were also involved in phenylalanine and tyrosine metabolism, purine metabolism, glutamate metabolism and phenylacetate metabolism (Figure S12).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolic profiles of extracts from\u003c/b\u003e \u003cb\u003efmo\u003c/b\u003e \u003cb\u003emutants at day 6 post-hatching\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt day 6 post-hatching, PCA of NMR spectra of the extracts of WT and all \u003cem\u003efmo\u003c/em\u003e mutants combined showed no sample overlaps between the WT and \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-2\u003c/em\u003e OE and \u003cem\u003efmo-3\u003c/em\u003e KO on PC1 (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). \u003cem\u003efmo-1\u003c/em\u003e KO, in contrast, showed a degree of separation but with some overlap with WT. Conducting a PCA of WT separately versus each \u003cem\u003efmo\u003c/em\u003e mutant no sample overlaps with \u003cem\u003efmo-3\u003c/em\u003e KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; Figure S13), \u003cem\u003efmo-2\u003c/em\u003e KO (Figure S14) and \u003cem\u003efmo-2\u003c/em\u003e OE (Figure S15) on PC1. PCA of \u003cem\u003efmo-4\u003c/em\u003e KO (Figure S16) versus WT showed complete overlap on PC1 and some overlap on PC2, and the PCA of \u003cem\u003efmo-1\u003c/em\u003e KO (Figure S17) versus WT also showed some overlaps on PC1 and PC2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe metabolome of each mutant at day 6 post-hatching was once again evaluated for discriminating metabolites using PCA loadings and ANOVA (Table S2). The discriminating metabolites of each strain allowed enriched pathways to be constructed for each strain (Figures S18-19). In the two strains with increased lifespan (\u003cem\u003efmo-3\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE) the discriminating metabolites were from pathways including the urea cycle and phenylacetate metabolism (Figure S19\u003cb\u003eError! Reference source not found.\u003c/b\u003e). \u003cem\u003efmo-2\u003c/em\u003e OE also had discriminating metabolites involved in pyrimidine metabolism and nicotinate and nicotinamide metabolism (Figure S19\u003cb\u003eError! Reference source not found.\u003c/b\u003e) and those for \u003cem\u003efmo-2\u003c/em\u003e KO included betaine metabolism, and transfer of acyl groups into mitochondria (Figure S18).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolic profiles of extracts of\u003c/b\u003e \u003cb\u003efmo\u003c/b\u003e \u003cb\u003emutants at day 9 post-hatching\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe metabonomics study extended to an additional timepoint for \u003cem\u003efmo-4\u003c/em\u003e KO vs WT, at day 9 post-hatching, following its significant metabolic differences vs WT at day 3, and because \u003cem\u003efmo-4\u003c/em\u003e KO had a significant lifespan extension over WT (Said et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). At day 9 post-hatching, PCA once again showed that \u003cem\u003efmo-4\u003c/em\u003e KO and WT had distinct metabolic profiles (Fig.\u0026nbsp;4\u003cb\u003eError! Reference source not found.\u003c/b\u003e, Figure S20) with increased levels of tryptophan, choline and phosphorylcholine, but decreased glutamine, asparagine, cystathionine, aspartate, agmatine, 5\u0026prime;-UMP, trehalose and 5'-guanosine monophosphate (GMP) in \u003cem\u003efmo-4\u003c/em\u003e KO (Table S3\u003cb\u003eError! Reference source not found.\u003c/b\u003e). The discriminating metabolites of \u003cem\u003efmo-4\u003c/em\u003e KO at day 9 post-hatching were involved in several pathways including aspartate metabolism, valine, leucine and isoleucine metabolism, phosphatidylcholine metabolism and betaine metabolism (\u003c/p\u003e\u003cp\u003eS21\u003cb\u003eError! Reference source not found.\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolic profile changes as a result of ageing in WT and\u003c/b\u003e \u003cb\u003efmo\u003c/b\u003e \u003cb\u003emutant worms\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHaving characterised WT and \u003cem\u003efmo-4\u003c/em\u003e KO worms at three different stages (day 3, 6 and 9 post-hatching), PCA of NMR-based metabolite extract profiles determined the metabolic trajectories of both strains over time. Both strains followed a similar metabolic trajectory from day 3 to day 6 post-hatching, with both groups moving to lower PC1 values and becoming more diffuse and overlapped (Figure S22). Notably, from day 6 to day 9, the WT spectral profiles did not move whereas the \u003cem\u003efmo-4\u003c/em\u003e KO worm metabolic profiles moved to lower PC1 and PC2 values and became distinct again from the WT (Figure S22\u003cb\u003eError! Reference source not found.\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThe metabolic changes that occurred with ageing were identified by comparing \u003csup\u003e1\u003c/sup\u003eH NMR metabolic profiles of WT and mutant worms at day 3 post-hatching relative to those at day 6 and day 9 post-hatching stages (Table S4-S11). The levels of alanine, lactate, asparagine, agmatine, succinate, cystathionine, histidine, 5\u0026prime;-AMP, 5\u0026prime;-ATP and 5\u0026prime;-UMP were decreased in day 6 post hatching WT worms whereas D-glucose levels were increased at days 6 and 9 compared with day 3 post-hatching (Table S12).\u003c/p\u003e\u003cp\u003eMost of the \u003cem\u003efmo\u003c/em\u003e mutants exhibited patterns of ageing progression that largely or partially overlapped with that of the WT worms, with changes particularly in for glucose, alanine, lactate, succinate and agmatine levels as the worms aged from day 3 to day 6 post-hatching (Table S12\u003cb\u003eError! Reference source not found.\u003c/b\u003e). The most notable differences in the long-lived lines were the increased glutamine, asparagine, aspartate and glutamate levels in \u003cem\u003efmo-2\u003c/em\u003e OE and increased tryptophan in \u003cem\u003efmo-4\u003c/em\u003e KO, \u003cem\u003efmo-3\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE strains.\u003c/p\u003e\u003cp\u003eDifferences were seen between \u003cem\u003efmo-4\u003c/em\u003e KO and WT lines at day 9 post-hatching (see Figure S20-21), with glutamine, glutamate, leucine, citrate and 5\u0026prime;-GMP decreased, and the levels of formate, tryptophan, choline, phosphoryl choline, ethanol and methanol, increased in the day-9, \u003cem\u003efmo-4\u003c/em\u003e KO strain (Table S12\u003cb\u003eError! Reference source not found.\u003c/b\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious work already showed that the absence of \u003cem\u003eFmo5\u003c/em\u003e in mouse had a significant impact upon ageing, fat metabolism and other metabolic processes.(Malagon et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) This raised the question as to whether the inactivation of \u003cem\u003efmo\u003c/em\u003e genes in \u003cem\u003eC. elegans\u003c/em\u003e would alter the metabolism associated with ageing. The systematic study of phenotypic and metabolic impacts of the \u003cem\u003efmo\u003c/em\u003es was particularly of interest with reference to \u003cem\u003eC. elegans\u003c/em\u003e, whose five \u003cem\u003efmo\u003c/em\u003e sequences all appear to be related to human (and mouse) FMO5 (Huang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Metabonomics was applied to investigate the metabolic profiles of a series of \u003cem\u003efmo\u003c/em\u003e mutant worms after our observation of lifespan extension in \u003cem\u003efmo-1\u003c/em\u003e KO, \u003cem\u003efmo-3\u003c/em\u003e KO, \u003cem\u003efmo-2\u003c/em\u003e OE and \u003cem\u003efmo-4\u003c/em\u003e KO mutants (Said et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We investigated whether these long-lived mutants had common changes to their metabolism, or if they were mutant-specific.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhenotypic changes due to genetic mutation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn a previous study, the loss of \u003cem\u003efmo-1\u003c/em\u003e, \u003cem\u003efmo-3\u003c/em\u003e and \u003cem\u003efmo-4\u003c/em\u003e and the overexpression of \u003cem\u003efmo-2\u003c/em\u003e increased longevity compared with WT \u003cem\u003eC. elegans\u003c/em\u003e worms (Said et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, we showed that \u003cem\u003efmo-4\u003c/em\u003e KO, \u003cem\u003efmo-2\u003c/em\u003e KO and \u003cem\u003efmo-3\u003c/em\u003e KO worms exhibited a delayed developmental phenotype, reaching the adult stage about 4\u0026ndash;5 h later than WT, timed from the beginning of egg production. \u003cem\u003eFmo-1\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE mutants did not show the same developmental delay. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results indicated that FMO enzymes have a role in \u003cem\u003eC. elegans\u003c/em\u003e development and in extending \u003cem\u003eC. elegans\u003c/em\u003e lifespan. Although another recent study reported that \u003cem\u003efmo-4\u003c/em\u003e KO had no effect on \u003cem\u003eC. elegans\u003c/em\u003e lifespan (Tuckowski et al., 2024), feeding levels were higher in the latter study, a difference in experimental conditions that could affect this model, which is highly sensitive to environment and diet (Ezcurra et al 2011).\u003c/p\u003e\u003cp\u003eA role for \u003cem\u003efmo-1\u003c/em\u003e and \u003cem\u003efmo-4\u003c/em\u003e in development is suggested by their transcriptional upregulation in \u003cem\u003eC. elegans\u003c/em\u003e at larvae stage versus adult (Said et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, phenotype was not correlated with \u003cem\u003efmo\u003c/em\u003e transcriptional regulation at larval versus embryo and adult stages (Said et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), suggesting that individual \u003cem\u003efmo\u003c/em\u003e genes play distinct and nonredundant roles that are not all correlated with worm development.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003efmo-2\u003c/em\u003e KO was the only \u003cem\u003eC. elegans\u003c/em\u003e mutant studied here that showed a distinctive behaviour phenotype, of swarming or aggregation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Swarming is one of the most complex social behaviours exhibited by \u003cem\u003eC. elegans\u003c/em\u003e (Avery et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It was reported that starvation in larvae (L1) induced swarming (Artyukhin et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eC. elegans\u003c/em\u003e was reported to exhibit a strong behavioural preference for 5\u0026ndash;12% oxygen, avoiding lower or higher levels of oxygen, and a link with both swarming and starvation is found in the observation that social feeding occurred only when oxygen exceeded the preferred level (Gray et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Mutation in the \u003cem\u003enpr-1\u003c/em\u003e gene, which encodes a predicted G protein-coupled receptor similar to neuropeptide Y receptors, causes a solitary strain to take on social behaviour (de Bono and Bargmann, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), suggesting the inclusion of Npr1 in further experiments to explain swarming behaviour upon loss of \u003cem\u003efmo-2\u003c/em\u003e.\u003c/p\u003e\u003cp\u003ePhenotypic analysis supports an important role in \u003cem\u003eC. elegans\u003c/em\u003e for \u003cem\u003efmo-4\u003c/em\u003e as its loss both increased sensitivity to bleach treatment (Figure S3) and increased embryo lethality relative to WT. The decreased rate of egg hatching was not a result of decreased egg formation rate (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Figure S3). The increased sensitivity to bleach treatment agrees with a reported osmoregulatory role for \u003cem\u003efmo-4\u003c/em\u003e, loss of which affects clearance of excess water during hypotonicity (Hirani et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A role for \u003cem\u003efmo-4\u003c/em\u003e is plausible in the synthesis of an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells (Hirani et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Alternatively, loss of \u003cem\u003efmo-4\u003c/em\u003e could affect composition of the cuticle of the worm, which provides the first line of defence against chemical and microbial stressors (Page and Johnstone, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). As disruption of cuticle collagen activates osmolyte- and antimicrobial- response genes (Dodd et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), this aspect of inactivation of \u003cem\u003efmo-4\u003c/em\u003e requires further investigation for multiple aspects of the phenotype.\u003c/p\u003e\u003cp\u003eThere may be other, redundant functions for \u003cem\u003efmo-4\u003c/em\u003e that, at least partially, overlap with \u003cem\u003efmo-2\u003c/em\u003e, since inactivation of \u003cem\u003efmo-4\u003c/em\u003e led to up-regulation of \u003cem\u003efmo-2\u003c/em\u003e by ca. 15- to 30-fold (Said et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Shared \u003cem\u003efmo-2\u003c/em\u003e functions would therefore be interesting regarding the \u003cem\u003efmo-4\u003c/em\u003e KO\u0026rsquo;s differences in egg hatching and worm development (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was reported that mutation of \u003cem\u003eC. elegans drp-1\u003c/em\u003e (dynamin related protein 1; mitochondrial pro-fission gene) is associated with defects in mitochondria segregation in gonads, which could be associated with increased embryonic lethality (Labrousse et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) but also with ageing, as mitochondrial size may be related with ageing in \u003cem\u003eC. elegans\u003c/em\u003e and other organisms. Therefore, a future investigation of the effect of \u003cem\u003efmo-4\u003c/em\u003e KO on the transcription level of \u003cem\u003edrp-1\u003c/em\u003e gene is also suggested.\u003c/p\u003e\u003cp\u003eThis study uncovered several new potential endogenous roles of \u003cem\u003eC. elegans fmo\u003c/em\u003es. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the different phenotypes associated with different \u003cem\u003efmo\u003c/em\u003e mutant strains.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of phenotypes of \u003cem\u003efmo\u003c/em\u003e mutant \u003cem\u003eC. elegans\u003c/em\u003e worms\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003ePhenotypes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLifespan (Said et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDevelopment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEgg hatching\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDistinctive social behaviour\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003efmo-1\u003c/b\u003e \u003cb\u003eKO\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExtended lifespan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo difference observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003efmo\u003c/b\u003e\u003cem\u003e-\u003c/em\u003e\u003cb\u003e2\u003c/b\u003e \u003cb\u003eKO\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDelayed development\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo difference observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eaggregation and grouped feeding behaviour\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003efmo-3\u003c/b\u003e \u003cb\u003eKO\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExtended lifespan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDelayed development\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo difference observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003efmo-4\u003c/b\u003e \u003cb\u003eKO\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExtended lifespan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDelayed development\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDecreased hatching rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003efmo-5\u003c/b\u003e \u003cb\u003eKO\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo difference observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003efmo-2\u003c/b\u003e \u003cb\u003eOE\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExtend lifespan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo difference observed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo effect\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eChanges in metabolite profiles of adult worms resulting from genetic mutation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe metabolome of each strain was analysed at day 3 post-hatching to compare the effect of each \u003cem\u003efmo\u003c/em\u003e mutation on the adult stage worm composition. Multivariate analysis (PCA) revealed that all \u003cem\u003efmo\u003c/em\u003e mutant strains possessed a distinct metabolic profile compared with WT, except for \u003cem\u003efmo-1\u003c/em\u003e KO, which had a similar although more diffuse metabolome (Figure S8).\u003c/p\u003e\u003cp\u003eHigher levels of BCAAs were observed in \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-4\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE strains compared to WT (day 3; by PCA and ANOVA, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Like other animals, \u003cem\u003eC. elegans\u003c/em\u003e cannot synthesise BCAAs (Payne and Loomis, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and so any difference in their relative concentrations must be due to a change in either protein turnover or BCAA catabolism (Brosnan and Brosnan, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Down-regulation of the branched-chain α-ketoacid dehydrogenase complex was hypothesised to be responsible for increased BCAA pool sizes in a mutant in a developmental arrest gene, \u003cem\u003edaf-2\u003c/em\u003e (Fuchs et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) which was linked with longevity, and affects fertility and embryonic development. The increased day-3 BCAAs levels in the \u003cem\u003efmo-4\u003c/em\u003e KO, which had effects in all these aspects of the \u003cem\u003eC elegans\u003c/em\u003e life cycle, may be caused by the same mechanism.\u003c/p\u003e\u003cp\u003e\u003cem\u003efmo-4\u003c/em\u003e KO also showed increased levels of threonine, phenylalanine and tyrosine at day 3 post-hatching compared with WT. These changes of amino acid level in the \u003cem\u003efmo\u003c/em\u003e mutants could also result from a role in development for \u003cem\u003efmo-4\u003c/em\u003e because these amino acids are the building blocks of proteins formed during growth and development of an organism (Edwards et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, the levels of agmatine, phosphorylcholine (PCho) and choline were decreased in both KO and OE strains of \u003cem\u003efmo-2\u003c/em\u003e. PCho is produced from choline phosphorylation by choline kinase. Meanwhile, endoplasmic reticulum (ER) stress activated choline kinase (CKB-2) expression, which was linked with ageing, and low phosphocholine (PCho) was correlated with high life expectancy (Pontoizeau et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The decreased level of PCho in these \u003cem\u003efmo-2\u003c/em\u003e mutant worms tallies with this and could therefore be related to effects on or from CKB-2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChanges of metabolite levels due to ageing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIf the metabolome is causally linked to lifespan extension, then the metabolomes of \u003cem\u003efmo-1\u003c/em\u003e KO, \u003cem\u003efmo-3\u003c/em\u003e KO, \u003cem\u003efmo-4\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE mutants should be different from that of wild-type at some, or even all, stages of the \u003cem\u003eC. elegans\u003c/em\u003e life cycle.\u003c/p\u003e\u003cp\u003eMultivariate analysis by PCA showed a distinct metabolic profile compared with WT for the \u003cem\u003efmo-4\u003c/em\u003e KO \u003cem\u003eC. elegans\u003c/em\u003e mutant at days 3 and 9 but not at day 6 post-hatching. The \u003cem\u003efmo-1\u003c/em\u003e KO was the only lifespan-extended strain that did not show any metabolic separation at day 3 or 6 post-hatching (Figures S8 and S17). The lack of effect of removing \u003cem\u003efmo-1\u003c/em\u003e on \u003csup\u003e1\u003c/sup\u003eH NMR-detectable \u003cem\u003eC. elegans\u003c/em\u003e metabolite profiles suggests that lifespan extension may not always be correlated with patent metabolite profiles changes. The number of significantly different metabolites between \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-2\u003c/em\u003e OE and \u003cem\u003efmo-3\u003c/em\u003e KO relative to WT, however, increased with ageing (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e \u0026amp;S2).\u003c/p\u003e\u003cp\u003eThere were no significant differences in the BCAA levels at day 6 post-hatching between WT and any \u003cem\u003efmo\u003c/em\u003e mutants (Table S2) but BCAA levels were down-regulated in \u003cem\u003efmo-4\u003c/em\u003e KO at day 9 post-hatching (Table S3). The same was also seen in two other long-lived strains, namely \u003cem\u003eeat-2\u003c/em\u003e(\u003cem\u003ead465\u003c/em\u003e) and \u003cem\u003eslcf-1\u003c/em\u003e(\u003cem\u003etm2258\u003c/em\u003e), both of which had decreased leucine vs WT in 7-day-old adult worms (Pontoizeau et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although the metabolomes of both \u003cem\u003eglp-1\u003c/em\u003e KO and the long-lived \u003cem\u003edaf-2\u003c/em\u003e mutant, mentioned above, showed elevated levels of BCAAs at 10 days\u0026rsquo; of age (Fuchs et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e); (Wan et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe abundance of PCho and choline pathway activation with age (Pontoizeau et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) means that the increased level of PCho in 9-day, \u003cem\u003efmo-4\u003c/em\u003e KO worms could be due to CKB-2 activation, perhaps to compensate for stress from ageing. This contrasts, however, with a previous study of other long-lived mutants which concluded that low PCho levels correlated with high life expectancy in \u003cem\u003eC. elegans\u003c/em\u003e (Pontoizeau et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) (Fuchs et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The results for \u003cem\u003efmo-4\u003c/em\u003e KO at day 9 post-hatching, with increased levels of PCho, choline and trimethylglycine, are consistent with reports of the WT dauer metabolome which shows elevated levels of phosphoserine, hydroxyproline and choline compounds (Fuchs et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These changes were also correlated for long lived mutants in this study and also for dauer stage nematodes, grown in the same conditions (Fuchs et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe long-lived mutants, \u003cem\u003efmo-3\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE at day 6 and \u003cem\u003efmo-4\u003c/em\u003e KO at day 9 post-hatching, showed higher levels of tryptophan relative to WT, seen previously in dauer stage and long-lived mutants \u003cem\u003edaf-2\u003c/em\u003e and \u003cem\u003eife-2\u003c/em\u003e (Fuchs et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In \u003cem\u003eC. elegans\u003c/em\u003e, blocking tryptophan catabolism may extend lifespan via regulation of proteotoxicity (accumulation of damaged or misfolded proteins) (van der Goot et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) whereas in human serum, tryptophan levels decreased with ageing (Yu et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and toxic tryptophan catabolites increased (Ramos-Ch\u0026aacute;vez et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). An increase in tryptophan catabolism with ageing may result from increased levels of the enzyme indoleamine-2, 3-dioxygenase (IDO) whereas depletion of tryptophan 2, 3-dioxygenase (\u003cem\u003etdo-2\u003c/em\u003e) increased tryptophan levels (Ramos-Ch\u0026aacute;vez et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Literature reports (Edwards et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e;\u003csup\u003e\u0026minus;\u003c/sup\u003eBennett and Kaeberlein, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Bennett et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), support a role for the increased tryptophan levels seen in \u003cem\u003efmo-3\u003c/em\u003e KO, \u003cem\u003efmo-4\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE mutants in extending lifespan in these strains.\u003c/p\u003e\u003cp\u003eAlthough there were some differences between the results of the present work and previous ageing studies, metabolomes are sensitive to precise experimental conditions and genetic changes, the exact life-stage targeted, and to extraction and analysis methods. Furthermore, using FUdR resulted in genotype-specific effects (in \u003cem\u003edaf-2\u003c/em\u003e mutant vs WT nematodes) on levels of eight specific metabolites (Davies, Leroi and Bundy, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), but collecting sufficient biomass for metabolite analysis would not be possible without the use of FUdR, or a similar intervention, to synchronise cultures (Davies, Leroi and Bundy, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).Therefore, we developed a gravity worm filtering protocol (Said \u003cem\u003eet al.\u003c/em\u003e, manuscript in preparation) removing the need for use of FUdR. It is likely therefore that there would be some variation between the results in this study compared with those studies that made use of chemically synchronised samples.\u003c/p\u003e\u003cp\u003e\u003cb\u003eA proposed mechanism of lifespan extension for\u003c/b\u003e \u003cb\u003efmo\u003c/b\u003e \u003cb\u003emutants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOne of the major functions of HLH-30 is to regulate autophagy and promote longevity in \u003cem\u003eC. elegans\u003c/em\u003e (Lapierre et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; O'Rourke and Ruvkun, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). \u003cem\u003efmo-2\u003c/em\u003e is a target of HLH-30, and was induced both by hypoxia and starvation (complete bacterial food source removal) and induction by starvation was dependent on HLH-30 (Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, reduction in \u003cem\u003etald-1\u003c/em\u003e activity by RNAi knockdown also resulted in high lifespan worms that lacked the hypoxic response transcription factor HIF-1 (Bennett et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The life-extending effects of hypoxia in \u003cem\u003eC. elegans\u003c/em\u003e begin in neurons with HIF-1-upregulated transcription and increased serotonergic signalling (Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These effects increased production of FMO-2 in the intestine, and increased longevity, whereas knocking out \u003cem\u003efmo-2\u003c/em\u003e did not affect \u003cem\u003eC. elegans\u003c/em\u003e lifespan (Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The latter findings (Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) were similar to those in the present study.\u003c/p\u003e\u003cp\u003eA new endogenous metabolic pathway of FMOs that relates to ageing processes was found to involve oxygenation of tryptophan, forming \u003cem\u003eN\u003c/em\u003e-formyl-kynurenine, which was then converted to kynurenine by formamidase (Choi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Formate is produced as a byproduct when kynurenine is synthesised from \u003cem\u003eN\u003c/em\u003e-formyl-kynurenine by formamidase (Brosnan and Brosnan, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A hypothesis whereby increased levels of formate confer stress resistance and lifespan extension under metabolically stressful conditions, such as hypoxia or DR (Choi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), could explain increased tryptophan and formate in \u003cem\u003efmo-4\u003c/em\u003e KO at day 9 post-hatching, and could link \u003cem\u003eC. elegans\u003c/em\u003e lifespan extension in \u003cem\u003efmo-4\u003c/em\u003e KO with that in the \u003cem\u003efmo-2\u003c/em\u003e OE and \u003cem\u003efmo-3\u003c/em\u003e KO mutants (Choi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, \u003cem\u003efmo-1\u003c/em\u003e KO was a long-lived mutant that did not show elevated tryptophan with ageing. Thus, we propose that \u003cem\u003efmo-1\u003c/em\u003e KO extends worm lifespan via a different mechanism. This is also supported by the previous study with the same lines, in which \u003cem\u003efmo-2\u003c/em\u003e transcription was upregulated 15- to 30-fold compared with WT upon loss of \u003cem\u003efmo-4\u003c/em\u003e, whereas loss of \u003cem\u003efmo-1\u003c/em\u003e up-regulated \u003cem\u003efmo-2\u003c/em\u003e by only approx. threefold (Said et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast, long-lived \u003cem\u003etald-1\u003c/em\u003e mutant \u003cem\u003eC. elegans\u003c/em\u003e had 30-40-fold increased \u003cem\u003efmo-2\u003c/em\u003e transcription relative to WT (Bennett et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, the up-regulation of \u003cem\u003efmo-2\u003c/em\u003e upon loss of \u003cem\u003efmo-4\u003c/em\u003e could be the cause of lifespan extension in the \u003cem\u003efmo-4\u003c/em\u003e KO strain.\u003c/p\u003e\u003cp\u003eAs the overexpression of \u003cem\u003efmo-2\u003c/em\u003e was reported to extend \u003cem\u003eC. elegans\u003c/em\u003e lifespan in four different conditions including hypoxia, DR (Leiser et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), MAPK and HLH-30 activation, (Bennett et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), an important future experiment would be to determine which environmental condition or transcription factor is responsible for \u003cem\u003efmo-2\u003c/em\u003e up-regulation in \u003cem\u003efmo-4\u003c/em\u003e KO. The proposed, linked mechanisms of action in \u003cem\u003efmo-4\u003c/em\u003e KO resulting in extended \u003cem\u003eC. elegans\u003c/em\u003e lifespan are shown in Fig.\u0026nbsp;5\u003cb\u003eError! Reference source not found.\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study indicated that there are interlinked but non-redundant roles for \u003cem\u003eC. elegans fmo\u003c/em\u003es, and revealed phenotypic differences between \u003cem\u003eC. elegans fmo\u003c/em\u003e mutants. Loss of \u003cem\u003efmo-4\u003c/em\u003e significantly affected on egg hatching and sensitivity to bleach, but, for example, the \u003cem\u003efmo-2\u003c/em\u003e KO strain showed distinct swarming and aggregation behaviour. The metabolome of the long-lived \u003cem\u003efmo-3\u003c/em\u003e KO, \u003cem\u003efmo-4\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE lines displayed higher tryptophan levels during ageing. \u003cem\u003efmo-3\u003c/em\u003e KO and \u003cem\u003efmo-4\u003c/em\u003e KO, but not \u003cem\u003efmo-1\u003c/em\u003e KO, may therefore extend the \u003cem\u003eC. elegans\u003c/em\u003e lifespan via the same mechanism as \u003cem\u003efmo-2\u003c/em\u003e OE. Because \u003cem\u003efmo-4\u003c/em\u003e KO showed significant upregulation of \u003cem\u003efmo-2\u003c/em\u003e over WT levels, it was hypothesised that overexpression of \u003cem\u003efmo-2\u003c/em\u003e in \u003cem\u003efmo-4\u003c/em\u003e KO was responsible for \u003cem\u003efmo-4\u003c/em\u003e lifespan extension. Further experiments are needed, including identifying transcription factor involvement, to confirm whether \u003cem\u003efmo-4\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE do indeed act by the same mechanism.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003estrains and maintenance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnimals were cultures at 20\u0026deg;C and maintained on OP50 seeded NGM plates. \u003cem\u003eC. elegans\u003c/em\u003e strains were purchased from the \u003cem\u003eCaenorhabditis\u003c/em\u003e Genetic Center (CGC, Minnesota, USA). \u003cem\u003eC. elegans\u003c/em\u003e strains used in this study: Bristol (N2) strain as the wild-type strain, \u003cem\u003efmo-1\u003c/em\u003e KO (RB671 [\u003cem\u003efmo-1\u003c/em\u003e(\u003cem\u003eok405\u003c/em\u003e) \u003cem\u003eIV\u003c/em\u003e]), \u003cem\u003efmo-2\u003c/em\u003e KO (VC1668 [\u003cem\u003efmo-2\u003c/em\u003e(\u003cem\u003eok2147\u003c/em\u003e) \u003cem\u003eIV\u003c/em\u003e]), \u003cem\u003efmo-3\u003c/em\u003e KO (RB686[\u003cem\u003efmo-3\u003c/em\u003e(ok354) III]), \u003cem\u003efmo-4\u003c/em\u003e KO (RB562 [\u003cem\u003efmo-4\u003c/em\u003e(\u003cem\u003eok294\u003c/em\u003e) \u003cem\u003eV\u003c/em\u003e]), \u003cem\u003efmo-5\u003c/em\u003e KO (tm2438) and \u003cem\u003efmo-2\u003c/em\u003e OE (KAE10 [\u003cem\u003eseaSi40 I; unc-119(ed3) III\u003c/em\u003e]).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWorm length measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSynchronised cultures of WT and \u003cem\u003efmo\u003c/em\u003e mutants (\u003cem\u003efmo-1\u003c/em\u003e KO, \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-3\u003c/em\u003e KO and \u003cem\u003efmo-4\u003c/em\u003e KO) were obtained using our developed egg preparation protocol. Egg of each strains were transferred to a new seeded NGM plate and left for 3 days until they reached the adult stage. 15\u0026ndash;20 worms of each strain were immobilised using 40 \u0026micro;l of 1mM tetramisole hydrochloride (levamisole; Sigma). Images were taken using an M80 stereomicroscope (Leica, Wetzlar, Germany). Worm length was measured using Image J (Schindelin et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The analysis was done using One-way ANOVA. Worm length of WT and \u003cem\u003efmo-5\u003c/em\u003e KO was also measured using an AI software (\u003cem\u003eC. elegans\u003c/em\u003e length measurement; Rapid Biolabs).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBleach time test (cuticle sensitivity test)\u003c/b\u003e\u003c/p\u003e\u003cp\u003e0.2 ml worm pellet of each strain were re-suspended in 7 ml M9 buffer, and 2 ml of 1M NaOH and 3 ml of 7.5% sodium hypochlorite (bleach) were added to each sample. The time for 95\u0026ndash;100% of worms to be disrupted of each strain was counted. Each experiment consisted of three biological replicates and the analysis was done using student t-test.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEgg hatching rate\u003c/b\u003e\u003c/p\u003e\u003cp\u003e100\u0026ndash;150 eggs were transferred to each of three plates for each strain. Eggs were obtained using egg preparation protocol or by picking from the original plates. All plates were incubated at 20\u0026deg;C for three days, then adult hatched worms were counted on each plate. The percentage of the hatched worms on each plate was calculated. The mean and SEM of the three repeats for each strain were calculated and plotted using Graphpad Prism 6.0 software (La Jolla, California, USA). The data were analysed using student t-test. Images of each plate was taken to double check the number of adult worms and to determine the visual differences between strains.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEgg laying rate\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNormally this assay was done by letting number of L4/adult worms to lay eggs for 12 hrs and repetitive transfer to new plates every 12 h and let eggs laid on each plate to hatch, two days later, number of worms per each plate was counted. This normal assay is not appropriate to the strains with reduced egg hatching rate as the count of hatched eggs will not be indicative for the total number of eggs laid. Modified egg laying rate was developed to overcome this problem, this assay aimed to count eggs instead of counting worms post hatching. 3\u0026ndash;5 adult (1st day of adulthood) worms were transferred to 35mm NGM plates seeded with OP50. All plates were left for 12 h. Number of eggs and hatched larvae were counted and recorded. The advantages of the modified assay was being suitable for the stains with reduced egg hatching rate, it was a combination of egg hatching and egg laying rate as results could be divided into number of laid eggs in 12 h and the rate of eggs hatched could be calculated from the ratio of hatched worms to the total number of laid eggs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMaintaining and growing\u003c/b\u003e \u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003eto the required stage\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. elegans\u003c/em\u003e WT and different \u003cem\u003efmo\u003c/em\u003e mutants (\u003cem\u003efmo-1\u003c/em\u003e KO, \u003cem\u003efmo-2\u003c/em\u003e KO, \u003cem\u003efmo-3\u003c/em\u003e KO, \u003cem\u003efmo-4\u003c/em\u003e KO, \u003cem\u003efmo-5\u003c/em\u003e KO and \u003cem\u003efmo-2\u003c/em\u003e OE) were tested metabolically at day 3 and day 6 post-hatching, in addition WT and \u003cem\u003efmo-4\u003c/em\u003e KO were also tested at day 9 post-hatching. Different strains were maintained and the synchronised cultures were obtained by using the developed egg preparation protocol. 100 \u0026micro;l egg pellet was transferred to each OP50 seeded NGM plate, hatched larvae were checked under the microscope daily for growth. For each strain, 5 different biological repeats were obtained at each tested stage. Worms were combined from three individual 90 mm NGM plates to make a single replicate. For day 3 post-hatching stage, once they reach the adulthood stage (defined as the point where there were eggs seen on the plates but no new-generation worms had hatched), they were ready for metabolite extraction. The adult worms of each strain were collected in 15 ml Falcon tubes using M9 buffer, all tubes were spun at 1,300 \u003cem\u003eg\u003c/em\u003e for 1 min. The supernatant was discarded, worm pellets were washed three times with 10 ml M9 buffer to get rid of the \u003cem\u003eE. coli\u003c/em\u003e. Samples were snap frozen and stored at -80\u0026deg;C until metabolite extraction.\u003c/p\u003e\u003cp\u003eFor the day 6 and 9 post-hatching stages, worms of each strain after they reached the adult stage (day 3 post-hatching) were washed and filtered from the larvae worms (new progeny) daily using our developed gravity worm filter protocol (Said \u003cem\u003eet al.\u003c/em\u003e, manuscript\u003c/p\u003e\u003cp\u003ein preparation). Mother worms were transferred to new seeded plates to supply the worms with OP50. At each stage, worms were collected and harvested using the same method as day 3 stage. Metabolite extraction of \u003cem\u003eC. elegans\u003c/em\u003e is critical, and required sufficient worms to give a 0.3\u0026ndash;0.5 ml pellet of synchronised adult worms, so these steps were repeated several times to obtain the ideal mass of the required worms.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolite extraction and sample preparation for NMR analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMetabolite extraction from different \u003cem\u003eC. elegans\u003c/em\u003e strains was performed using methanol and worms were disrupted using zirconium beads (Sigma, Dorset, UK) for 5 min in TissueLyser II (Qiagen) at 30 Hz. The metabolite extracts of all \u003cem\u003eC. elegans\u003c/em\u003e strains were dried overnight in a RapidVap Vertex Evaporator (LABCONCO, UK) before NMR analysis. Dried extracts were vortexed and resuspended with 240 \u0026micro;l of phosphate buffer (pH. 7.4; 0.93 g NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (Fisher), 1.04 g K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e(Fisher), 0.86 mg TSP (Sigma) and 5.85 mg sodium azide (Fisher), all dissolved in 10 ml D\u003csub\u003e2\u003c/sub\u003eO (Sigma)). Eppendorf tubes were centrifuged for 10 min at 12,000 \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C. 200 \u0026micro;l of each extract was transferred to new 3mm diameter NMR tubes (SampleJet Tube 3.0x103.5mm) by using eVol XR electronic syringe (SGE-Analytical Science, UK).\u003c/p\u003e\u003cp\u003e\u003cb\u003eNuclear Magnetic Resonance Spectroscopy.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll sample were analysed at 300.0 K by 600 MHz \u003csup\u003e1\u003c/sup\u003eH NMR (Bruker Spectrospin) using a cooled Bruker Sample Jet to store the samples prior to pre-heating and then insertion in the magnet. 1D \u003csup\u003e1\u003c/sup\u003eH NMR spectra were acquired with the Bruker 1D NOESY water suppression pulse sequence noesygppr1d. 128 scans were accumulated into 32K data points with a sweep widthe of ca 20 ppm (12,019 Hz) and a relaxation delay of 4.0 s. The \u003csup\u003e1\u003c/sup\u003eH NMR free induction decays were zero-filled to 128 K data points and apodised with a line broadening of 0.3 Hz, prior to Fourier transformation, manual phasing and baseline correction where necessary using MNova version 12.0.1.20560 (Mestrelab, 2018). Various 2D \u003csup\u003e1\u003c/sup\u003eH NMR experiments were acquired as previously described (Veeravalli et al, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolite identification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMetabolites were identified by three complementary methods: (I) comparison with reference spectra from the human metabolome database (HMDB; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.hmdb.ca/\u003c/span\u003e\u003cspan address=\"http://www.hmdb.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Wishart \u003cem\u003eet al.\u003c/em\u003e, 2007) and the biological magnetic resonance data bank (BMRB; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bmrb.wisc.edu/metabolomics/\u003c/span\u003e\u003cspan address=\"http://www.bmrb.wisc.edu/metabolomics/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e); (II) analysis of previously published data; and (III) the interpretation of a series of two-dimensional (2D) spectra such as \u003csup\u003e1\u003c/sup\u003eH COSY, JRES and HSQC, using published methods (Dona et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMultivariate Statistical Analyses.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRegions of the \u003csup\u003e1\u003c/sup\u003eH NMR spectra without signals (upfield of 0.5 ppm and downfield of 9.5 ppm) and the region containing residual water signals at ca 4.8 ppm were removed. The spectra were normalised to a total signal area of 100 and thensuperimposed on one another by stacking in MNova (Mestrelab, 2018). The spectra were saved as a csv file (transposed comma separated variable) and then imported into Matlab 2018b (Mathworks, UK) for processing as a cellular array.\u003c/p\u003e\u003cp\u003eThe data were carefully aligned using the isoshifft algorithm (Tomasi et al, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), the relevant sample metadata (sample name, sample genotype, sample age) were imported and the spectra were bucketed into segments typically of 0.04 ppm spectral width. Further statistical analysis was performed in PLS-Toolbox 9.3 (Eigenvector USA) using standard methods such as principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eAdditional Information\u003c/h2\u003e\u003cp\u003eSupplementary information accompanies this paper at \u0026hellip;\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eDr M. Said is a founder and CSO of Rapid Biolabs, an AI company involved in C. elegans and other imaging. The other authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eWe thank the University of Greenwich for support for the PhD studentship of MS. JIS-C\u0026rsquo;s position is funded by the Medical Research Council (MRC).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor ContributionsConceived and design experiments: MS, ET, JEPerformed the experiments: MS, JS-CAnalysed the data: MS, JE, RFWrote the paper: MS, JEReviewed, modified and approved the manuscript: MS, FC, JS-C, ET, JE\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Dr Beatriz Jimenez and the staff at Imperial College for access to and assistance with the 600 MHz NMR facility.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe original NMR spectroscopy data from this study has been deposited at MetaboLights (Yurekten et al, 2023): https://www.ebi.ac.uk/metabolights/ at deposit MTBLS11908.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltun, Z. F. and Hall, D. H. 2009. Introduction. 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(2023) MetaboLights: open data repository for metabolomics, \u003cem\u003eNucleic Acids Research\u003c/em\u003e, \u003cu\u003e52\u003c/u\u003e(D1) D640-D646; doi.org/10.1093/nar/gkad1045 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"metabolomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mebo","sideBox":"Learn more about [Metabolomics](http://link.springer.com/journal/11306)","snPcode":"11306","submissionUrl":"https://submission.nature.com/new-submission/11306/3","title":"Metabolomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7122670/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7122670/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Flavin-Containing Monooxygenases (FMO) are widely conserved, xenobiotic-detoxifying enzymes whose additional endogenous functions have been revealed in recent studies. Those roles include the regulation of longevity in the model nematode \u003cem\u003eCaenorhabtidis elegans.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003eThe purpose of this study was to compare aspects of the phenotypes of \u003cem\u003eC. elegans\u003c/em\u003e worms with mutations in all \u003cem\u003efmo\u003c/em\u003egenes, particularly focusing on the metabolome and its relationship with lifespan-extension and the worm life cycle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e NMR Spectroscopic analysis of the extracts of metabolites from \u003cem\u003eC. elegans\u003c/em\u003e worms of different ages and \u003cem\u003efmo \u003c/em\u003egenotypes was used to compare metabolite profiles of \u003cem\u003eC. elegans\u003c/em\u003eworms and determine how these changed with genotype and ageing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eLoss of both \u003cem\u003efmo-4\u003c/em\u003e and \u003cem\u003efmo-3 \u003c/em\u003eand over-expression of \u003cem\u003efmo-\u003c/em\u003e2, resulted in increased levels of tryptophan in the metabolome, which correlated with an extended lifespan in these mutants. Loss of \u003cem\u003efmo-4\u003c/em\u003e also led to decreased embryo hatching, along with increased sensitivity to bleach during sterilisation protocols. In contrast, in the extended lifespan \u003cem\u003efmo-1\u003c/em\u003e knockout worm, the metabolome did not reveal any significant metabolite changes and therefore lifespan effects may occur through another mechanism, or hidden metabolic changes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Genetic interventions coupled with metabolome profiling in \u003cem\u003eC. elegans\u003c/em\u003e can provide insights into biological mechanisms in ageing that might lead to strategies for healthy lifespan extension in human old age.\u003c/p\u003e","manuscriptTitle":"Phenotypic and metabonomics studies of FMOs in C. elegans and their roles in lifespan extension","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 17:50:19","doi":"10.21203/rs.3.rs-7122670/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-27T05:53:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-27T01:46:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111280925290164604226974933719079638362","date":"2025-08-29T11:42:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-28T15:21:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"241239374984036872249489855511918198731","date":"2025-07-31T14:09:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-15T15:29:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-15T05:54:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-15T05:53:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Metabolomics","date":"2025-07-14T15:09:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"metabolomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mebo","sideBox":"Learn more about [Metabolomics](http://link.springer.com/journal/11306)","snPcode":"11306","submissionUrl":"https://submission.nature.com/new-submission/11306/3","title":"Metabolomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6aa2f051-8d5d-4b6d-a30e-677061b67a0b","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:03:08+00:00","versionOfRecord":{"articleIdentity":"rs-7122670","link":"https://doi.org/10.1007/s11306-025-02367-4","journal":{"identity":"metabolomics","isVorOnly":false,"title":"Metabolomics"},"publishedOn":"2025-11-15 15:58:23","publishedOnDateReadable":"November 15th, 2025"},"versionCreatedAt":"2025-07-17 17:50:19","video":"","vorDoi":"10.1007/s11306-025-02367-4","vorDoiUrl":"https://doi.org/10.1007/s11306-025-02367-4","workflowStages":[]},"version":"v1","identity":"rs-7122670","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7122670","identity":"rs-7122670","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}