Akagi, A.E. et al. (2017) International Worm Meeting "Tissue-specific ascaroside production in the nematode Caenorhabditis elegans."

Schroeder, F.C., Akagi, A.E., Sternberg, P.W., Artyukhin, A.B.

[International Worm Meeting, 2017]

Ascarosides are a family of small molecule signals that regulate a variety of important behaviors in C. elegans and other nematodes including mate attraction, aggregation, repulsion, and entry into dauer diapause under stressful conditions. Structurally, ascarosides are modular glycosides of the dideoxysugar ascarylose. Small structural changes in the fatty acid moiety attached to the first position of the sugar as well as the addition of other metabolically-derived components to the fourth position have been shown to elicit different behaviors. DAF-22, a thiolase responsible for the last step of peroxisomal beta -oxidation of the ascaroside lipid side chain, is necessary for the production of biologically active ascarosides and is expressed in three different tissues in the worm: the intestine, the body wall muscle, and the hypodermis. We have transgenically rescued the expression of DAF-22 in each tissue individually within a DAF-22 knockout background. Although the intestine was initially hypothesized as the site of ascaroside biosynthesis, we have found that ascarosides are produced in all three tissues and rescue ascaroside-regulated behaviors.

Ludewig, A.H. et al. (2017) International Worm Meeting "Pheromones control lifespan via sirtuins by generating oxidative stress."

Pulido, D.C., Schroeder, F.C., Thornton, S.N., Ludewig, A.H.

[International Worm Meeting, 2017]

Previous work showed that chemosensation of hermaphrodite produced pheromones ascr#2 and ascr#3 increases C. elegans adult lifespan and stress resistance (Ludewig 2013), whereas male-produced pheromones of then unknown identity shorten hermaphrodite lifespan (Gems 2000, Maures 2014). We show that the male-produced ascaroside ascr#10 underlies male-induced hermaphrodite progeria, and that pheromone-mediated lifespan increase and decrease depend on several different sirtuins (Ludewig, 2013; Ludewig, unpublished). Sirtuins, a family of NAD+ dependent histone deacetylases, have been implied as conserved regulators of lifespan and stress resistance in various species ranging from yeast to vertebrates. However, the mechanisms by which sirtuins influence lifespan are purely understood. We show that sensing of lifespan-increasing ascarosides transiently increases reactive oxygen species (ROS) levels in the mitochondria and requires the transcription factor skn-1/Nrf, a general regulator of stress responses. Antioxidants abolish the effects of ascarosides on lifespan, demonstrating that ROS production is necessary for sirtuin-dependent longevity. Mitochondrial ROS production is sirtuin-dependent and requires an intact nicotinamide catabolic pathway, indicating that ascaroside-mediated effects on lifespan depend on conversion of NAD+ into nicotinamide by sirtuins, similar to previously reported effects of sir-2.1 over expression (Schmeisser 2013). Our research uncovered an endogenous lifespan regulatory system based on antagonism of longevity-promoting and progeria-inducing ascarosides that act by modulating sirtuin-dependent ROS production. Furthermore, our work suggests that the intensely discussed variability of sirtuin dependent lifespan phenotypes in C. elegans may be due to the confounding effects of worm-produced ascarosides accumulating on experimental plates, in conjunction with other environmental stimuli activating skn-1/Nrf.

Manohar, M. et al. (2019) International Worm Meeting "Nematode-derived metabolite ascr#18 mediates plant-nematode interactions."

Chen, S., Wang, X., Tenjo, F.J., Schroeder, F.C., Manohar, M., Klessig, D.F.

[International Worm Meeting, 2019]

Plant-parasitic nematodes cause billions of dollars' worth of agricultural damage annually. Although resistant cultivars and nematicides have been used to control nematodes, there exists a need for more effective treatment strategies. Plants are known to respond to plant-parasitic nematodes similar to other pathogens; however, the underlying molecular mechanism remains elusive. Nematodes use molecules from a conserved pheromone family, the ascarosides, to regulate developmental and social behaviors. Previously, we have shown that ascr#18, the major ascaroside in the plant-parasitic nematodes, elicits plant defense response in monocots and dicots, improving their resistance to nematodes, bacterial and fungal pathogens. In recent work, we demonstrated that ascr#18 is taken up by Arabidopsis and tomato, metabolized into shorter side-chained ascarosides, and secreted into the rhizosphere via the roots. The identity of ascr#18 and metabolized ascarosides was confirmed by LC-MS/MS and using 13C-labeled derivatives. Since nematodes use peroxisomal ?-oxidation pathway to metabolize ascarosides, we investigated ascaroside metabolism in several Arabidopsis mutants defective in this pathway. Interestingly, a mutant defective in production of two acyl-CoA oxidases, acx1/acx5, is unable to metabolize ascr#18. In addition, we have shown that acx1/acx5 mutant is compromised in ascr#18-mediated enhanced resistance to root-knot nematodes. Our results suggest that plants use conserved metabolic pathways to process ascr#18 into its derivatives, thereby modifying the composition of their root exudates to regulate their interaction with nematodes

Ludewig, A.H. et al. (2017) International Worm Meeting "Larval crowding accelerates C. elegans development and reduces lifespan."

Ludewig, A.H., Schroeder, F.C., Panda, O., Thornton, S.N., Pulido, D.C., Artyukhin, A.B.

[International Worm Meeting, 2017]

Environmental conditions experienced during animal development have sustained impact on maturation and adult lifespan. We recently showed that in C. elegans developmental rate and adult lifespan depend on larval population density and that this effect is mediated by excreted small molecules. We established the time point of first egg laying as ultimate and easy accessible marker of developmental pace and for full maturity (= egg laying assay). We found that wildtype hermaphrodites raised under high density conditions developed significantly faster than animals raised in isolation. Furthermore, larval population density but not population density during adulthood, strongly affected C. elegans lifespan. Population-density dependent developmental acceleration (Pdda) was dramatically enhanced in fatty acid beta -oxidation mutants that are defective in the production of dauer-inducing ascarosides. In contrast, Pdda is abolished by synthetic ascarosides or steroid hormones that act on the nuclear hormone receptor DAF-12. However neither ascarosides nor any of the known steroid hormones are required for Pdda, instead it is mediated by another larval population density-dependent signal (Ludewig 2017, in press) that antagonizes the effects of the dauer pheromone. We further show that the effects of high larval population density persist through adulthood, as C. elegans larvae raised at high densities exhibit significantly reduced adult lifespan and respond differently to exogenous chemical signals compared to larvae raised at low densities, independent of density during adulthood. Our results demonstrate how inter-organismal signaling during development regulates reproductive maturation and longevity.

Gudibanda, P. et al. (2017) International Worm Meeting "Cryptic S.M.S. (Small Molecule Signaling) between Caenorhabditis and Pristionchus ."

Movahed, N., Gudibanda, P., Antebi, A., Schroeder, F.C., Loi, P., Sommer, R.J.

[International Worm Meeting, 2017]

The primary mechanism for organisms to react to rapidly changing environments is the alternation of gene transcription profiles by modulating extra- and intracellular small molecule signaling (SMS). The formation of dauers, a stress-resistant, developmental arrest stage that is induced under unfavorable growth conditions in both Caenorhabditis elegans (free living nematode) and Pristionchus pacificus (necromenic nematode), represents an evolutionarily conserved strategy to counter adverse environmental conditions. Although transcriptional profiling suggests a high degree of conservation of the underlying signaling networks, including the roles of transcription factors DAF-16 and DAF-12, it is unclear whether the chemical structures of the steroid hormones regulating C. elegans development are broadly conserved in nematodes. In this study, we identify the endogenous small molecule ligands of the NHR Ppa-DAF-12 using an unbiased metabolomics approach, which demonstrates conserved steroid-based SMS in C. elegans and P. pacificus. For our unbiased, endogenous ligand screen, metabolome fractions generated from WT and daf-22 mixed-stage P. pacificus extracts were tested for activation of a hybrid NHR, consisting of the ligand binding domain of Ppa-DAF-12 and the DNA-binding domain of Cel-DAF-12, in an HEK-cell culture based luciferase assay. Using high-resolution mass spectrometry, we determined that active fractions contained the exact same three dafachronic acids (DAs), D7DA, D1,7DA and 3aOH-D7DA, that had previously been characterized in C. elegans. The de novo biosynthesis of these DAs in P. pacificus was confirmed by 13C2-labeled cholesterol feeding experiments. We further showed that, like in C. elegans, accumulation of dauer-inducing ascarosides in P. pacificus liquid culture counteracts DA biosynthesis. As a result, dauer pheromone-defective Ppa-daf-22 mutant worms produce larger quantities of DAs than Ppa WT, like in Cel-daf-22 mutant worms. Taken together, these results demonstrate extensive conservation of SMS between C. elegans and P. pacificus, including the DAF-12 ligand's chemical structures and the regulation of their biosynthesis via ascarosides. We further show that DAs are excreted in physiologically relevant concentrations by both C. elegans and P. pacificus, raising the possibility of inter-species regulation of development. We currently investigate potential conservation of other components of DAF-12 signaling, including interactions with corepressors (e.g. the Ppa homolog of Cel-DIN-1) and other NHRs (e.g. NHR-8).

Ludewig, A.H. et al. (2015) International Worm Meeting "Population density in Caenorhabditis elegans controls developmental rate via a small molecule-based push-pull strategy ."

Micikas, R.M., Gimond, C., Braendle, C., Ludewig, A.H., Schroeder, F.C., Doering, F., Judkins, J.J.

[International Worm Meeting, 2015]

Adequate sensing and processing of information about the environment is crucial for organismal survival. The nematode C. elegans is a particularly useful model for investigating responses to changes in environmental parameters such as temperature, food availability, and crowding. Here we show that timing of C. elegans reproductive development is controlled by a push-pull mechanism based on two different types of small molecule signals. By using the time point of first egg laying as a marker for full maturity, we found that animals raised under high density conditions developed up to 10 hours faster than animals raised in isolation. Mutant analyses revealed that population-density dependent onset of egg laying (pdde) is controlled by chemosensation of different two groups of endogenously produced small molecule signals. Fatty acid beta-oxidation mutants that are defective in the production of a family of glycolipids, the ascarosides, which previously have been shown to regulate formation of stress-resistant dauer larvae, showed dramatically enhanced pdde, whereas pdde was abolished or greatly reduced in mutants defective in evolutionarily conserved steroid hormone signaling. Correspondingly, addition of synthetic ascarosides or steroid hormones diminished or abolished pdde by either reducing or accelerating developmental rate. We confirmed pdde in two different C. elegans wild-type strains as well as in a related species, C. briggsae. Taken together, our results how that developmental rate in C. elegans is controlled by the interplay of a known class of interorganismal signals, the ascarosides, and bile acid-like steroids called dafachronic acids that previously had been identified as ligands of the nuclear hormone receptor DAF-12, a homolog of vertebrate vitamin D and pregnane-X receptors. Our work shows that the dafachronic acids also function as interorganismal signals that act via chemosensory mechanisms. Moreover, we suggest that measuring onset of egg laying provides an efficient method to measure the effect of environmental changes on developmental rate.

Androwski, Rebecca J et al. (2013) International Worm Meeting "Dendritic arborization in dauer IL2 neurons: Genetic and bioinformatic analyses."

Androwski, Rebecca J, Rashid, Alina, Barr, Maureen M, Schroeder, Nathan E, Ritter, Tom

[International Worm Meeting, 2013]

Under adverse environmental conditions, C. elegans enters a stress resistant dauer stage. We discovered a wild-type dauer-specific phenotype wherein six inner labial sensory neurons (IL2) undergo dramatic reorganization including extensive arborization of the IL2 quadrant (IL2Q) neurons (see N. Schroeder et al abstract). To identify genes involved in dauer-specific IL2 remodeling , we used a candidate gene approach. Representative genes were examined from several molecular pathways: dauer formation, ciliogenesis and intraflagellar transport, Notch signaling, cell fate and axon guidance. For example, both UNC-86 and LIN-32 are transcription factors necessary for IL2 cell fate. Most unc-86 alleles are defective in IL2 formation. However, unc-86(n848) mutants show normal IL2 morphology in non-dauers, but defects in dauer-specific IL2 arbors. Interestingly, lin-32 mutants that form IL2 neurons show no obvious defects in arborization. The RFX-transcription factor DAF-19, is a master regulator of ciliogenesis. daf-19(m86) mutants lack all sensory cilia, but show extranumerary branching in the lateral IL2s suggesting a role for cilia in inhibiting arborization. Through a forward genetic screen we identified kpc-1, a kex2-like proprotein convertase and furin homolog, as required for organized arborization in both dauer IL2 neurons (See N Schroeder et al abstract) and adult PVD and FLP multidendritic neurons (see Rashid et al abstract). To identify potential KPC-1 substrates, we cross referenced genes upregulated in PVDs (1), genes upregulated during dauer (2), and genes containing putative proprotein cleavage sites (3). We have assembled a list of 159 potential regulators of dendritic branching which we are beginning to characterize. 1.Smith et al. Dev Bio. 345 (2010) 18-33. 2.Jeong et al. PLoS One. (2009 Jan) 4(1) e4162. 3.Duckert et al. Protein Eng Des Sel. 17 (2004) 107-112.

Evan Z Macosko et al. (2007) International Worm Meeting "Regulation of C. elegans roaming behavior by a multi-component pheromone."

Jon Clardy, Rebecca Butcher, Jim Thomas, Evan Z Macosko, Cori Bargmann

[International Worm Meeting, 2007]

Overall movement during animal foraging can take two general strategies: exploration of a wide area (roaming) and restriction to a local area (dwelling). C. elegans, when feeding on a bacterial lawn, has been shown to alternate between roaming and dwelling states.i Recordings of worm movements on food indicate that the behavioral state changes are arrhythmic and can be modified by food quality. We found that a multi-component pheromone released from incised or lysed worms dramatically inhibits C. elegans roaming behavior. The same worm lysate also acts as a potent repellent, suggesting that this mixture could be analogous to an alarm pheromone. Our chemical analysis indicates that there are at least two important components of the worm lysate that inhibit roaming: (1) a derivative of the sugar ascarylose, which is also a component of dauer pheromoneii and (2) a small indole-containing compound. We are currently asking if the same compounds also induce acute avoidance behavior. To determine the neuronal circuitry that regulates roaming and dwelling, we killed individual cells by laser ablation. Loss of either ADL, ASK, or AWC increased roaming in the presence of pheromone. In contrast, ablation of ASI resulted in a constitutive dwelling phenotype. The interneurons AIZ and AIY are important for proper roaming and dwelling behavior, while AIB and AIA are less important. Finally, though AVA is a crucial command interneuron for many locomotory behaviors, we found that AVA-ablated animals roam relatively normally. We also tested candidate mutants for sensitivity to pheromone. Mutations in egl-4, a gene previously shown to antagonize roaming,i reduce pheromone sensitivity. Rescue of egl-4 using heterologous promoters indicates that the EGL-4 gene product functions in chemosensory neurons to promote dwelling. iFujiwara M., P. Sengupta and S.L. McIntire, 2002 Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-Dependent Protein Kinase. Neuron 36: 1091-1102. iiButcher R.A., M. Fujita, F.C. Schroeder, J. Clardy, Small Molecule Signaling of Dauer Formation in C. elegans, 2007 International Worm Meeting.

Ludewig, Andreas H. et al. (2013) International Worm Meeting "Low population density increases lifespan and delays egg laying of C. elegans hermaphrodites."

Doering, Frank, Schroeder, Frank C., Ludewig, Andreas H.

[International Worm Meeting, 2013]

In C. elegans, high population density (overcrowding) has been shown to trigger developmental arrest and dauer formation within early larval stages (Golden and Riddle 1982). The actual population density signal sensed by the worms includes a synergistic mixture of ascaroside pheromones as well as small signalling peptides (Ludewig and Schroeder, 2013; Yamada et al., 2010). However, many aspects of population density-dependent signaling events remain poorly understood. Here, we compared low and intermediate dense C. elegans populations grown under ad libidum feeding conditions. We found that the number of worms per plate strictly determined their average lifespan whereat the number of worms was inverse correlated to mean lifespan. Isolated worms lived around 4 (!) days longer than animals grown up in larger groups. In 1999, Gems and Riddle demonstrated strong dependence of lifespan on population density for males but not for hermaphrodites. However, in those assays worms were placed on experimental plates as L4 larvae, whereas our assays started with eggs, hinting on some defining events during developmental stages L1-L3 in hermaphrodites. We also found that the time point of first egg lay was correlated with the number of worms per plate. Worms that grew up in isolation laid their first egg 9-11 hours later than worms grown up in groups of 25 or more animals per plate. The overall time course of egg laying from isolated worms was shifted towards later time points and the total number of eggs was reduced by 45%. This population dependent early egg laying (= eel) phenotype is completely abolished in daf-16(mu86) mutants and partially abolished in daf-12(rh61; rh411) mutants. Unexpectedly, we also found precocious first egg laying under mild dietary restriction (DR). Evidence from previous studies suggests that small-molecule signalling may trigger DR early egg laying and population dependent longevity (Ludewig and Schroeder, 2013; Yamada et al., 2010; Yzraelit, 2012). To test this hypothesis, we currently examine candidate small molecules for their influence on those phenomena.

Chiang, Y. et al. (2017) International Worm Meeting "Wild-isolates of C. elegans exhibit variable attraction to the sex-pheromone mimicry compound produced by the nematode-trapping fungus A. oligospora."

Chiang, Y., Hsueh, Y.

[International Worm Meeting, 2017]

The nematode-trapping fungus A. oligospora produces odors that mimic sex and food cues to attract many nematode species (Hsueh et al. 2017). One of the compounds (MMB) that likely mimics male pheromone in Canerhbiditis nematodes is highly attractive to two lab strains of C. elegans (N2 and Hawaiian). To investigate whether this attraction is highly conserved among the wild-isolates, we analyzed the MMB chemotaxis behavior among the C. elegans wild isolates from the CeNDR collection. We found that MMB-attraction is highly polymorphic trait. Six out of forty wild-isolates tested exhibited weak attraction to MMB. Genetic analysis showed that this trait is controlled by QTL in some strains but a single locus in others. The high incidence of MMB-insensitive strains among the wild-isolates suggests that lost of MMB attraction might offer benefit to C. elegans in the natural environments. Hsueh YP, Gronquist MR, Schwarz EM, Nath RD, Lee CH, Gharib S, Schroeder FC, Sternberg PW. 2017. Nematophagous fungus Arthrobotrys oligospora mimics olfactory cues of sex and food to lure its nematode prey. eLife 6.