Edison AS et al. (1995) International C. elegans Meeting "Cloning and Sequencing of Multiple Transcripts of a FMRFamide-like Neuropeptide Gene in the Nematode Ascaris suum"

Messinger LA, Edison AS, Stretton AOW

[International C. elegans Meeting, 1995]

Five transcripts of a gene encoding a novel sub-family of six FMRFamide-like neuropeptides in the nematode Ascaris suum have been cloned and sequenced. The translated product of these transcripts is a precursor protein containing two main halves: a relatively hydrophobic region with no obvious peptides and a series of peptides separated by characteristic processing sites. The mature peptides share the C-terminal sequence PGVLRFamide but have different N-terminal sequences. Three of the peptides were previously isolated by immunocytochemistry [Cowden and Stretton, Peptides, in press] and three others are novel sequences. Of the transcripts, four have identical translated regions but differ in the 5' or 3' untranslated regions. A fifth transcript encodes a precursor protein with only the peptide-containing C-terminal domain.

Bifarin, O.O. et al. (2017) International Worm Meeting "Systems Biology of Caenorhabditis elegans Glycosyltransferases."

Zhang, S., Gouveia, G., Tayyari, F., Bifarin, O.O., Edison, A.S., Taujale, R.

[International Worm Meeting, 2017]

Glycosyltransferases (GTs) catalyze the transfer of glycosyl groups from sugar substrate donors to a host of acceptor substrates - oligosaccharides, monosaccharides, proteins, lipids, nucleic acids, and other small molecules; effectively GTs catalyze glycosidic bond formation between these multifarious possibilities of molecules. Numerous studies show that GTs are important for developmental and physiological processes in C. elegans (Berninsone, 2006). However, most GTs have not yet been empirically validated. C. elegans utilizes GTs for critical functions, including pheromone signaling with ascarosides and the detoxification pathways, though identities of GTs regulating these functions remain unknown. C. elegans controls much of its behavior and development through the use of ascarosides, which are also present in many free-living and parasitic nematodes (Choe et al 2012). Some of the phenotypes mediated by ascarosides include aggregation, olfactory plasticity, dauer formation, attraction behavior, and hermaphrodite behavior. (Srinivasan et al 2008; Edison, 2009; Ludewig & Schroeder, 2013) Chemically, ascarosides are glycosides of the dideoxy sugar ascarylose, attached to a fatty acid side chain, thus implicating GTs in their biosynthesis. In addition, the innate immune system in C. elegans utilizes a wide range of immune effectors and enzymes (including GTs) for microbial defenses and xenobiotics detoxification (Lindblom & Dodd, 2006; Stupp et al 2012). Stupp et al 2012 showed that C. elegans can detoxify two bacterial toxins, 1-hydroxyphenazine (1-HP), and indole via N- and O-glycosylation. Our research aims to discover the roles of specific GTs in biological processes like these. In this exploratory study, we selected a dozen GT mutant strains, the majority of which belong to the GT-A fold protein (families 2, 7, 21, 27, and 13). 6 replicates of each strains (L1 stage) were cultured to about a population of 100-200 thousand for NMR metabolomics measurements, and animals were randomly selected from each sample for the measurement of population distribution using the large particle flow cytometer COPAS Biosorter. The Biosorter measures the extinction and time of flight of individual nematode which is used as a descriptor of developmental stages. Analysis of metabolic changes of some of the GT mutants and the association with the population distribution will be reported.

Taujale, R. et al. (2017) International Worm Meeting "An evolutionary systems approach to an integrative study of the proteome and metabolome of Glycosyltransferases in C. elegans."

Kannan, N., Taujale, R., Bifarin, O.O., Edison, A.S.

[International Worm Meeting, 2017]

Glycosyltransferases (GTs) are a broad class of proteins involved in the transfer of a glycosyl group from a donor molecule to an acceptor. As such, they are involved in a wide range of biological functions through their roles in glycosylation modifications, synthesis of cellular receptors, biosynthesis of polysaccharides, glycolipids and glycoproteins. In C. elegans, GTs have been implicated to play roles in development, cuticle formation, detoxification, signaling and other pathways. However, only around 20% of the more than 260 GTs have been characterized. The large expansion of some of the GT families, presence of unique sugars and pathways in worms and the unknown specific roles of GTs in the implicated pathways pose specific questions and challenges that require a deeper understanding of the functional units of these GTs in C. elegans. We used a Bayesian statistical approach to align and classify more than 250,000 GT sequences across all taxonomic groups into functional categories based on the patterns of conservation and variation in large multiple sequence alignments. We use patterns unique to each GT family as a conceptual starting point for investigating their sequence-structure-function relationships. Implementing these methods, we can further pinpoint contrasting, similar and co-evolved features that differentiate, associate and functionally relate the GT families respectively. We will present an initial analysis that highlights the presence of co-conserved features that distinguish multiple GT families, highlighting GTs in worms. We have generated a phylogenetic classification based on these features and mapped phenotypic associations for these families based on literature. Our analysis reveals several expanded and unique GT families in C. elegans with limited information that we hypothesize might be involved in detoxification, signaling and other pathways unique to worms. Using the co-conserved features as a starting point, we further investigate structural data to pinpoint targets for mutational and metabolomic studies. An initial metabolomic analysis by others in the lab of select GT mutants in C. elegans is revealing specific features that are statistically different, suggesting family specific phenotypic changes. Informatic analysis layered with multiple data sources from literature serve as a tool to identify targets and derive hypotheses to conduct deeper metabolomic studies that can help elucidate the functional changes and biological activity of the associated GT families.

Schroeder F C et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Endogenous Small Molecules Increase Lifespan of Caenorhabditis elegans via SIR-2.1"

Fox B W, Bethke A, Pungaliya C, Malik R U, Schroeder F C, Ludewig A H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Lifespan in metazoans is regulated by several conserved signaling pathways, including the insulin/insulin-like growth factor and sirtuin pathways. W e have found that components of the dauer pheromone, the ascarosides (Edison 2009), regulate C. elegans adult lifespan and stress resistance. Ascarosides increased lifespan and thermotolerance of wild-type worms by up to 56% and 25%, respectively, without reducing fecundity or feeding rate. These lifespan increases are completely abolished by loss of the histone deacetylase SIR-2.1 or loss of components of peroxisomal fatty acid beta-oxidation, but do not require insulin signaling via the FOXO-homolog DAF-16 or TGF-beta signaling. Our findings establish endogenous small molecules as modulators of sirtuin-dependent pathways that connect longevity and stress resistance with peroxisomal fat metabolism. A. S. Edison, Curr. Opin. Neurobiol. 19(4), 378 (2009).

Malik R U et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Ascarosides ascr#2 and ascr#3 Increase Thermotolerance of Adult C. elegans"

Fox B W, Malik R U, Pungaliya C, Schroeder F C, Ludewig A H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

We investigated whether the ascarosides, major components of the C. elegans dauer pheromone (Edison, 2009), affect stress resistance of adult worms. We found that ascarosides markedly increased survival under oxidative stress and resistance to heat stress (thermotolerance at 35 degC). We further measured pharyngeal pumping rates under heat stress and found that pumping rates of worms on ascaroside plates were significantly higher than on control plates. Next, we asked whether nutritional conditions influence the observed ascaroside-mediated increases of stress resistance. For thermotolerance assays under caloric restriction (CR) conditions, we transferred worms to plates without bacteria before exposure to heat stress. Mean heat stress survival time under CR conditions was higher than for worms with bacteria, in accordance with previous studies demonstrating increased stress resistance under starvation conditions. Notably, addition of ascarosides did not further increase thermotolerance of CR worms. These results show that the worms' metabolic state influences the efficacy of ascarosides in increasing thermotolerance. A. S. Edison, Curr. Opin. Neurobiol. 19(4), 378 (2009).

Carter, T. et al. (2017) International Worm Meeting "NMR-based metabolomic characterization of ascaroside production in recombinant inbred lines (RILs) of Caenorhabditis elegans."

Edison, A.S., Tayyari, F., Gouveia, G., Ponce, F., Andersen, E., Carter, T.

[International Worm Meeting, 2017]

Organisms from all kingdoms of life communicate in extremely diverse ways. One such example is the unique and complex chemical signaling of small molecules used by Caenorhabditis elegans, known as ascarosides. Ascarosides are glycosidic derivatives of 3,6-dideoxyascarylose that are attached to fatty acid side chains that can be oxidized to various lengths, as well as many other modifications. Many ascarosides work synergistically at dose-dependent concentrations in concert with one another to communicate a variety of signals. Adding to this complexity is the fact that variable concentrations of the same ascaroside can elicit different behaviors. Although ascarosides are conserved in nematodes, different strains produce and respond to different mixtures ascarosides and the variable "bouquet" of concentrations that work together have yet to be fully addressed. We analyzed a subset (10) of the hundreds of existing recombinant inbred lines (RILs) of C. elegans and their parent strains, the laboratory strain (N2) and a wild isolate from Hawaii (CB4856). Our primary goal in analyzing the RILs and parental N2 and CB4856 strains is to relate metabolite profiles and ascaroside variation to the complex phenotypic variation. Using an NMR (Nuclear Magnetic Resonance) Spectroscopy based metabolomic approach we will present the preliminary analysis revealing global metabolic changes among the parent strains and their recombinant progeny. These results will determine the feasibility of extending these studies to larger numbers of RILs. With a more robust sample size, we aim to isolate and identify ascaroside variance within and between the RILs using both NMR and LC-MS (Liquid Chromatography-Mass Spectrometry). Once isolated, the ascarosides will be used to test the behavioral response of N2 and CB4856 to the different RIL ascaroside bouquets using simple bioassays.

Messinger LA et al. (1995) International C. elegans Meeting "Evolutionary Patterns in FMRFamide Genes: Implications for Nematode Neurophysiology."

Edison AS, Nelson DL, Messinger LA, Stretton AOW

[International C. elegans Meeting, 1995]

We have sequenced an Ascaris suum gene encoding six peptides related to molluscan FMRFamide neuropeptides (Edison et al., in preparation). As in other FMRFamide-like genes, the peptides are processed from a precursor protein containing multiple peptides. We compared the A. suum sequence to other available FMRFamide-like sequences. Although the sequences of the A. suum and Caenorhabtidis elegans peptides are similar, a phylogenetic analysis of the genes finds no evidence of homology. These and other FMRFamide-like genes appear to have evolved independently through internal reiterations rather than by gene duplication. This study reveals potential patterns of functional diversification in nematode neuropeptides.

Chuman, Tatsuji et al. (2011) International Worm Meeting "Identification of a New Ascaroside, Female Attracting Pheromone in Panagrellus redivivus."

Edison, Arthur S., Choe, Andrea, von Reuss, Stephan, Schroeder, Frank C., Chuman, Tatsuji, Sternberg, Paul W., Kaplan, Fatma, Ajredini, Ramadan, Alborn, Hans

[International Worm Meeting, 2011]

Panagrellus redivivus, a free-living nematode related to the well-known model organism, Caenorhabiditis elegans, has been studied in the laboratory for decades and is therefore useful for comparative biological studies with C. elegans. P. redivivus can be easily cultured in the laboratory using conditions similar to those used for C. elegans, and the two species share many desirable traits such as short generation time. Whereas C. elegans has self-fertilizing hermaphrodites and males, P. redivivus has females and males and requires mating for reproduction. P. redivivus females can specifically attract males and males can specifically attract females but the chemical nature of this attraction has until now not been known. We used a protocol, previously developed for C. elegans, to collect large volume liquid co-cultures with bacterial food as well as biologically active worm water samples of P. redivivus. In addition we developed a robust bioassay to test for female attraction using the worm water samples. By activity-guided fractionation, in combination with NMR and LC-MS analyses, we found a pheromone component, component-1, as a female attractant from its worm water sample. Component-1 is a new ascaroside compound and its structure is elucidated by MS and NMR analyses after purification. The synthesis of component-1 for confirmation of the proposed structure is now undergoing. These results suggest a highly conserved and complex system of nematode pheromones and may one day lead to new approaches to the control of parasitic species1,2). References 1.Srinivasan, J., Kaplan, F., Ajredini, R., Zachariah, C., Alborn, H. T., Teal, P. E., Malik, R. U., Edison, A. S., Sternberg, P. W., and Schroeder, F. C. 2008. A blend of small molecules regulates both mating and development in Caenorhabditits elegans. Nature. 454:1115-1118. 2.Edison, A. S. 2009. Caenorhabditis elegans pheromones regulate multiple complex behaviors, Curr Opin Neurobiol 19, 378-388.

Choe, A. et al. (2011) International Worm Meeting "Identification of ascr#1 as the female sex pheromone in the free-living nematode Panagrellus redivivus."

Kaplan, F., Sternberg, P.W., Von Reuss, H., Chuman, T., Schroeder, F., Choe, A., Kogan, D., Ajredini, R., Alborn, H., Edison, A.S., Dossey, A.T.

[International Worm Meeting, 2011]

Nematodes produce and respond to pheromones in order to help them form an appropriate behavioral or developmental response to their environment. Pheromones have been studied in the free-living nematode Caenorhabditis elegans (Clade 9A) , helping us to understand how small-molecules convey information about population density and mate availability. Panagrellus redivivus is a free-living gonochoristic nematode (Clade 10B) that serves as a useful comparative model to the hermaphroditic species C. elegans. We have developed a robust, semi-automated bioassay to study the attraction and repulsion of P. redivivus males and females to purified worm-secretions. We have performed activity-guided fractionation of P. redivivus liquid cultures, in combination with NMR and LCMS analysis, to identify gender specific sex pheromones. Here we report the isolation of the natural ascaroside, ascr#1, from the free-living nematode P. redivivus by large-scale purification. This is the same pheromone that plays a small role in dauer-formation in C. elegans, but does not play any role in C. elegans male or hermaphrodite attraction. We have evidence that ascr#1 not only attracts P. redivivus males, but also repels P. redivivus females, indicating that the same pheromone can be used across multiple genera for different purposes both between species and within species. *The first two authors contributed equally to this work.

Lassegue, Circe et al. (2011) International Worm Meeting "Do ascarosides affect egg-laying behavior in Caenorhabditis elegans?"

Lassegue, Circe, Edison, Arthur, Nimalendran, Rathika, Ajredini, Ramadan

[International Worm Meeting, 2011]

Caenorhabditis elegans, a small transparent nematode that lives in temperate soil environments, is one of the simplest eukaryotic organisms with a nervous system to be studied in great detail. Over recent years, a large number of ascarosides have been identified as signaling molecules in C. elegans (Edison, 2009). Ascaroside levels are affected by worm concentration and available food when developed in "worm water". Ascarosides have been shown to regulate a large number of behaviors in C. elegans including dauer formation (Butcher, et al., 2007), mating behavior ((Srinivasan, et al., 2008), aggregation (Macosko, et al., 2009), and olfaction (Yamada, et al., 2010). Additionally, environmental and homeostatic cues are now being explored to see how these affect nematode egg-laying habits (Schafer et al., 2001). We studied the modulatory effect of several ascarosides on egg-laying behavior and brood size in adult female C. elegans. This study aims to determine the effect of ascarosides on egg-laying behavior in adult C. elegans. A range of concentrations of several synthetic ascarosides as well as natural worm water produced by C. elegans were studied. Standard egg-laying assays and known positive and negative controls were utilized (Koelle, 2004).