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.

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).

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.

Bifarin, O. et al. (2019) International Worm Meeting "UDP-Glycosyltransferases in Caenorhabditis elegans: Insights into Roles in Xenobiotics Detoxification."

Shaver, A., Edison, A., Bifarin, O.

[International Worm Meeting, 2019]

Uridine 5'-diphospho glucuronosyl or glycosyl transferases (UGTs) are involved in phase II xenobiotics metabolism, where they aid the detoxification of xenobiotics via glycosylation of small molecules. While some UGTs have been implicated in several transcriptomics studies when Caenorhabditis elegans are challenged with xenobiotics, there is very little knowledge of the modifications carried out by specific UGTs. In an effort to improve our knowledge of the roles of UGTs in C. elegans, we sort to identify glycosylation (UGT) genes involved in the detoxification of selected xenobiotics and their respective modifications. In this light, we have developed a high-throughput toxicity and defective glycosylation assay (H-TDGA). H-TDGA incorporates a dead/alive toxicity assay using SYTOX green nucleic acid stain; and assays for defective glycosylation using High Performance Liquid Chromatography-Ultra Violet detector (HPLC-UV), High-Resolution Mass Spectrometry (HRMS), and Nuclear Magnetic Resonance (NMR) experiments, all for the detection of potentially glycosylated metabolites and structural elucidation. Using H-TDGA, we have exposed candidate UGT mutant lines to indole, a metabolite produced by Escherichia coli. Indole is toxic to C. elegans at high concentrations, and wild type animals are able to detoxify the xenobiotic via an N-linked glycosylation. Results of assays with UGT mutant lines will be presented.

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).

Goldberg, Hailey V. et al. (2019) International Worm Meeting "Development of a UGT Mutant Library in Caenorhabditis elegans."

Edison, Arthur S., Asif, Muhammad Z., Doll, Jack, Roth, Julia, Goldberg, Hailey V.

[International Worm Meeting, 2019]

The purpose of this study is to create a Caenorhabditis elegans (C. elegans) mutant library by using CRISPR/Cas9 to knock out UGT genes. This library will be comprised of the existing UGT mutants in order to provide us with the needed information to peruse other non-explored UGT genes to knock out in the future. In C. elegans, UGT genes regulate the glycosylation of environmental toxins allowing for survival of the nematode[1]. CRISPR/Cas9 is a powerful gene-editing system allowing for a Cas9 endonuclease to induce a double strand break in the DNA, rendering non-homologous end joining between the broken DNA[2]. As a result, that particular gene in the DNA is knocked out and a mutant is created. As part of the Vertically Integrated Projects (VIP) undergraduate research team at UGA, we have developed a workflow that will allow us to create this mutant library[3]. Upon completion, this library will allow us to test the effects of different xenobiotics and natural compounds on UGT knockout mutants which will allow us to better understand the role of these genes and their associated proteins in the glycosylation and drug resistance pathways of C. elegans; this provides us with a model which can be later be tested in parasitic nematodes. Additionally, the CRISPR/Cas9 protocols established for UGT knockouts will allow future undergraduate students to partake in CRISPR/Cas9 genetic research through the VIP program in the Edison Lab to continue producing UGT mutants for metabolomics analysis.

Shaver, A. et al. (2019) International Worm Meeting "Utilizing the Genetic Diversity of C. elegans for Unknown Metabolite Identification."

Edison, A., Shaver, A., Kirby, P., Andersen, E., McIntyre, L., Garcia, B., Gouveia, G.

[International Worm Meeting, 2019]

C. elegans has been and remains a valuable genetic model organism utilized to study conserved mechanisms in regulating metabolism, longevity, and age-related diseases. The extensive genetic work and tools now available for C. elegans make it an ideal candidate organism to help develop, test, and validate a pipeline to identify unknown metabolites. Today the field of metabolomics has the ability to collect tens of thousands of metabolite features within a single sample with great resolution, however, most of these features remain unknown as identifying them has proven challenging. We are utilizing the existing wealth of genetic and phenotypic data to use C. elegans as a model system for characterization of complex metabolomes and in turn a pipeline for semi-automated compound identification. We are utilizing available genetic mutants from the Caenorhabditis Genetics Center (CGC) and the fully sequenced natural isolates available through the Caenorhabditis elegans Natural Diversity Resource (CeNDR) to cover much of the genetic diversity in the species. To generate a high-throughput growth assay suitable for downstream metabolomic analysis, we developed a new method to cleanly culture millions of mixed stage C. elegans within a given sample. Our protocol allows us to spend minimal handling time on each culture while generating a sample to be utilized for both phenotypic assays (i.e. population distribution and body size) and metabolic workflows at multiple institutions (i.e. LC-MS/MS). Utilizing this workflow, we can extract C. elegans stage-specific information from each strain that will allow us to uncover detailed growth and phenotypic data previously not described and allow us to associate specific metabolic features with a given life-cycle stage. This method can be utilized in many experiments across the C. elegans field to enhance the phenotypic data extracted from a given sample.

Zhang, S. et al. (2017) International Worm Meeting "An approach to integrate 2D flow cytometry data with Nuclear Magnetic Resonance data in Caenorhabditis elegans developmental studies."

Taujale, R., Gouveia, G., Tayyari, F., Edison, A., Borges, R., Zhang, S., Ponce, F. V.

[International Worm Meeting, 2017]

Nuclear magnetic resonance (NMR) is a useful tool to study changes in Caenorhabditis elegans metabolomics as a function of development. NMR is highly reproducible and provides atom-specific information that helps in the identification of unknown compounds. We typically use between 50k-200k worms per sample. When we add biological replicates for statistical significance, it is difficult to investigate each developmental stage independently. Therefore, we have developed an approach that integrates data from the large particle?nematode?Biosorter with NMR data. This method allows for the correlation of a specific NMR feature with a specific developmental stage. We transform the flow cytometry dataset into the same dimension as NMR spectra dataset. This is accomplished by creating a 1D vector that contains the experimental worm population distribution along the axis length (TOF) and optical density (EXT) axes. Upon doing this, we can use Statistical Total Correlation Spectroscopy (STOCSY), a widely used statistical tool for finding correlated features in NMR spectra. In our application, we obtain correlations between NMR and biosorter datasets. The result is a correlation of a subset of worm population with a specific peak, which can also show whether a metabolite changes its concentration during the organism's development or not. We have tested this method by integrating a dataset from flow cytometry and NMR from the same samples. The dataset came from 28 samples of C. elegans collected at from 5 time points. After picking peaks from ascarosides that showed up in specific developmental stages as driver peaks for STOCSY, we constructed a statistical biosorter map that indicated how highly correlated (positively or negatively) the NMR peak was with the distribution. This approach is general and should be easily applied to other flow-cytometry-based quantitative analysis.