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).

Stupp, Gregory S. et al. (2011) Worm Breeder's Gazette "WormBox: a parallelized webcam recording system for nematode bioassays"

Stupp, Gregory S., Edison, Arthur S.

[Worm Breeder's Gazette, 2011]

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.

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.

Izrayelit, Yevgeniy et al. (2011) International Worm Meeting "Connecting genotype with metabotype in C. elegans: 2D-NMR based metabolomics uncovers small molecules dependent on peroxisomal beta-oxidation via DAF-22."

Edison, Arthur, Schroeder, Frank, Robinette, Steven, Izrayelit, Yevgeniy

[International Worm Meeting, 2011]

We present a novel approach to 2D-NMR based metabolomics and demonstrate its utility for the identification of diverse sets of small molecules that differentiate genetically distinct C. elegans strains. We show the value of this method by comparing two daf-22 alleles to wild type (N2) worms. daf-22 catalyzes the final step in peroxisomal fatty acid beta-oxidation to produce ascarosides, a class of small molecules that regulate dauer formation as well as several different social behaviors. Many of these ascarosides were identified by comparing 2D-NMR spectra of wild-type and daf-22 worms using a simple overlay technique (DANS)1. However, DANS was limited to detecting highly conspicuous metabolic changes. Here we report a semi-automated method for comprehensive comparison of metabolomes that combines 2D-NMR with statistical analysis. To investigate changes in the metabolomes associated with the daf-22 mutation, we grew liquid cultures of wild type and two different daf-22 mutant alleles. dqfCOSY spectra, a particularly information-rich form of 2D-NMR spectra, were acquired for both the wild-type and mutant metabolomes. A peak recognition algorithm and principle component analysis (PCA) were utilized to compare the spectra from the three C. elegans strains. Spectral back projections of principle components (PC) were visualized as COSY-like 2D spectra to connect statistical analysis with molecular structure. PCA effectively separated the spectral data sets of the three genotypes; PC1 differentiated both daf-22 mutant alleles from wild-type C. elegans, whereas PC2 separated the two daf-22 alleles from each other. Significantly, back projection of PC loadings onto the COSY spectra enabled identification of a large number of metabolites that were up or down regulated in the daf-22 mutant sets. The analysis provides insight into the role of peroxisomal beta-oxidation and ascaroside signaling in C. elegans metabolism. We highlight the wide applicability of this metabolomics approach. (1) Pungaliya C, Srinivasan J, Fox BW, Malik RU, Ludewig AH, Sternberg PW, Schroeder FC. A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7708-13.

Stupp, Gregory et al. (2011) International Worm Meeting "Responses of C. elegans to Pathogenic Challenge."

Stupp, Gregory, Edison, Arthur S., Ajredini, Ramadan, Gulig, Paul A., Robinette, Steven L.

[International Worm Meeting, 2011]

C. elegans releases and responds to many different chemicals necessary for regulating important behaviors such as mating attraction, dauer formation, aggregation, and recognition and differentiation of food and pathogens. The nematode can sense bacterial populations through small-molecule messengers such as acyl-homoserine lactones, and are able to interfere with certain quorum sensing systems. Additionally, C. elegans has a complex olfactory system which allows the nematode to avoid detrimental conditions such as areas of high population density or osmolarity and areas containing pathogenic bacteria. Moreover, this system, which is affected by released pheromones, exhibits behavioral plasticity that allows the nematode to learn and adapt, for example, to avoid an odorant associated with harmful conditions. This study seeks to determine changes in C. elegans behavior and exometabolome, the set of small molecules released into the environment by the worms, in the presence of a pathogen. We collected exudates from a synchronous population of C. elegans under standard conditions and in the presence of synthetic compounds produced by Pseudomonas aeruginosa, for the purpose of identifying worm responses to pathogenic conditions. We acquired 2D NMR spectra of the exudates, and used a novel method developed in our lab for 2D NMR alignment and pattern recognition (Robinette et al., 2011) to identify NMR peaks correlated with worm responses. We also quantified behavioral responses to these pathogen-challenged exudates using custom software that tracks individual nematodes, quantifies reversal frequency and calculates average speed for a set of nematodes on an agar plate in order to identify potential alarm responses. Additionally, we are working on a high-throughput version of the bioassay apparatus that consists of six webcams mounted on a custom lighting unit which will enable recording and analysis of six bioassays simultaneously. The availability of libraries of mutants for both C. elegans and P. aeruginosa makes this an exciting project to probe mechanisms of interspecies chemical interactions. 1. Edison AS. Current opinion in neurobiology. 2009;19(4):378-88. 2. Beale E, et al. Applied and environmental microbiology. 2006;72(7):5135-7. 3. Kaplan F, et al. Journal of chemical ecology. 2009;35(8):878-92. 4. Schulenburg H, Ewbank JJ. Molecular microbiology. 2007;66(3):563-70. 5. Yamada K, et al. Science. 2010;329(5999):1647-1650. 6. Robinette SL, et al. In Press, Analytical Chemistry (2011).

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).

Chism, Kyra et al. (2021) International Worm Meeting "Computational Analysis of UDP-glycosyltransferase Variation across Strains of Caenorhabditis elegans"

Taujale, Rahil, Asif, Muhammad Zaka, Benveniste, Maci, Chism, Kyra, Tucker, Niyelle, Watkins, Rockford, Edison, Arthur, Nicolas, Bailey, Levin, Ari, Johnson, Aleya

[International Worm Meeting, 2021]

Caenorhabditis elegans are simple non-parasitic nematodes with a relatively short life cycle and a wealth of genomic information across multiple databases, making them ideal model organisms. However, little is known about the UDP-glycosyltransferases (UGTs) responsible for their innate detoxification response. UGTs are a large family of phase II enzymes responsible for the glycosylation of small molecules across organisms, thus interacting with small molecules such as toxins in the worms' immediate environment. The Edison Vertically Integrated Projects (VIP) Computational Team is a group of undergraduate students who are working to identify the diversity that exists in UGTs across C. elegans isolates from different geographical locations found in the Caenorhabditis elegans Natural Diversity Resource (CeNDR) database in order to make inferences about their evolutionary relationships and functions. The CeNDR database is a collection of wild isolates of C. elegans and their genomic data found globally used by researchers worldwide. Out of the 250 glycotransferases are responsible for transferring sugar molecules to various substrates, there are about 79 UGTs that transfer sugar molecules to small molecules including toxins. Two approaches were implemented to identify UGTs and make inferences based on their variation. First, we created a catalog of UGTs in the N2 reference strain and used them to create a phylogenetic tree that allowed us to depict the relationships between the UGT protein sequences. For our second approach, we quantified UGT variation using the strains found in the CeNDR database. The results and inferences from this research will help us explore possible functions of UGT genes and improve our understanding of UGT variation in C. elegans.

Asif, Muhammad Zaka et al. (2021) International Worm Meeting "Determining the Effects of 1-Hydroxyphenazine Exposure on UGT Mutants in Caenorhabditis elegans"

Shah, Man, Hafeez, Zaki, Nocilla, Kelsey, Asif, Muhammad Zaka, Edison, Arthur

[International Worm Meeting, 2021]

Caenorhabditis elegans is an ideal model organism for studying the xenobiotic detoxification pathways of various natural and synthetic toxins. One such toxin that has been shown to cause death in C. elegans is 1-hydroxyphenazine (1-HP), a molecule produced by the bacterium Pseudomonas aeruginosa. Prior research in our lab has shown the median lethal dose (LD50) for 1-HP in C elegans is 179 muM in PD1074 and between 150-200muM in N2.(Asif et al., 2021; Stupp et al., 2013). Prior research has also shown that C. elegans detoxifies 1-HP by glycosylating it with one, two, or three glucose molecules in N2 worms.(Stupp et al., 2013) We hypothesize that UDP-Glucuronosyltransferase (UGT) enzymes are responsible for glycosylating 1-HP in C. elegans. To identify UGT enzymes implicated in the glycosylation of 1-HP in PD1074, we have implemented our plate-based toxicity assay developed in our prior work on available UGT strains. We began by testing eight UGT mutants, UGT-1, UGT-6, UGT-9, UGT-23, UGT-49, UGT-60, UGT-62, and UGT-66, at the LD50 concentration of 1-HP in PD1074. We screened for mutants with a different mortality rate to N2 and PD1074 worms. Additionally, we will perform HPLC/UV analysis and NMR analysis in order to describe the differences in glycosylation patterns and the ratios of glycosylated and unglycosylated products in mutant strains with differential susceptibility to 1-HP than N2 and PD1074 worms. This could help explain the variation in mortality rates between the different strains and help us understand the complexity of UGTs in C. elegans.