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).

Long Truong et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Purification of hermaphrodite-derived social signals"

Erik Jorgensen, Fatma Kaplan, Jamie White, Long Truong, Hans Alborn, Peter Teal, Art Edison

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Signals released into the environment by C. elegans hermaphrodites elicit sex-specific social behaviors: males are attracted to the signals and hermaphrodites avoid them. We are interested in the chemical nature of these signals. The purpose of this work is to first purify the factors in hermaphrodite-conditioned media (HCM) that elicit sex-specific attraction and avoidance behaviors, and then determine their chemical structure. Here, we focus on the attraction activity. Our first purification step used reverse-phase solid phase extraction chromatography (SPEC) to separate based on hydrophobicity. HCM was loaded onto a C18 cartridge and eluted with increasing methanol concentration; fractions were collected and tested for attraction behavior. A single highly hydrophobic fraction showed very strong attraction behavior and no avoidance behavior. A less hydrophobic fraction elicited attraction behavior and a mild avoidance response. Second, we used ion-exchange SPEC to further separate the active C18 fractions. Coupled cation and anion exchange cartridges were loaded with the active hydrophobic fraction. We collected the flow-through as a neutral fraction, and then decoupled the cartridges and eluted them separately with increasing potassium chloride concentration. Two ion-exchange fractions specifically gave strong attraction behavior: a fraction from the cation-exchange cartridge that eluted at 250mM KCl, and the neutral fraction that was not retained by either cartridge. Third, reverse-phase high performance liquid chromatography (HPLC) coupled with mass spectroscopy (MS) was used to analyze both active and inactive ion-exchange fractions. We detected unique species in active fractions that are not present in the inactive fractions. We will continue our purification until we detect activities in single HPLC fractions that correspond to a single species by MS. Structures will be determined by magnetic resonance spectroscopy. To summarize our current results, it seems likely that more than one factor that can elicit the attraction behavior in C. elegans, and they can be separated into distinct fractions based on hydrophobicity and charge.

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.

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).

Paix, A. et al. (2017) International Worm Meeting "Genome editing using Cas9 ribonucleoprotein complexes and linear repair templates."

Seydoux, G., Paix, A., Folkmann, A.

[International Worm Meeting, 2017]

The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates. The protocol requires no cloning or selection, and can be used to generate base and gene-size edits in just 4 days. Point mutations, insertions, deletions and gene replacements can all be created using the same experimental pipeline. In this workshop, we will present our method and the rules governing successful genome editing in C. elegans

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.

Hwang, H. et al. (2019) International Worm Meeting "Plasticity of ascr#3 avoidance behavior in C. elegans."

Hwang, H., Kim, K.

[International Worm Meeting, 2019]

Experiences or environmental cues can influence neuronal functions and behaviors. However, the neuronal and molecular mechanisms of behavioral plasticity are not fully understood. Caenorhabditis elegans secretes a mixture of small molecules referred as ascarosides that sense environmental conditions including population density and influence many aspects of development and behavior of C. elegans (Edison, 2009; Srinivasan et al., 2012). Wild-type hermaphrodites are repulsive to ascr#3(asc-?9, C9) while wild-type males are attractive to it (Macosko et al., 2009, Jang et al 2012). Previously, we have shown that ascr#3 avoidance behaviors are modulated by early experience of ascr#3 and feeding state, indicating that ascr#3 avoidance behaviors are plastic (Hong et al., 2017, Ryu et al., 2018). Here, we next investigated whether ascr#3 response is habituated by repeated exposure of ascr#3. We found that wild-type hermaphrodites exhibit normal ascr#3 avoidance to each exposure up to ten times, suggesting that ascr#3 avoidance behavior is not habituated. However, we found that the ascr#3-sensing ADL neurons shows decreased Ca2+ response to ascr#3 upon repeated exposure of ascr#3, indicating that adapted ADL responses to repeated ascr#3 exposure do not lead to habituation of ascr#3 avoidance behavior. In addition, we examined the effects of mating on ascr#3 avoidance and found that mated hermaphrodites appear to exhibit increased ascr#3 avoidance. Understanding how the behaviors are altered upon various experience or condition is the essential step to dissect molecular and neuronal mechanisms of behavioral plasticity.

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.