Zuckerman BM et al. (1991) International C. elegans Meeting "A MICROBIAL EGG-LAYING INHIBITING FACTOR FOR CAENORHABDITIS ELEGANS."

Zuckerman BM, Willett JW, Dicklow MB

[International C. elegans Meeting, 1991]

A microbial exometabolite (ME) has been isolated which inhibits egg- laying in Caenorhabditis elegans. Nematodes exposed to active concentrations of the factor, and maintained at 22C in axenic culture ( liver extract medium), mature and grow normally. Eggs are fertilized and larvae develop and move within eggs as in untreated nematodes. However, eggs are not laid, though occasionally hatch occurs within the gonad. In shake culture of the microbe, active yellow-pigmented ME appears at 3-4 days. A concentration of 8 parts ME: 1 part liver extract completely inhibits egg-laying. Concentrations of 4 ME: 1 liver extract and 2 ME: 1 liver extract, inhibit egg-laying about 50 and 25%, respectively. Sections from nematodes exposed to extracts of a microbe showing similar properties examined by transmission electron microscopy indicated that the constrictor and dilator muscles associated with vulval function in egg-laying appear normal. ME is thermostable (at 100C for 5 min.), and the active fraction does not pass through 6,000-8,000 MW dialysis tubing. About one-half of the activity is lost when ME is dialyzed through 12,000 14,000 MW membrane tubing, suggesting the presence of at least two active fractions. Trypsinization obliterated activity. Thus far, the double control experiment with trypsin inhibitor has not yielded definitive results. SDS gel electrophoresis of boiled ME, with molecules below 8,000 MW removed by dialysis, revealed several strong protein bands, one or more of which may be ME. ME purification and characterization studies continue.

Tsukita Sh et al. (2000) Japanese Worm Meeting "CLC-1, a homologue of vertebrate claudin, is present at cell-cell junction of pharynx and has some barrier function"

Asano K, Tsukita Sh, Sasaki H, Furuse M, Asano A

[Japanese Worm Meeting, 2000]

Several homologues of claudin, major integral protein of tight junction, were found in the genomic database of C. elegans. Among them, CLC-1 (formerly claudin-CE1) was studied first. It is a 182 amino acid protein with four transmembrane domains. CLC-1::GFP is localized cell-cell junctions between myoepithelial cells of pharynx. CLC-1::DsRed was co-expressed with JAM-1::GFP in pharynx of SU93 strain which is stably expressing GFP tagged JAM-1 at cell-cell junctions, although expression of the former was not detected in hypodermal cells. Incubation of the worms in a solution containing TRITC-dextran (MW=10,000) showed that this molecule can go into gullet and gut as incubation progresses, but no penetration of the molecule into tissues was observed. RNAi experiments with full length CLC-1 cDNA showed that this barrier for the high molecular weight dye was damaged by RNAi, and penetration of the dye into pharynx and some other tissues were observed.

Willett JD et al. (1991) International C. elegans Meeting "SEROTONIN AND AGING IN CAENORHABDITIS ELEGANS."

Madan S, Oates KK, Willett JD, Hong C, Zuckerman BM

[International C. elegans Meeting, 1991]

Alterations in neuroendocrine function are fundamental to altered physiological responses to stimuli observed in aging organisms. In mammalian systems the neurotransmitter serotonin declines in the senescent central nervous system. Such changes are believed relevant to functional declines in learning, memory and behavior. Since C. eleqans is an ideal system in which to explore the molecular mechanisms of age-dependent alterations in serotonin levels, studies have been undertaken on serotonin levels throughout the nematode life cycle. While serotonin is present in C. eleqans and 'serotonergic' cells have been identified using immunofluorescent techniques, its functional role in these organisms remains obscure. As in mammalian systems, a decline in serotonin levels in the senescent nematode is observed. We have also found an indolealkylamine of unknown structure present in amounts greater than that of serotonin. This substance appears to be a 5-oxygenated indolealkylamine with a MW of 234. Work is underway to fully characterize this material.

Asif, M. et al. (2019) International Worm Meeting "Implementation of a Model for Team Based Undergraduate Research: Toxicity studies on UGT mutants of Caenorhabditis elegans."

Rahmatullah, T., Asif, M., Maina, S., Roth, J., Guo, J., Edison , A., Goldberg, H., Doll, J., VanDerGaag, T.

[International Worm Meeting, 2019]

Engaging undergraduate students in life science research promises substantial benefits, but those benefits often aren't realized due to reduced access, improper training, and lack of awareness of the needs of undergraduate researchers1. The Vertically Integrated Projects (VIP) model of undergraduate research was developed by researchers at Purdue University in 20012. According to this model, teams of undergraduate researchers work for a minimum of one year on a pre-defined project. They are overseen by a graduate student mentor and experienced members of the team are responsible for training new members of the team. We have developed a training regimen and a team design that allows for two teams of undergraduate students (3-4 students per team) to work on different projects which link together forming a cohesive, publishable research study. In this case, the teams are studying the effects of the toxin 1-Hydroxyphenazine (1-HP) on Uridine 5'-diphospho-glucuronosyltransferase (UGT) knockout mutants, which prior research suggests are responsible for xenobiotic detoxification in parasitic nematodes3-4. One team is responsible for developing and conducting assays in order to observe the difference in xenobiotic resistance in the different knockout mutants while the other team is responsible for creating the necessary knockout mutants using the CRISPR gene editing method5-6. We have identified six key areas of research, which each team is trained for. They are rearing of the model organism, study design, method development, data collection, data handling and analysis, and writing for publication. We are also working on developing measures to test the proficiency of each team in these tasks as we deem them necessary for a successful research endeavor.

Fares H et al. (1996) East Coast Worm Meeting "The coelomocytes and immunity of Caenorhabditis elegans"

Greenwald IS, Fares H

[East Coast Worm Meeting, 1996]

The coelomocytes in C. elegans are large cells full of vesicles that are situated in the pseudocoelomic space of the worms. Adult hermaphrodites have six of these cells while adult males have five. Cells homologous/analogous to these coelomocytes in other organisms are involved in their immune response. Furthermore, some aspects of immunity have been found to be conserved among different organisms throughout evolution. Understanding the function of coelomocytes and immunity in the worm C. elegans might give us some insights into some aspects of immunity in bigger organisms. To begin studying the biology of the coelomocytes in C. elegans, we are first trying to determine the types of compounds that can be internalized by these cells. So far, we have found that Bovine Albumin (BA)-FITC, Lipopolysaccharide (LPS)-FITC, and Dextran-rhodamine (MW 40,000 Da) injected into the pseudocoelomic space are efficiently taken up by the coelomocytes, while similar injections with FITC or Dextran-FITC (MW 2,000,000 Da) are not. The compounds taken up by the coelomocytes are probably membrane bound (punctate fluorescence in the coelomocytes). Furthermore, the fluorescent signal inside the coelomocytes from the BA-FITC and LPS-FITC appears yellowish in time indicating a more acidic milieu. Also, the fluorescent signal from the BA-FITC in the coelomocytes, but not from BA-FITC injected into body wall muscle, disappears in time, suggesting that it (at least the FITC) is being degraded/inactivated in the coelomocytes. The coelomocytes internalize GFP in transgenic worms carrying DNA expressing GFP secreted into the pseudocoelomic space. Interestingly, we have found that the fusion proteins from constructs expressing the first 80 amino acids of SEL-1 (which includes a signal sequence) fused to GFP are more efficiently internalized by coelomocytes than ones expressing only the signal sequence of SEL-1 fused to GFP. We are currently trying to determine the molecular reason for this. We are also setting up a large scale genetic screen for mutants defective in the uptake of secreted GFP. A question to be addressed is whether the coelomocytes, in addition to a scavenging function, are able to mediate immunity in worms. To that effect, we have found so far that worms injected in their pseudocoelomic space with Escherichia coli (concentration of 2x107 ml; ~100 E. coli per worm) eventually die. By lowering the concentration of bacteria injected, we will be able to determine whether worms lacking coelomocytes (by laser ablation or mutation) are susceptible to concentrations of bacteria that wild type worms can tolerate.

Kawazoe, Yuya et al. (2011) International Worm Meeting "A multi-worm optogenetic system."

Kimura, Kotaro, Yawo, Hiromu, Kawazoe, Yuya

[International Worm Meeting, 2011]

Optogenetic techniques are extremely useful for proving that the particular activity pattern of neuron(s) is the causal reason for a specific neural function. One of the major problems associated with optogenetics, however, is the requirement of a strong light for the activation. To analyze C. elegans behavior with optogenetics, most methods use a microscope with a strong light source to obtain enough light intensity and a computer-controlled motorized stage to keep the worm in the field of view. However, such methods limit the observation to one animal per assay, require multiple expensive equipments, and may not be suitable for experiments with delicate signal gradients such as odor or temperature. To overcome the limitations associated with such methods, we have established a simple and easy-to-use optogenetic system in combination with a strong LED ring and an improved channelrhodopsin (ChR). First, we developed a strong LED ring that allows exposure of an entire 9-cm plate at 0.5 mW/mm2 with 470 nm light; the intensity is comparable to the strongest light intensity obtained from a mercury lamp for GFP excitation. The LED can also be used to expose a small area of the plate at 1.2 mW/mm2. We have also developed a custom-made program with LabVIEW to control LED intensity in synchronization with capturing the images via a USB camera. Because of the high resolution of the camera (approximately 2.5 k x 2 k), the system does not need a microscope or a motorized stage, and we can record the behavior of multiple worms on a 9-cm plate simultaneously. Thus, using this system we are able to efficiently conduct optogenetic behavioral analysis for delicate signal gradients. In addition, this system is relatively inexpensive: the total cost, including the LED ring, camera, and PC is approximately $14,000. To prove that the LED can actually activate the optogenetic ion channel, we expressed an improved ChR, ChRGR (Wen et al., PLoS ONE, 2010), in the body wall muscles with a myo-3 promoter. Upon light stimulation, the movement of transgenic animals was significantly inhibited in the presence of all-trans-retinal, indicating that the LED intensity was strong enough to activate ChRGR. We are currently trying to express ChRGR in the AWB sensory neurons to understand how the activity of AWB regulates avoidance behavior to repulsive odor 2-nonanone, which is enhanced by preexposure to the odor in a dopamine-dependent manner (Kimura et al., J. Neurosci., 2010).

Ewbank JJ et al. (2000) European Worm Meeting "Using C. elegans to identify Serratia marcescens virulence factors"

Ewbank JJ, Kurz CL, Finlay BB, Celli J

[European Worm Meeting, 2000]

Tan and Ausubel, working with the enterobacterium Pseudomonasaeruginosa, have established C. elegans as a model for the study of pathogenesis and host defences. Clearly, the use of the worm as a model for studying pathogenicity will be limited to those pathogens that are able to infect the worm. Luckily, in this respect, its susceptibility to P. aeruginosaappears not to be an isolated case. A second opportunistic human pathogen, Serratia marcescens, is also capable of infecting C. elegans. Like P. aeruginosa, S. marcescens is able to infect a broad range of plant and animal hosts and has been used as a model pathogen in studies of Drosophila innate immunity. Using a strain of S. marcescensthat expresses GFP, we have been able to follow the infection process. The bacteria are able to survive within the usually hostile environment of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens. We are currently screening these mutant bacterial clones individually for those showing reduced virulence.Of the first 2000 mutants screened, 18 showing markedly reduced virulence have been retained for further study. The molecular characterization of these mutants may reveal novel virulence factors that represent potential drug targets. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. S: 29-34.

C Leopold Kurz et al. (2001) International C. elegans Meeting "Identifying universal Serratia marcescens virulence factors using C. elegans."

Jonathan Ewbank, Jean Celli, Dominique Ferrandon, B Brett Finlay, Emmanuel Andres, Sophie Chauvet, C Leopold Kurz, Mitchell Uh

[International C. elegans Meeting, 2001]

Tan and Ausubel, working with the enterobacterium Pseudomonas aeruginosa , have established C. elegans a s a model for the study of pathogenesis and host defences . A second opportunistic human pathogen, Serratia marcescens , is also capable of infect ing C. elegan s . Like P. aeruginosa , S. marcescens is able to infect a broad range of plant and animal hosts. Using a strain of S. marcescens that expresses GFP, we have been able to follow the infection process. T he bacteria are able to survive within the usually hostile environmen t of the nematode intestine, proliferate and kill the host. Under standard assay conditions, the progression of the infection is highly reproducible. We have used a transposon mutagenesis system to create a library of insertion mutants of S. marcescens . We are currently screening these mutant bacterial clones individually for those showing reduced virulence . Of the first 2000 mutants screened, 9 showing markedly reduced virulence have been retained for further study. T he molecular characterization of these mutants has revealed novel virulence factors. In order to determine whether these virulence factors are specific to the infection of the nematode, we have also tested them in a Drosophila infection model and found that 4 of them are attenuated for their virulence. Tests in a mammalian model will reveal whether we have identified virulence factors that are important irrespective of the host. Tan MW, Ausubel FM. (2000) Caenorhabditis elegans : a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr Opin Microbiol. 3: 29-34.

Centonze VE et al. (1995) International C. elegans Meeting "CALCIUM OSCILLATIONS DURING EARLY C. ELEGANS DEVELOPMENT"

White JG, Centonze VE

[International C. elegans Meeting, 1995]

Transient increases in intracellular calcium ions have been observed during fertilization and subsequent stages of the first cell cycle in oocytes, eggs and zygotes of many animal species. These calcium spikes are thought to modulate reorganizations in cellular structure and function during meiosis, mitosis, and cytokinesis. Using techniques to obtain images at high spacial and temporal resolution, we are attempting to document oscillations of intracellular calcium during the early stages of C. elegans development. Calcium indicator dyes, Calcium Green-1 dextran (10,000 MW) or Calcium Crimson (Molecular Probes, Eugene, OR) are injected into the germline or oocytes of C. elegans. Early embryoes containing dye are later dissected from the worm and mounted on an agar pad for observation by light microscopy. Standard confocal microscopy has been used to record in time-lapse the calcium dynamics at a single focal plane in an embryos loaded with Calcium Green. Our preliminary evidence indicates that there is a rise in intracellular calcium coincident with the onset of mitosis and that elevated calcium levels are also observed in the region of the cytokinetic furrow. Additionally, there are areas of conspicuously low calcium in regions from which there is known to be cytoplasmic streaming of P-granules. This may be evidence for a calcium sink signaling cell polarization. We hope to obtain a more complete view of calcium dynamics by collecting time-lapsed 3D data sets. This will be done using the 2-photon excitation laser scanning system being developed at the IMR.

Fouad, A.D. et al. (2017) International Worm Meeting "Efficient and precise ablation of C. elegans cells using pulsed infrared laser cauterization."

Ji, H., Fang-Yen, C., Teng, S., Mark, J.R., Fouad, A.D., Liu, A.

[International Worm Meeting, 2017]

Laser microsurgery has long been a powerful tool for creating specific cellular or circuit lesions in C. elegans. Laser ablations have been typically performed by using a nanosecond or femtosecond pulsed laser to induce local plasma formation in the target cell. In our experience, aberrations in microscope objectives limit our ability to ablate deep structures in animals larger than L2 larvae using traditional laser ablation systems. In addition, the small size of plasma formation makes it difficult to lesion larger structures such as the ventral nerve cord. Here, we demonstrate that C. elegans cells can be efficiently ablated by using a pulsed infrared laser to damage tissues via locally elevated temperature. This method, while less spatially precise than ablation by nanosecond or femtosecond lasers, is highly effective in thick samples like L4 and adult C. elegans. We demonstrate that a single, 0.8 ms pulse from a laser with a wavelength of 1480 nm and peak power of 400 mW, is ideal for killing a targeted neuron but not its immediate neighbors. At this dosage, a neuron 2.5 m away from the target has a ~45% chance of being killed, and a neuron 5 m away has a ~10% chance of being killed. We show that a short train of pulses can reliably sever the ventral nerve cord in adult C. elegans. Hence, our method is a tool that can be used to lesion cells and other structures in C. elegans in cases where traditional microsurgery methods are not practical.