Saito, R. et al. (2017) International Worm Meeting "The molecular mechanism regulating quick avoidance behavior to pathogenic bacteria P. aeruginosa."

Saito, R., Shinkai, Y., Doi, M.

[International Worm Meeting, 2017]

Avoidance from unfavorable environments is one of important survival strategies in many animals. As for Caenorhabditis elegans, avoidance from pathogenic bacteria is required to select its better living environment. Worms show this avoidance behavior to Pseudomonas aeruginosa which is a common Gram-negative pathogenic bacterium and is known to affect health condition in many animals including humans. For this reason, C. elegans which fed P. aeruginosa shows an associative learning behavior between odors (secondary metabolites from the bacteria) and several uncomfort in the body. The components of secondary metabolites, which are perceived by single class of sensory neuron, have been identified. However, it is not clear what kind of uncomfort signals is detected in the body, and how those signals are integrated into the nervous system to regulate learning behavior. Therefore, in this study, we are trying to elucidate the molecular and cellular mechanism which acts to detect an uncomfort signal from P. aeruginosa in the body. To identify the genes involved in the detection of bacteria as an unfavorable signal, we searched mutants which show altered response to P. aeruginosa (PA14). The immune response systems in C. elegans is known to be activated from 4 hours after worms fed PA14. We found that spending more than 6 hours on the PA14 lawn was required to retain an associative learning in the bacteria choice assay. We also examined how quickly worms respond to PA14, and found that more than 80% of wild-type animals showed avoidance behavior to PA14 within 1 hour. Therefore, we screened mutants which did not show quick avoidance behavior to PA14 in this period. We successfully isolated a candidate mutant allele (ta218) which show a significantly lower quick avoidance behavior to PA14 (only 20% avoidance in 1 hour). We mapped the ta218 mutation at the central region of the 4th chromosome. Simultaneously, we focused on the immune response pathway in C. elegans whether some genes are responsible for quick avoidance behavior or not. Using avoidance assay, we found that some mutants such as age-1 and sek-1, both are known to function for the worm immune response, did not show quick avoidance behavior to PA14, suggesting that not all but some genes may be acting to detect pathogenic bacteria for quick avoidance. We are now trying to identify the corresponding gene for the ta218 mutant by using both SNP mapping and next-generation sequence analysis. We hope that our analyses will identify the molecular mechanism for the pathogenic bacteria detection, separated from existing immune response pathway in C. elegans.

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.

Li-Wei Lee et al. (2007) International Worm Meeting "Expression of hepatitis D viral antigen by bicistronic construct in Caenorhabditis elegan."

Chi-Ruei Huang, Li-Wei Lee, Hsiao-Wen Lo, Keng-Poo Tan, Szecheng J. Lo

[International Worm Meeting, 2007]

To investigate whether a viral protein which is dominate present in the nucleolus will affect the nucleolus structure of Caenorhabditis elegans, we developed a bicistronic construct derived from C. elegans operon CEOP5428 containing (fib-1) fibrillarin and rps-16 genes. When a construct containing pfib-1::GFP-rps-16::Cheery was expressed in transgenic worms, both GFP and cheery signal were detected. We then substituted the rps-16::cheery into DesRed::LD (large antigen of hepatitis D viral antigen) and obtained transgenic worms expressing both signals in the nucleus. The preliminary results showed that no significant phenotypes appeared in transgenic worm. However, this bicistronic construct can be employed to express different uncharacterized viral genes or oncogenes for studying their effect on phenotype as a quick functional screen.

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.

Carmona, Christopher et al. (2015) International Worm Meeting "Understanding Neurodynamics Through Light Field Microscopy."

Arisaka, Katsushi, Polanco, Edward, Carmona, Christopher, Madruga, Blake, Hammond, Addam

[International Worm Meeting, 2015]

Light field microscopy is a new technique that allows for quick volumetric imaging of fluorescent specimens. It utilizes a microlens array (MLA) as a key optical component that produces a light field and trades spatial resolution against angular resolution or axial resolution. The MLA is a matrix of lenses with diameters of 130mum that each resolve a visual perspective of a specimen being imaged at relative distances from the native object plane. As a result, recorded light-fields can be computationally reconstructed into full volumes. The volume reconstruction is formulated as an inverse linear transformation that is modeled using wave optics theory. Here, an epifluorescence light field microscope is designed and configured in order to resolve the neural activity of C. elegans during real-time inculcated behavior. The theoretical limits of the microscope's lateral resolution in relation to optical design choices are discussed and compared with experimental results. The primary focus of this investigation is the utilization of light field microscopy in conjunction with computationally intensive image processing methods as a useful tool for analyzing the behavior and corresponding brain activity of C. elegans. Light field microcopy has the potential to offer real-time 3-D video data of the unrestrained behavior of C. elegans.

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.

Park, H. et al. (2019) International Worm Meeting "Highly efficient genome editing strategy for new null mutant using "STOP-IN" CRISPR/Cas9."

Liu, J., Wang, H., Sternberg, P.W., Park, H., Tan, M.

[International Worm Meeting, 2019]

Introducing null mutations into a gene of interest is essential for understanding its function. We developed a simple and highly-efficient CRISPR/Cas9 genome editing protocol for new null C. elegans mutants. We use a 43 basepair "STOP-IN" cassette with multiple stop codons in all three reading frames. This method is cloning-free and using the simple mixture of preassembled Cas9 protein and repair-ssDNA oligonucleotides for direct injection into gonads. A unique sequence harbored in the "STOP-IN" cassette facilitates quick detection of successful knock-in events via PCR. Also, an additional exogenous Cas9 target sequence in the cassette allows further genome editing. We have used this "STOP-IN" CRISPR/Cas9 method to generate new null mutants for over 70 genes, including nlp (Neuropeptide-Like Protein) and other well-conserved but uncharacterized genes. Our knock-in rate averages 57%, based on the animals we screened for a co-CRISPR knockin. We also successfully reverted the mutants back to the wild-type using same method targeting the knock-in sequence; this allows construction of isogenic control strains that are useful for quantitative phenotypes. The overall procedure from injection to confirmation can be finished within 12 days per gene. We suggest that this "STOP-IN" CRISPR/Cas9 technique will be quite useful to build new a null mutant library to understand gene or gene family function in C. elegans and other model organisms.