K Bull et al. (2004) European Worm Meeting "FURTHER INVESTIGATIONS TO CHARACTERISE THE PHYSIOLOGICAL EFFECTS OF THE NOVEL ANTHELMINTIC EMODEPSIDE ON C. ELEGANS"

A Harder, K Bull, R Walker, L Holden-Dye

[European Worm Meeting, 2004]

The cyclodepsipeptide, emodepside, is a novel broad spectrum anthelmintic with a distinct mode of action. Previous research in our laboratory, using the model nematode Caenorhabditis elegans, has demonstrated that emodepside potently paralyses pharyngeal and body wall muscle by binding to a latrophilin-like receptor at the neuromuscular junction, leading to activation of a G protein signalling pathway and the exocytosis of, possibly, an inhibitory neuropeptide(s). The present study reports on the effects of emodepside on C. elegans growth and development to obtain a better understanding of the physiological effects of this novel anthelmintic. The action of emodepside on the growth and locomotion of a synchronized population of C. elegans (N2 Bristol strain) was determined. Eggs were isolated from gravid adult hermaphrodite worms by the alkaline hypochlorite method (Lewis & Fleming, 1995) and were resuspended in M9 buffer (composition in g/litre: 6g Na2HPO4, 3g KH2PO4, 5g NaCl, 0.25g MgSO4.H2O). Approximately 100 eggs were placed on agar plates containing OP50 E. coli and either emodepside (10nM to 500nM) or vehicle control (1% ethanol). The plates were incubated for 5 days at 20C and the developmental progress of the worms, from hatching to fertile adulthood, was monitored. Locomotion was quantified by counting body bends/minute. Emodepside possesses a clear effect on the locomotion of C. elegans larval stage four (L4), with a concentration-dependent effect on normal sinusoidal movements. The IC50 for this effect on L4 worms was ~10nM. At an emodepside concentration of 50nM or more, the majority of L4 worms appeared unable to produce body bends in their anterior region, although they were still capable of forward locomotion via shallow and irregular sinusoidal movement in their posterior region. It was also observed that at 10nM or higher, emodepside retarded C. elegans growth to adulthood. Worm development in the presence of emodepside (10nM to 500nM) was measured quantitatively 43 and 64 hours after transfer of synchronised egg population onto the agar plates. Emodepside also appears to have a significant concentration-dependent impact on C. elegans egg laying. At 100nM or more, hermaphrodite adult worms became bloated with unlaid eggs, and at 500nM adult C. elegans appeared completely incapable of egg laying, eventually rupturing due to hatching of progeny within the parent worm.

K Amliwala et al. (2002) European Worm Meeting "A comparison of the action of ivermectin and emodepside on C.elegans"

K Amliwala, R J Walker, A Harder, J Willson, L Holden-Dye

[European Worm Meeting, 2002]

The cyclodepsipeptide, emodepside, is a novel broad spectrum anthelmintic with a distinct mode of action. Here we report preliminary studies comparing the commercially available broad spectrum anthelmintic ivermectin with emodepside. The free-living nematode Caenorhabditis elegans was used to identify the effective concentration window of emodepside inhibitory action on the growth and development of C.elegans. This information will subsequently be utilized in forward genetic strategies to identify emodepside resistant phenotypes, with the aim of identifying genetic determinants of emodepside action. The action of ivermectin and emodepside on the growth and locomotion of a synchronized population of C.elegans (N2 Bristol strain) was determined. Eggs were isolated from gravid adult hermaphrodite worms by the alkaline hypochlorite method (Lewis, Fleming, 1995) resuspended in M9 buffer (composition in g/liter Na2HPO4 6, KH2PO4 3, NaCl 5, MgSO4.7H2O 0.25). Approximately 100 eggs were placed on agar plates containing OP50-E.coli and either emodepside (100pM 950nM), ivermectin (100pm-9.5nM) or vehicle control (1% ethanol). The plates were incubated for 5 days at 20C and the developmental progress of the worms, through larval stages, to fertile adulthood, monitored. Locomotion was quantified by counting body bends/min. Ivermectin inhibits the development of larval stages to fertile adults at concentrations as low as 2.25nM. There exists a concentration dependent inhibition of larval development to adulthood up to 9.5nM, at which point 90% of juvenile worms are arrested at L1 stage (n=2). Emodepside has no observable effect on larval stages of C.elegans at any of the concentrations tested (n=4). However, in mature fertile adults there was a concentration-dependent effect on locomotion. The threshold for this effect was observed at 4.5nM (13 2 compared to control 25 1, body bends/min, n=4), and at 90nM locomotion was dramatically decreased to 1.3 0.3 body bends/min. It was observed that above 180nM emodepside growth to adulthood was retarded, on average by 24 hrs, when compared with worms on lower emodepside concentration and control plates. Furthermore, at 950nM hermaphrodite adults were arrested before egg laying could occur (n=2). In conclusion, these data suggest that emodepside has a distinctly different anthelmintic mechanism to ivermectin, in that it interacts with a target that is expressed, or functionally important, in the adult stage only. Furthermore, this target is involved in motor control.

J Willson et al. (2002) European Worm Meeting "An investigation into the mechanism of action of the novel anthelmintic emodepside using A.suum"

J Willson, A Harder, L Holden-Dye, R J Walker, K Amliwala

[European Worm Meeting, 2002]

Resistance of parasitic nematodes to existing anthelmintics has encouraged the search for novel compounds. A new compound, emodepside, a 24 membered cyclic depsipeptide, acts as a potent, broad-spectrum anthelmintic. It causes fast onset paralysis of nematodes, favouring the view that it is neuropharmalogically active. The aim of this project is to define and characterise the site of action of emodepside. The action of emodepside on the body wall muscle of the nematode Ascaris suum was investigated. It was shown that emodepside relaxes the muscle in a concentration-dependent and irreversible manner. Dorsal muscle strips (DMS) were bathed in artificial perienteric fluid (APF). The DMS was contracted using acetylcholine (ACh; 30M, 30secs). The amplitude of this contraction after pretreatment of the DMS with emodepside (10M) was expressed as a percentage of the control contraction (i.e. without emodepside pretreatment). Emodepside reduced the contraction by 39% 4 (n=9). Emodepside was also shown to relax the DMS following prolonged contraction with ACh. The DMS was contracted with ACh (30M, 10mins). A slow relaxation of the DMS was then observed over a period of 10mins. Addition of 10M emodepside at the point of maximum contraction, caused a significantly (P<0.005) faster relaxation of the DMS, 9.0% min-1 0.3 (n=5), compared to control (6.1% min-1 0.6, n=5). The action of emodepside was similar to that of an inhibitory neuropeptide PF2 (SADPNFLRFamide), and differed from that of the inhibitory neurotransmitter GABA. Application of PF2 (1M) at the maximum point of ACh contraction caused a faster relaxation of the DMS (9.2%min-1 0.4 n=6) compared to ACh control (7.4% min-1 0.3, n=6), which was similar to emodepside (9% min-1 0.4, n=6). In contrast to the slow relaxation of the muscle observed with emodepside and PF2, the response to GABA was rapid and returned to base line tension. To elucidate whether emodepside acts pre-or post-synaptically at the neuromuscular junction, the effect of emodepside was investigated on a denervated muscle strip preparation. Application of emodepside (10M) to the denervated muscle strip caused no reduction in ACh induced contraction (n=7). These data suggest that emodepside may inhibit nematode motility by triggering the release of an inhibitory neuropeptide at the neuromuscular junction. Emodepside therefore acts pre-synaptically to cause a relaxation of worm muscle. Emodepside action is similar to that of the inhibitory peptide PF2 and differs from that of GABA. Currently I am determining how emodepside acts pre-synaptically to cause release of an inhibitory peptide. To this end the effect of emodepside on c.elegans will be investigated, to give an insight into the molecular mechanisms of emodepside action.

Marcus Guest et al. (2006) European Worm Meeting "Investigations into the Mode of Action of the Novel Anthelmintic Emodepside"

Marcus Guest, Kiran Amliwala, Robert Walker, Lindy Holden-Dye, Kathryn Bull, James Wilson, Neil A. Hopper

[European Worm Meeting, 2006]

Emodepside is a novel cyclo-octadepsipeptide that paralyses nematodes (1). A number of different lines of evidence indicated that the effect of emodepside may involve an effect on neurotransmitter release including the following: it causes de-staining of neurones labelled with FM4-64; goa-1(n1134) and egl-30(tg26), both of which are predicted to have increased ACh release at the neuromuscular junction, are hypersensitive to emodepside in two assays, locomotion and pharyngeal pumping; strains with mutations in various synaptic proteins involved in vesicular-mediated release of neurotransmitter are less susceptible to the paralytic actions of emodepside (2).. A mutagenesis screen has been performed to isolate highly resistant animals. These resistant animals show at least a hundred-fold less sensitivity to emodepside than wild-type and intriguingly show hyper-sensitivity to the acetylcholine esterase inhibitor aldicarb. This suggests that emodepside may reduce acetylcholine release at the neuromuscular junction, in contrast to the previous model of increased transmitter release. Aldicarb is being used as a tool to address the effects of emodepside on acetylcholine release and to resolve these conflicting results. Recent data indicate that treatment of wild-type animals with a low concentration of emodepside increases the time to paralysis in the presence of aldicarb. Currently we are testing whether sensitivity to levamisole is also affected by pretreatment with emodepside.. (1) Harder et al., 2005 Parasitol Res 97, S1-S10. (2) Willson et al., 2004 Current Biology 14, 1374-1379. Supported by BBSRC CASE and Bayer. We are grateful to the C. elegans knockout consortium (USA and Japan) for the provision of strains.

Audrey Bellier et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Mutation confers resistance to Bacillus thuringiensis Cry toxins in C. elegans"

Chang-Shi Chen, Audrey Bellier, Raffi Aroian

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis (Bt) are pore-forming toxins that specifically target insects and nematodes. To better understand the toxicity of the Cry toxin and the resistance pathways of the target animals, we are studying Bt toxin action and resistance in C. elegans. A previous genetic screen in the laboratory for C. elegans resistance mutants to Cry5B intoxication led to the identification of glycolipid carbohydrates as key receptors for Cry5B toxin. We have been studying another Cry toxin -Cry21A- that is also highly toxic to C. elegans. Interestingly, the mutants resistant to Cry5B appear to be sensitive to Cry21A, suggesting that Cry5B and Cry21A proteins use different receptors or different mechanisms to intoxicate the nematodes. Thus, studying resistance to Cry21A is predicted to help elucidate new ways nematodes can use to resist Bt toxins. We have developed an efficient genetic screen for recovering mutants resistant to Cry21A. Those mutants appear with a much lower frequency than Cry5B resistant mutants, suggesting that Cry21A is harder to resist. One of the mutants found in that resistance screen presents an early stop codon in a previously identified gene. Loss-of-function mutation in this gene has already been reported to confer resistance to pathogenic attack. Interestingly, we found that this mutant is also resistant to other Cry toxins -Cry5B and Cry6A-, showing for the first time a broad resistance mechanism to different Cry toxins. Moreover, we found that this mutant shows resistance to various other stresses such as heat, pathogenic bacteria and has an increased lifespan. We will present here the first mutant resistant to Cry toxins that is not related to a Cry toxin specific mechanism (receptor, proteases..), but is a broadly applicable mechanism.

Audrey Bellier et al. (2007) International Worm Meeting "Isolating resistance mutants to Bacillus thuringiensis Cry21A toxin in C. elegans."

Audrey Bellier, Raffi Aroian, Tim Tucey

[International Worm Meeting, 2007]

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes. They are used around the world as natural insecticides and in millions of hectares or transgenic crops. The greatest threat to the use of this technology is the development of pest resistance. To better understand the toxicity of the Cry toxin and the resistance pathways of the target animals and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. A previous genetic screen in the laboratory for C. elegans resistance mutants to Cry5B intoxication led to the identification of glycolipid carbohydrates as key receptors for Cry5B and Cry14A toxins. We have been studying another Cry toxin -Cry21A- that is also highly toxic to C. elegans. Interestingly, the mutants resistant to Cry5B appear to be sensitive to Cry21A, suggesting that Cry5B and Cry21A proteins use different receptors or different mechanisms to intoxicate the nematodes. Thus, studying resistance to Cry21A is predicted to help elucidate new ways nematodes can resist Bt toxins. We have developed an efficient genetic screen for recovering mutants resistant to Cry21A. Those mutants appear with a much lower frequency than Cry5B resistant mutants, suggesting that Cry21A is harder to resist. We used Cry21A toxin produced in Bt for the initial screen and we retested the resistance mutants candidates with Cry21A produced in E. coli, confirming that way that those mutants are resistant to Cry21A and not to other components produced by Bt. We found two mutants that are much healthier than N2 when exposed to a low dose of Cry21A toxin, either produced in Bt or E. coli. Those mutants have been outcrossed and we are looking at the effects of the toxin on their viability and fertility. The characterization and mapping of those mutations is under progress.

Hideaki Hiraki et al. (2003) International Worm Meeting "Semi-automatic system for creation of cell shape model in C. elegans embryogenesis"

Hideaki Hiraki, Yuji KOHARA

[International Worm Meeting, 2003]

Cells change their shapes and arrangements in embryogenesis. 4D microscopy of C. elegans is useful to observe the process. Time-lapse recording of multiple optical sections with Nomarski DIC microscope and tools to analyse the results have been developed and used successfully, especially for cell lineage analysis because cell nuclei of C. elegans are evident in DIC images. Though cell shape models of early embryo can be reconstructed from DIC images, delineating cell boundaries become harder as the cell number increases. On the other hand, cell shapes can be directly visualized by confocal fluorescent microscopy with vital stain of plasma membranes. We have been developing a computer system to create cell shape models from a time series of confocal images of plasma membranes. In the system, cell shapes are automatically calculated by a seeded region growing algorithm from a 3D image and a set of seed point coordinates. The algorithm segment the image volume to enclosed regions one per a seed. Here a cell boundary is detected as ridges of high fluorescent signal between two seed points. In many cases, the seed points can be automatically placed using results of another time point. But manual editing of the seed sets is necessary where signal to noise ratio is low. The system constructs a pseudo cell lineage from the overlay relationships among the segmented regions over the time course. The inconsistency in the pseudo lineage indicates where editing is required and the editing is assisted by the graphical user interface. We have tested the system on the "dub" data set in WORM 4D CDs, 6/17/97, Bill Mohler, UW-Madison, that is of N2 embryo in 24-200 cell stages. Changes in physical parameters of cell shapes and arrangements, such as cell volumes and cell-to-cell contacting areas, could be estimated from the resultant shape model of a whole embryo under gastrulation. We are planning to apply this system to compare mutant embryos and the embryos from other species closely related to C. elegans.

Hideaki Hiraki et al. (2005) International Worm Meeting "Semi-automatic system for the creation of cell shape models in C. elegans embryogenesis"

Yuji KOHARA, Hideaki Hiraki

[International Worm Meeting, 2005]

Cell to cell interactions play critical roles in early embryogenesis, therefore, it is very important to have information about the arrangements of cells, cell shapes and the contact among them. Early embryogenesis of C. elegans can be analyzed utilizing time-lapse recording of multiple optical sections with a Nomarski DIC microscope. Though cell shape models of early embryo can be reconstructed from DIC images, delineating cell boundaries becomes harder as the cell number increases. Conversely, cell shapes can be directly visualized by confocal fluorescent microscopy with vital stain of plasma membranes. We have been developing a computer system to create cell shape models from a time series of confocal images of plasma membranes. In this system, cell shapes are automatically calculated by a seeded region growing algorithm from a 3D image and a set of seed point coordinates. Manual editing of the seed coordinates is assisted by a graphical user interface. The region growing algorithm segments the image volume in such a way that one region contains one seed. A cell boundary is detected as ridges of high fluorescent signal between two seed points. We tested the system on the "dub" data set in WORM 4D CDs kindly provided by Bill Mohler, and obtained cell shape models successfully for the most part. However, we encountered a problem that some contours of cells weren't delineated correctly where the signal to noise ratio was low. This time, we reinforced the system with the following two methods to fix the problem. One is to merge some regions after the region growing step. By this method, a cell could be represented by a few regions and the noise-derived false region boundary became minimized. Another one is a correction method based on the comparisons of the segmentation results among neighboring timepoints. Even if the cell shape is delineated incorrectly, the images of its neighboring timepoints can be sometimes processed correctly. However, because cells deform during development, comparing cell shapes among the neighbor timepoints is not straightforward. Therefore, we devised a program to calculate a smooth deformation field between two timepoints assuming that cells deformed continuously. Some errors of the segmentation could be corrected by voting among the candidate shapes that were generated by deformation of the segmentation results from the neighbor timepoints. We are improving the system more, and plan to apply this system to compare the cellular arrangements and the cell-to-cell contacts among mutant embryos and the embryos from other species closely related to C. elegans.

Zarkower D et al. (1991) International C. elegans Meeting "THE tra-1 GENE ENCODES TWO ZINC FINGER PROTEINS."

Hodgkin JA, Zarkower D

[International C. elegans Meeting, 1991]

The tra-l gene is the terminal gene in the regulatory pathway controlling sexual differentiation in C. elegans. Mutational studies have suggested that tra-l is necessary and sufficient for female somatic development and that it has a complex role in the germ line. We have examined transcription from a region shown to contain tra-l based on the detection by clones from the region of rearrangements that cosegregate with several tra- 1 mutations. These rearrangements suggest that the gene spans at least 25 kb. The tra-l region encodes at least two transcripts. One is a 1.4 kb RNA that is present only during L2. The second is a 5 kb RNA present during all hermaphrodite stages and in adult males. Both RNAs are of low abundance. There may also be a third RNA of 1 kb in adult males. We have not yet examined larval males. Northern blot analysis suggests that the 1.4 kb RNA is spliced from a primary transcript of about 13 kb and the 5 kb RNA from a primary transcript of at least 23 kb. We have isolated and sequenced cDNAs representing the 1.4 kb RNA from the Kim library and by PCR. The 1.4 kb RNA can encode a protein of about 340 amino acids with two C- terminal 'zinc-finger' motifs. The fingers are 45% identical to those of GLI, a human gene amplified in certain glioblastomas, and to cubitus interruptus Dominant, a Drosophila segment polarity gene. The non-finger sequence is serine-rich and shows no similarity to other proteins in the databases. Genomic sequencing and PCR suggest that the 5 kb RNA is colinear with the 1.4 kb RNA until the end of the second finger. It is then spliced to include two more fingers that are 80% identical to the corresponding GLI and cubitus interruptus fingers. By analogy to the other two genes, there is likely to be a fifth finger in the tra-l product. cDNA clones of the 5 kb RNA have proved elusive. To help define the functions of the tra-l products, we have examined RNA from tra-l mutants. One mutation, el488, a rearrangement upstream of the predicted start of transcription, appears to reduce levels of the 1.4 kb RNA. Since el488 animals have a hermaphrodite gonad and germline in a masculinized soma, the product of the 1.4 kb RNA may function primarily in the nongonadal soma. We have also analyzed a number of stronger mutations in strains carrying eDp6 (a free duplication covering tra-l) and have detected transcripts of altered size. These are harder to interpret due to the presence of wild type transcripts from eDp6. To overcome this problem we are analyzing RNA from single homozygous animals by PCR.

Schwarz, E.M. et al. (2017) International Worm Meeting "De-completing the C. elegans genome."

Ichikawa, K., Fire, A.Z., Morishita, S., Edgley, M.L., Artiles, K.L., Gabdank, I., Schwarz, E.M., Yoshimura, J., Rougvie, A.E., Smith, C.L., Shoura, M., Wahba, L.

[International Worm Meeting, 2017]

By 2005, the C. elegans genome was sequenced to apparent completion from a mixture of divergent laboratory strains, all derivatives of Brenner's original "N2". The lack of a single worm strain matching this sequence creates challenges for genomics and systems biology. We thus chose to produce a updated reference genome for C. elegans from the strain VC2010, which was derived from a clonal isolate of N2. After sequencing to a depth of 115x coverage with trimmed, error-corrected PacBio and Oxford Nanopore reads having an average length of 11.8 kb, we assembled a updated reference genome with near-chromosomal contiguity. Half of the assembly falls into 10 blocks of continuous sequence (contigs) ranging from 3.6 Mb to 8.4 Mb in size, and the entire assembly is contained in only 82 contigs. Our assembly is very close to the previous reference assembly's quality (97.6% full-length CEGMA hits, versus 98.4% in previous N2). It is also slightly larger than the previous reference assembly (102.0 Mb versus 100.3 Mb in previous N2, a difference of 1.7%). We have closed some remaining gaps in the updated reference genome assembly with PacBio and Oxford Nanopore single reads, and used Hi-C to estimate gap sizes. This revealed 26 unlinkable regions in the updated reference genome of =130 kb, whose flanks contain highly repetitive DNA. In the official N2 genome, these regions have relatively few copies of repetitive DNA, and are ungapped. We suspect that the large gaps are real, but were lost during propagation and sequencing of cosmid clones during the original N2 genome project, making them a kind of genomic "dark matter". In other words, we think that we have de-completed the C. elegans genome. The apparent 1.7% increase in the updated reference's genome size may reflect other overlooked repeats that exist biologically in N2. Sequences in N2 that are missing from the official genome were recently described by Zhao et. al. (PubMed 26039588), with our observations expanding the discrepancy from 40 kb to 1.7 Mb. Such "dark matter" regions probably exist in other nematode genomes and make them harder to assemble. Since they have persisted in C. elegans, they may have biological functions; possibilities for such functions include generation of ncRNAs and aiding chromosomal segregation. Our consensus VC2010 genome assembly will be made available in Wormbase, with CGC distributing a VC2010-derived strain (PD1074) after July 2017.