Parts, Liisa et al. (2017) International Worm Meeting "Mechanisms and evolution of agrochemical resistance-associated natural variation in wild populations of C. elegans."

Woollard, Alison, Parts, Liisa, Flemming, Anthony

[International Worm Meeting, 2017]

Resistance to pesticides is a global food security problem and a rising issue for the agrochemical sector, analogous to the global health concerns caused by widespread antibacterial resistance. A number of compound families, both wide spectrum pesticides and more targeted nematicides, are available for agricultural use to combat the damage caused by pests that results in about 15% of global crop loss annually. Most of these compounds were introduced decades ago and reports of resistance exist for each class, yet little is known about the molecular mechanism and evolutionary biology of resistance. Sometimes resistance takes decades to evolve, sometimes just a few years and some species evolve resistance more commonly than others. We are evaluating the use of C. elegans as a model for understanding the mechanisms and evolution of pesticide resistance. Selection for resistance will act on natural variation in susceptibility in wild populations and such variation has been observed in C. elegans: for example the Hawaiian strain CB4856[1] is resistant to the nematicide avermectin through variation at the target protein. We have assessed natural variation in pesticide resistance by investigating the development of 25 highly divergent C. elegans wild isolates upon exposure to 29 bioactive insecticides, fungicides and nematicides and demonstrated both increased sensitivity as well as increased resistance to particular chemicals in different wild strains. We plan to identify the genetic basis of particular variation in resistance as well as to model the emergence of agrochemical resistance in an experimental evolution approach. References: [1] Ghosh, R., et al. (2012) Natural Variation in a Chloride Channel Subunit Confers Avermectin Resistance in C. elegans. Science, 335, p574-578.

Saunders, B. et al. (2017) International Worm Meeting "A function of mtDNA effects copy number regulation in C. elegans."

Saunders, B., Patel, M., Kirby, C.

[International Worm Meeting, 2017]

Eukaryotes contain both a nuclear genome and a mitochondrial genome (mtDNA). Generally organisms contain hundreds to thousands of copies of mtDNA per cell. It is known that a mechanism exists that closely regulates the copy number of mtDNA within the cell, but this mechanism is still not well understood. The mtDNA encodes essential parts of the electron transport chain (ETC). The ETC is responsible for generating most of the ATP within the cell. We have characterized C. elegans strains that contain a mixture of wildtype and mutant mtDNA in which some of these essential parts of the ETC are nonfunctional or are completely deleted from the genome. Some of these strains exhibit increased mtDNA copy number. Based on these data, we hypothesize that some function of mtDNA is coupled with its copy number regulation. I am currently developing assays to measure concentrations of a number of different metabolites that might play a role in mtDN copy number regulation. I will present data from these experiments.

Yusuke Hokii et al. (2004) East Asia Worm Meeting "Isolation of novel small RNAs from Caenorhabditis elegans"

Chisato Ushida, Risa Arai, Masayosi Shimoyama, Akiyosi Kubo, Akira Muto, Yusuke Hokii

[East Asia Worm Meeting, 2004]

We isolated novel small RNAs from C. elegans by using gel electrophoresis techniques. Sequences of the separated RNAs were determined by cloning and sequencing their cDNAs. RNAs of about 50 to 1,000 nt in length were prepared from the mixed stage worms and separated by the denaturing gel electrophoresis. 32 bands were detected and sequences of 107 cDNAs from the bands were determined. Eighty-seven cDNAs corresponded to parts of the known ncRNA gene sequences such as rRNAs, tRNAs and U snRNAs. Nine cDNAs had parts of exon sequences and eight had parts of intron sequences of protein coding genes. The remaining 3 cDNAs revealed sequences corresponded to the intergenic sequences of genome. RNAs smaller than 50 nt in length were also separated on the denaturing gel and fifteen bands were detected. Although the purified RNAs from the 15 bands were subjected to the enzymatic sequencing method of Donis-Keller, clear sequence could not be obtained. This is probably because each band contains more than one RNA species, since more than 100 species of tiny RNAs (19-23 nt) are detectable by Northern blotting (Ambros et al., 2003 and Lim et al., 2003). This small RNA fraction was further separated by a two dimensional (2D) gel electrophoresis, which resolved about 100 spots. By using this 2D-gel electrophoresis, we compared the expression pattern of embryonic small RNAs with small RNAs prepared from the mixed stage worms. Remarkable difference between the two was observed. The spots were classified into three groups, 1) spots which are detected only in the embryonic RNA preparation, 2) spots which are detected only in the mixed stage worm RNA preparation, 3) spots which are detected both in the embyonic and mixed stage worm RNA preparations. Each group had 85, 51 and 54 spots, respectively. Several cDNA seqeuences, which contained novel small non-coding RNA candidates, were obtained from each group.

Karin Kiontke et al. (2002) East Coast Worm Meeting "Evolutionary changes in stoma ultrastructure within the nematode taxon Diplogastridae"

Karin Kiontke, Alexander Furst v Lieven, Walter Sudhaus, Shu-Yi Chiou, David H A Fitch

[East Coast Worm Meeting, 2002]

Most free living nematodes feed on bacteria. Within Diplogastridae, however, diet changed repeatedly, as did stoma morphology. The evolution of a large dorsal tooth probably allowed the evolution of carnivory and fungivory within this taxon. Our aim was to understand which changes occurred in the diplogastrid stoma at the ultrastructural level. Ultrastructure was already known for some bacterivorous species (e.g. De Ley, P et al. 1995, Nematologica 41: 153) and for the partly carnivorous Diplogaster halicti with its derived shortened stoma and large dorsal tooth (Baldwin, J et al. 1997, Can J Zool 75: 407). We used TEM to investigate Diplogasteroides nasuensis, a bacterivorous species with a tube-shaped stoma, probably representing an early branch of the diplogastrid clade. The stoma of D. nasuensis is formed by cell processes whose number and arrangement corresponds to that observed in Caenorhabditis elegans, cephalobids and panagrolaimids. Some novelties evolved early in Diplogastridae: (1) The dorsal tooth is formed by two sets of cell processes (instead of one). (2) The channel of the dorsal pharynx gland lies between these cell processes, allowing the evolution of the tooth functioning as injection device. (3) Pharyngeal cell processes in the diplogastrid stoma are shortened and interlaced. (4) Some myofilaments are arranged longitudinally instead of radially, possibly allowing the kind of complex mobility of stoma parts observed in carnivorous species. In the past it was debated how to homologize stoma parts in Secernentea. We reject the attempt to use cell lineage in C. elegans and Cephalobus cubanensis as a basis for homologization (Dolinski, C et al. 1998, Dev Genes Evol 208: 495), which disregards the importance of cell-cell signaling and positional information in cell fate determination. More importantly, this hypothesis leads to non-parsimonious assumptions for evolutionary events, involving a double gain and subsequent loss of cell processes in the lineage leading to C. elegans. However, stoma parts in Cephalobidae, Panagrolaimidae, C. elegans and D. nasuensis can be unambiguously homologized based on the conserved spatial arrangement of the cell processes by which they are formed. We are currently using sequences of small subunit ribosomal RNA genes to independently test the phylogenetic relationships among these species and to trace evolutionary changes in stoma characters. Supported by the Deutsche Forschungsgemeinschaft (Su 198/2-2)

Barbara Meissner et al. (2007) International Worm Meeting "Mapping out a muscle cell - a first step towards a full description of protein localization within a single cell type."

Don Moerman, Mariana Veiga, Adam Lorch, Marco Marra, Aruna Somasiri, Steven Jones, Teresa Rogalski, Rick Zapf, Rebecca Fox, Barbara Meissner, Adam Warner, Kim Wong, David Miller

[International Worm Meeting, 2007]

The beautifully organized muscle sarcomeres within C. elegans body wall muscle offers an opportunity to identify the precise position of proteins within cell substructures and the possibility of determining how such structures are assembled. Over the past few years it has become abundantly clear that in order to tackle such a complex problem in cellular architecture one first needs a parts list and then one needs to determine where all the parts are located within a cell. Generating a parts list: We have been using several different techniques to identify all of the expressed genes within C. elegans muscle, including SAGE (Serial Analysis of Gene Expression) RNA microarray chips (in collaboration with the Miller lab), and protein mass spectrometry. From our SAGE studies we have identified over 8,000 genes expressed in muscle. A comparison of this list with Affymetrix GeneChip data identifies an overlap of at least 4,127 genes. Our protein studies have identified 1,142 proteins expressed in embryonic muscle, 73% of which are also identified by SAGE. These combined studies yield a list of about 4,500 genes that we have high confidence are expressed in muscle. We performed a comprehensive RNAi screen of 3,301 of these genes and identified 119 that affect sarcomere assembly and/or maintenance in adult muscle. Determining subcellular localization: We are using a Gateway vector to express protein::GFP fusions in muscle cells. At present we are focusing on genes which a) have tags in the muscle SAGE libraries and/or have been identified through our proteomics studies, b) show a phenotype in the RNAi screen and c) are orthologs or at least homologs of human genes. To date we have analyzed the expression of 45 genes, 22 of which display localized expression in the C. elegans body wall muscle (e.g. dense bodies, M-lines, golgi, mitochondria, cell membrane or nuclei) and 10 with expression in the cytoplasm. The localization of all proteins within a muscle cell will be an invaluable resource in our attempt to understand how proteins interact within muscle to form properly organized and regulated sarcomeres.

Bo-mi Song et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Serotonin as a modulator of isthmus peristalsis"

Bo-mi Song, Leon Avery

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

The pharynx of C. elegans, a neuromuscular organ responsible for feeding, consists of three muscle parts connected by gap junctions: the corpus, isthmus and terminal bulb. The isthmus is divided into two parts based upon their behavior: anterior and posterior isthmus. During feeding, the corpus and anterior isthmus pump to take bacteria into the pharynx. The posterior isthmus transfers the bacteria into terminal bulb by peristaltic movement. The terminal bulb grinds the bacteria for digestion. The goal of this study is to uncover the molecular mechanisms by which isthmus peristalsis is generated and modulated. This study is meaningful for two reasons; first, it will provide insights how neurons coordinate muscle movements. Pharyngeal muscle is interesting to study, in particular, because muscles can show different behaviors while connected by gap junctions. Second, it may lead us to find molecular mechanisms underlying peristaltic movement in vertebrates. Considering the difficulties of studying mammals, C. elegans can be a valuable model system to study this poorly understood problem. So far, two different assays have been developed and both forward and reverse genetic approaches have been taken. We found that 1. Serotonin is sufficient to induce isthmus peristalsis. 2. Serotonin signaling is not necessary for isthmus peristalsis in presence of food. 3. Serotonin signaling from pharyngeal neurons increases the frequency of isthmus peristalsis. 4. Cholinergic neurons provide at least in part of the serotonin signaling, which quite narrows down the candidate neurons responsible for the serotonin signaling. In addition, we got 6 different mutants which will provide useful information on different aspects of isthmus peristalsis.

Kleemann, Gunnar et al. (2013) International Worm Meeting "WormView: a library of modular Matlab functions for static and dynamic image analysis."

Murphy, Coleen, Parsons, Lance, Kleemann, Gunnar

[International Worm Meeting, 2013]

We present WormView, a library of Matlab functions, which can be interchangeably combined to conduct automated image analysis and data visualization. WormView is designed to be flexible and easy enough to use that users with little familiarity with programming can construct new applications. The modular architecture of WormView allows a user to rearrange interchangeable parts into novel applications without requiring new code generation. At the same time, the open source Matlab code can be easily modified by more advanced users to generate new functions without affecting the other parts of the program. The components of the WormView library fall into four progressive functional categories (1) particle identification, (2) particle verification (optional), (3) shape and time series analysis, and (4) data compilation and figure generation. In a typical analysis, single images or images in a series are passed into GetWorm. GetWorm collects worm data as binary thresholded images, as well as position, shape and size data. The GetWorm output is passed to through an optional quality assurance step involving manual perusal or automated verification by SVM (a support vector machine; machine learning). The processed dataset is then used collect data on the worm path (WormTrip), worm posture (SpineWorm), and movement patterns. Finally the processed files are passed into a final analysis for compilation of time course data, particle counts, shape and size analysis and figure generation. We will present sample configurations of the library, which can be used to measure aspects of worm shape, size, posture, position and color as they change over time.

Burns, Andrew et al. (2021) International Worm Meeting "Selective Control of Parasitic Nematodes with Bioactivated Nematicides"

Ross, Rachel, Hu, Chun, Castelli, Jack, Snider, Jamie, Palmeira, Bruna, Vaidya, Aditya, Lautens, Mark, Volpatti, Jonathan, Zasada, Inga, Stagljar, Igor, Kitner, Megan, Redman, Elizabeth, Dowling, James, MacParland, Sonya, Cowen, Leah, Xiao, Qi, Finney, Constance, Cutler, Sean, Marwah, Sagar, Burns, Andrew, Tiefenbach, Jens, Puumala, Emily, Krause, Henry, Meyer, Susan, MacDonald, Margaret, Chung, Sai, Roy, Peter, Gilleard, John

[International Worm Meeting, 2021]

Global food security is threatened as the world amasses 10 billion people amid limited arable land. While nematode pests are a major barrier to agricultural intensification, most traditional nematicides are now banned because of poor nematode-selectivity, leaving farmers with inadequate controls. Here, we describe a screen carried out in the model nematode Caenorhabditis elegans that enriches for selective nematicides by identifying molecules that are bioactivated by cytochrome P450s, which are phylogenetically diverse. We identify a family of structures, called nemactivins, that are robustly bioactivated to a toxic metabolite selectively in nematodes. At low parts-per-million concentrations, nemactivins perform comparably well with commercial nematicides at controlling infection by the world's most destructive plant-parasitic nematode Meloidogyne incognita. Hence, nemactivins are first-in-class bioactivated nematicides that provide much needed nematode-selectivity.

White, Amelia G et al. (2011) International Worm Meeting "Rapid and accurate phenotyping of embryonic lethality via image analysis of C. elegans developmental stages from high-throughput image data."

Gunsalus, Kristin C, Paaby, Annalise, Munarriz, Eliana, Cipriani, Patricia G, Geiger, Davi, Sontag, Eduardo, Piano, Fabio, White, Amelia G, Kao, Huey-Ling

[International Worm Meeting, 2011]

We present an automated image analysis system (DevStaR) for quantitative phenotyping of C. elegans embryonic lethality and sterility phenotypes. Our image analysis system counts each developmental stage in an image of a C. elegans population, allowing efficient high throughput calculation of C. elegans viability phenotypes. DevStaR is an object recognition machine comprising several hierarchical layers that build successively more sophisticated representations of the objects (developmental stages) to be classified. The algorithm segments the objects, decomposes the objects into parts, extracts features from these parts, and classifies them using an SVM (support Vector Machine) and global shape information. This enables correct classifications in the presence of complicated occlusions and deformations of the animals. Features of the classified objects are then used to obtain a count of each developmental stage. We are currently using this system to analyze phenotypic data from C. elegans high-throughput genetic screens, and have processed over one million images for lab users so far. Validation of DevStaR measurements will be shown by comparing DevStaR output to both manual counting of developmental stages and manual scores of quantitative phenotypes. DevStaR can provide an accurate measurement of quantitative phenotype and is comparable to manual scoring. DevStaR has been used to score a C. elegans genome wide RNAi screen with up to 30 repeats per clone tested at up to 5 temperatures per clone. The screen consists of over 600,000 images each scored by DevStaR, Analysis of these data illustrate the convenience of DevStaR scoring and the use of a quantitative phenotype. Our system overcomes a previous bottleneck in image analysis by achieving near real-time scoring of image data in a fully automated manner. Our system reduces the need for human evaluation of images and provides rapid quantitative output that is not feasible at high throughput by manual scoring.

Affolter M et al. (2000) European Worm Meeting "Control of the C. elegans hox gene ceh-13"

Kohler R, Schnabel R, Mueller F, Affolter M, Streit A, Tak K, Marty T, Belfiore M

[European Worm Meeting, 2000]

Null mutants of the C. eleganslabial homolog ceh-13 fail to form proper anterior structures and display cell adhesion defects1. ceh-13 is required zygotically and it is expressed in different cell lineages and at different times during development 1,2. A ceh-13::GFP reporter construct containing about 8kb of promoter sequence mimics normal ceh-13 expression, at least in the embryo 2. We will present data showing that separable parts of the ceh-13 promoter are responsible for the different temporal and spatial aspects of ceh-13 expression1. We have been able to define three non-overlapping regions each of which drives a reporter gene in a subset of the cells where the full length ceh-13::GFP is expressed. One fragment mediates expression in the early embryo in the ABxxxp cells, in the Ep cell and in their respective daughters. A second fragment contains an autoregulatory element and drives expression in different neuronal muscle and hypodermal cells in later embryogenesis. The third element leads to expression in parts of the adult nervous system and in the male tail. Important features of the transcriptional control appear to be conserved between flies and worms. The ceh-13 promoter is capable of inducing a reporter gene in the Drosophilaembryonic mid-gut in a pattern very reminiscent of endogenous labial expression. This expression is dependent on endogenous labial and on wingless signaling. We are currently characterizing the ceh-13 promoter in more detail, focusing mainly on the fragment, which is responsible for the early embryonic expression in C. elegans and for the wg responsiveness in Drosophila. Results will be presented. 1) Brunschwig et al. (1999) Development 126:1537-1546 2) Wittmann et al. (1997) Development 124:4194-4200