Witte H et al. (2015) Dev Genes Evol "Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus."

Sommer RJ, Streit A, ROdelsperger C, Moreno E, Kim J, Witte H

[Dev Genes Evol, 2015]

The diplogastrid nematode Pristionchus pacificus is a nematode model system for comparative studies to Caenorhabditis elegans and integrative evolutionary biology aiming for interdisciplinary approaches of evo-devo, population genetics, and ecology. For this, fieldwork can be combined with laboratory studies, and P. pacificus has a well-developed methodological toolkit of forward genetics, whole genome sequencing, DNA-mediated transformation, and various -omics platforms. Here, we establish CRISPR/Cas9-based gene inactivation and describe various boundary conditions of this methodology for P. pacificus. Specifically, we demonstrate that most mutations arise within the first 9 hours after injections. We systematically tested the efficiency of sgRNAs targeting different exons in Ppa-dpy-1 and characterized the molecular nature of the induced mutations. Finally, we provide a protocol that might also be useful for researchers working with other non-Caenorhabditis nematodes.

Jagan Srinivasan et al. (2002) Worm Breeder's Gazette "Towards a Pristionchus genome map II - Microevolutionary analysis of the nematode genus Pristionchus"

Ralf J Sommer, Min Zheng, Isabel Kipping, Arturo Gutierrez, Andre Pires da Silva, Hanh Witte, Jagan Srinivasan

[Worm Breeder's Gazette, 2002]

We describe the molecular characterization of laboratory strains of the nematode genus Pristionchus , which lays a foundation for microevolutionary analyses of vulva development. We isolated 13 laboratory strains of the Pristionchus genus that are derived from natural isolates from around the world. Mating experiments and ITS sequence analysis indicated that these 13 strains represent four different species; the gonochoristic species P. lheritieri and three hermaphroditic species, P. pacificus , P. maupasi and an as yet undescribed species Pristionchus sp., respectively. P. pacificus is represented by five different strains isolated from California, Washington, Hawaii, Ontario and Poland. Our working 'wild type' strain is the California strain. Since polymorphisms are becoming an important tool in modern day genomic analysis, which facilitate cloning of mutations, we decided to search for polymorphisms in the various Pristionchus pacificus strains. We performed amplified restriction fragment length polymorphism (AFLP) analyses of the different P. pacificus strains and found that the American strains are highly polymorphic (Srinivasan et al., 2001). We observed the largest genetic variation between the strain from California on the one hand and Washington and Hawaii on the other hand. In contrast, the developmentally distinct strain from Poland is identical to the Californian strain. Hence, we chose the Washington strain as our polymorphic strain for future experiments. These results provide us a framework for further studies on microevolution in developmental processes. Reference: Srinivasan, J., Pires-daSilva, A.; Gutierrez, A.; Jungblut, B.; Zheng, M.; Witte, H.; Schlak., I. & Ralf J. Sommer, Evolution & Development (2001) 3 : 229-240.

Gutierrez A et al. (2003) Development "Functional specificity of the nematode Hox gene mab-5."

Gutierrez A, Knoch L, Witte H, Sommer RJ

[Development, 2003]

Hox genes encode evolutionarily conserved transcription factors involved in morphological specification along the anteroposterior body axis of animals. The two most striking features of Hox genes are colinearity and the strong sequence conservation. Among all animals studied so far, the nematode Caenorhabditis elegans contains one of the most divergent Hox clusters. The core cluster contains only four members, which in part deviate from the colinearity rule. In addition, orthologous and paralogous nematode Hox sequences diverged substantially. Given these nematode-specific features, we asked how these Hox proteins evolved and how they provide functional specificity. We investigated the role of MAB-5 during ray formation and established an in vivo assay using Cel-mab5 regulatory elements to express orthologous, paralogous and chimeric cDNAs in a Cel-mab-5 mutant background. We show that the MAB-5 ortholog from Pristionchus pacificus, but not the C elegans paralogous Hox proteins can rescue Cel-mab-5. Experiments with chimeric, truncated and mutagenized Hox proteins suggest the specificity to be conferred by the N-terminal arm and helix 1, but not helix H of the

Thomas JH et al. (1994) Worm Breeder's Gazette "lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein"

Thomas JH, Horvitz HR

[Worm Breeder's Gazette, 1994]

lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein Jeffrey H. Thomas and H. Robert Horvitz, HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA

Sun S et al. (2022) Curr Biol "Chitin contributes to the formation of a feeding structure in a predatory nematode."

Witte H, Sommer RJ, Sun S

[Curr Biol, 2022]

Some nematode predators and parasites form teeth-like denticles that are histologically different from vertebrate teeth, but their biochemical composition remains elusive. Here, we show a role of chitin in the formation of teeth-like denticles in Pristionchus pacificus, a model system for studying predation and feeding structure plasticity. Pristionchus forms two alternative mouth morphs with one tooth or two teeth, respectively. The P. pacificus genome encodes two chitin synthases, with the highly conserved chs-2 gene being composed of 60 exons forming at least four isoforms. Generating CRISPR-Cas9-based gene knockouts, we found that Ppa-chs-2 mutations that eliminate the chitin-synthase domain are lethal. However, mutations in the C terminus result in viable but teethless worms, with severe malformation of the mouth. Similarly, treatment with the chitin-synthase inhibitor Nikkomycin Z also results in teethless animals. Teethless worms can feed on various bacterial food sources but are incapable of predation. High-resolution transcriptomics revealed that Ppa-chs-2 expression is controlled by the sulfatase-encoding developmental switch Ppa-eud-1. This study indicates a key role of chitin in the formation of teeth-like denticles and the complex feeding apparatus in nematodes.

Sun J et al. (2013) Exp Parasitol "Parasitism of secondary-stage juvenile of Heterodera glycines and four larva stages of Caenorhabditis elegans by Hirsutella spp."

Sun J, Xie H, Xiang M, Liu X, Qiu J

[Exp Parasitol, 2013]

The fungi Hirsutella rhossiliensis and Hirsutella minnesotensis generally parasitize only plant-parasitic nematodes in nature but parasitize the bacterivorous nematode Caenorhabditis elegans on agar plates. To establish a model system for studying the interaction between fungi and nematodes, we compared the parasitism of the first- to fourth-stage larvae (L1-L4) of C. elegans and second-stage juvenile (J2) of Heterodera glycines by twenty isolates of Hirsutella spp. Although parasitism differed substantially among isolates, both H. minnesotensis and H. rhossiliensis parasitized a higher percentage of H. glycines J2s than of C. elegans larvae. Parasitism of C. elegans L1s was correlated with parasitism of H. glycines J2s. Parasitism of C. elegans by H. rhossiliensis and H. minnesotensis was negatively correlated with larva size and motility, i.e., parasitism was higher for the younger stages. The C. elegans L1 is recommended for studying parasitism of nematodes by H. rhossiliensis and H. minnesotensis.

Fei YJ et al. (2000) J Biol Chem "A novel H(+)-coupled oligopeptide transporter (OPT3) from Caenorhabditis elegans with a predominant function as a H(+) channel and an exclusive expression in neurons."

Leibach FH, Romero MF, Krause MW, Huang W, Ganapathy V, Fei YJ, Liu JC

[J Biol Chem, 2000]

We have cloned and functionally characterized a novel, neuron-specific, H(+)-coupled oligopeptide transporter (OPT3) from Caenorhabditis elegans that functions predominantly as a H(+) channel. The opt3 gene is approximately 4.4 kilobases long and consists of 13 exons. The cDNA codes for a protein of 701 amino acids with 11 putative transmembrane domains. When expressed in mammalian cells and in Xenopus laevis oocytes, OPT3 cDNA induces H(+)-coupled transport of the dipeptide glycylsarcosine. Electrophysiological studies of the transport function of OPT3 in Xenopus oocytes show that this transporter, although capable of mediating H(+)-coupled peptide transport, functions predominantly as a H(+) channel. The H(+) channel activity of OPT3 is approximately 3-4-fold greater than the H(+)/peptide cotransport activity as determined by measurements of H(+) gradient-induced inward currents in the absence and presence of the dipeptide using the two-microelectrode voltage clamp technique. A downhill influx of H(+) was accompanied by a large intracellular acidification as evidenced from the changes in intracellular pH using an ion-selective microelectrode. The H(+) channel activity exhibits a K(0.5)(H) of 1.0 microM at a membrane potential of -50 mV. At the level of primary structure, OPT3 has moderate homology with OPT1 and OPT2, two other H(+)-coupled oligopeptide transporters previously cloned from C. elegans. Expression studies using the opt3::gfp fusion constructs in transgenic C. elegans demonstrate that opt3 gene is exclusively expressed in neurons. OPT3 may play an important physiological role as a pH balancer in the maintenance of H(+) homeostasis in C. elegans.

Hong RL et al. (2008) Proc Natl Acad Sci U S A "Natural variation in Pristionchus pacificus insect pheromone attraction involves the protein kinase EGL-4."

Hong RL, Witte H, Sommer RJ

[Proc Natl Acad Sci U S A, 2008]

The geographical mosaic theory of coevolution predicts that different local species interactions will shape population traits, but little is known about the molecular factors involved in mediating the specificity of these interactions. Pristionchus nematodes associate with different scarab beetles around the world, with Pristionchus pacificus isolated primarily from the oriental beetle in Japan. In particular, the constituent populations of P. pacificus represent a rare opportunity to study multiple specialized interactions and the mechanisms that influence population traits at the genetic level. We identified a component of the cGMP signaling pathway to be involved in the natural variation for sensing the insect pheromone ETDA, using targeted introgression lines, exogenous cGMP treatment, and a null egl-4 allele. Our data strongly implicate egl-4 as one of several loci involved in behavioral variation in P. pacificus populations. That EGL-4 homologs have been independently implicated for behavioral variations in other invertebrate models suggests that EGL-4 may act as a modulator for interspecies behavioral repertoires across large phylogenetic distances.

Imadzu H et al. (1994) Worm Breeder's Gazette "Two Tropomyosins Expressed in Body Wall and the Third Did In Pharynx of Caenorhabditis elegans"

Kagawa H, Sakube Y, Imadzu H

[Worm Breeder's Gazette, 1994]

TWO TROPOMYOSINS EXPRBSSBD IN BODY WALL AND THE THIRD DID IN PHARYNX OF CAENORHABDDITIS ELEGANS. H. Imadzu, Y. Sakube and H. gagawa. Department of Biology, Faculty of Science, Okayama University, Okayama, 700 Japan.

Wilson PA et al. (1982) Parasitology "Strongyloides ratti (homogonic): the time-course of early migration in the generalized host deduced from experiments in lactating rats."

Steven GE, Simpson NE, Wilson PA

[Parasitology, 1982]

Unsuckled mother rats given a 1 h suckling stimulus 3 h after subcutaneous injection of an exact dose of homogonic Strongyloides ratti allow fewer worms to develop in their intestines by day 9 than nulliparous rats (Wilson & Simpson, 1981). This effect is studied in more detail in terms of the length of time between weaning and stimulus (W leads to S) and injection and stimulus (I leads to S). It was observable with a W leads to S of 30 h but this and a period of 5 h were less effective than 24 h. With W leads to S constant at 24 h, significantly more worms developed in mothers when I leads to S was 24 h compared to 3 h and 10 h (P less than 0.005). The data, combined with those from nulliparous controls, are presented as a measure of the change with time of numbers of larvae in that compartment of the system which gives access to the stimulated mammary gland. It is argued that the particular compartment is the local lymph node draining the injection site and that the kinetics deduced are applicable to migration in the rat in general.