Three-dimensional reconstruction of the face and feeding apparatus of the predatory nematode Pristionchus pacificus

Pristionchus pacificus is a nematode model for the developmental genetics of morphological polyphenism, especially at the level of individual cells. The polyphenism of P. pacificus includes an evolutionary novelty, moveable teeth, which have enabled predatory feeding in this species and others in its family (Diplogastridae). From transmission electron micrographs of serial thin sections through an adult hermaphrodite of P. pacificus, we three-dimensionally reconstructed the 73 epithelial cells of its face, mouth, and pharynx. We found that the epithelia that produce the predatory morphology of P. pacificus are identical to Caenorhabditis elegans in the number of cell classes and nuclei. However, differences in cell form, connectivity, and nucleus position correlate with gross morphological differences from C. elegans and outgroups. Moreover, we identified fine-structural features, especially in the anteriormost pharyngeal muscles, that underlie the conspicuous, left-right asymmetry that characterizes the P. pacificus feeding apparatus. Our reconstruction provides an anatomical map for studying the genetics of polyphenism, feeding behaviour, and the development of novel form in a satellite model to C. elegans.

Analysis of meiosis in Pristionchus pacificus reveals plasticity in homolog pairing and synapsis in the nematode lineage

Meiosis is conserved across eukaryotes yet varies in the details of its execution. Here we describe a new comparative model system for molecular analysis of meiosis, the nematode Pristionchus pacificus, a distant relative of the widely studied model organism Caenorhabditis elegans. P. pacificus shares many anatomical and other features that facilitate analysis of meiosis in C. elegans. However, while C. elegans has lost the meiosis-specific recombinase Dmc1 and evolved a recombination-independent mechanism to synapse its chromosomes, P. pacificus expresses both DMC-1 and RAD-51. We find that SPO-11 and DMC-1 are required for stable homolog pairing, synapsis, and crossover formation, while RAD-51 is dispensable for these key meiotic processes. RAD-51 and DMC-1 localize sequentially to chromosomes during meiotic prophase and show nonoverlapping functions. We also present a new genetic map for P. pacificus that reveals a crossover landscape very similar to that of C. elegans, despite marked divergence in the regulation of synapsis and crossing-over between these lineages.

Influence of environmental temperature on mouth-form plasticity in Pristionchus pacificus acts through daf-11-dependent cGMP signaling

Mouth-form plasticity in the nematode Pristionchus pacificus has become a powerful system to identify the genetic and molecular mechanisms associated with phenotypic (developmental) plasticity. In particular, the identification of developmental switch genes that can sense environmental stimuli and reprogram developmental processes has confirmed long-standing evolutionary theory. Together with the associated gene regulatory networks, these developmental switch genes have been important to show that plasticity is consistent with the Modern Synthesis of evolution. However, how these genes are involved in the direct sensing of the environment, or if the switch genes act downstream of another, primary environmental sensing mechanism, remains currently unknown. Here, we study the influence of environmental temperature on mouth-form plasticity. Using forward and reverse genetic technology including CRISPR/Cas9, we show that mutations in the guanylyl cyclase Ppa-daf-11, the Ppa-daf-25/AnkMy2 and the cyclic nucleotide-gated channel Ppa-tax-2 eliminate the response to elevated temperatures. Together, our study indicates that DAF-11, DAF-25 and TAX-2 have been co-opted for environmental sensing during mouth-form plasticity regulation in P. pacificus. This work suggests that developmental switch genes integrate environmental signals including perception by cGMP signaling.

Steroid Hormone Pathways Coordinate Developmental Diapause and Olfactory Remodeling in Pristionchus pacificus

Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate development and host-seeking behavior, we used the entomophilic nematode Pristionchus pacificus to characterize dauer-constitutive mutants (Daf-c) that inappropriately enter developmental diapause to become dauer larvae. We found two Daf-c loci with dauer-constitutive and cuticle exsheathment phenotypes that can be rescued by the feeding of Δ7-dafachronic acid, and that are dependent on the conserved canonical steroid hormone receptor Ppa-DAF-12. Specifically at one locus, deletions in the sole HydroxySteroid Dehydrogenase (HSD) in P. pacificus resulted in Daf-c phenotypes. Ppa-hsd-2 is expressed in the canal neurons (CAN) and excretory cells whose homologous cells in C. elegans are not known to be involved in the dauer decision. While in wildtype only dauer larvae are attracted to host odors, hsd-2 mutant adults show enhanced attraction to the host beetle pheromone, along with ectopic activation of a marker for putative olfactory neurons, Ppa-odr-3. Surprisingly, this enhanced odor attraction acts independently of the Δ7-DA/DAF-12 module, suggesting that Ppa-HSD-2 may be responsible for several steroid hormone products involved in coordinating the dauer decision and host-seeking behavior in P. pacificus.

The community-curated Pristionchus pacificus genome facilitates automated gene annotation improvement in related nematodes

Background The nematode Pristionchus pacificus is an established model organism for comparative studies with Caenorhabditis elegans. Over the past years, it developed into an independent animal model organism for elucidating the genetic basis of phenotypic plasticity. Community-based curations were employed recently to improve the quality of gene annotations of P. pacificus and to more easily facilitate reverse genetic studies using candidate genes from C. elegans. Results Here, I demonstrate that the reannotation of phylogenomic data from nine related nematode species using the community-curated P. pacificus gene set as homology data substantially improves the quality of gene annotations. Benchmarking of universal single copy orthologs (BUSCO) estimates a median completeness of 84% which corresponds to a 9% increase over previous annotations. Nevertheless, the ability to infer gene models based on homology already drops beyond the genus level reflecting the rapid evolution of nematode lineages. This also indicates that the highly curated C. elegans genome is not optimally suited for annotating non-Caenorhabditis genomes based on homology. Furthermore, comparative genomic analysis of apparently missing BUSCO genes indicates a failure of ortholog detection by the BUSCO pipeline due to the insufficient sample size and phylogenetic breadth of the underlying OrthoDB data set. As a consequence, the quality of multiple divergent nematode genomes might be underestimated. Conclusions This study highlights the need for optimizing gene annotation protocols and it demonstrates the benefit of a high quality genome for phylogenomic data of related species.

Different combinations of serotonin receptors regulate predatory and bacterial feeding behaviors in the nematode Pristionchus pacificus

Feeding behavior is one of the most fundamental behaviors in animals, and regulation of this behavior is critical for proper food intake. The nematode Pristionchus pacificus exhibits dimorphism in feeding behavior, bacterial feeding and predatory feeding on other nematodes, and the latter behavior is assumed to be an evolutionarily novel behavior. Both types of feeding behavior are modulated by serotonin; however, the downstream mechanism that modulates these behaviors is still to be clarified. Here, we focused on serotonin receptors and examined their expression patterns in P. pacificus. We also generated knockout mutants of the serotonin receptors using the CRISPR/Cas9 system and examined feeding behaviors. We found that Ppa-ser-5 mutants and the Ppa-ser-1; Ppa-ser-7 double mutant decreased predation. Detailed observation of the pharyngeal movement revealed that the Ppa-ser-1; Ppa-ser-7 double mutant reduces tooth movement, which is required for efficient predatory feeding. Conversely, Ppa-ser-7 and Ppa-mod-1 mutants decreased bacterial feeding. This study revealed that specific combinations of serotonin receptors are essential for the modulation of these distinct feeding behaviors, providing insight into the evolution of neural pathways to regulate novel feeding behavior.

A simplified workflow for the analysis of whole-genome sequencing data from Pristionchus pacificus mutant lines

Nematodes are attractive model systems to understand the genetic basis of various biological processes ranging from development to complex behaviors. In particular, mutagenesis experiments combined with whole-genome sequencing has been proven as one of the most effective methods to identify core players of multiple biological pathways. To enable experimentalists to apply such integrative genetic and bioinformatic analysis in the case of the satellite model organism Pristionchus pacificus, I present a simplified workflow for the analysis of whole-genome data from mutant lines and corresponding mapping panels. Individual components are based on well-maintained and widely used software packages and are extended by 50 lines of code for the analysis and visualization of allele frequencies. The effectiveness of this workflow is demonstrated by an application to recently generated data of a P. pacificus mutant line, where it reduced the number of candidate mutations from an initial set of 3,500 single nucleotide variants to ten.