Nematode Pristionchus Pacificus Research Articles

The Role of Epigenetic Switches in Polyphenism Control: Implications from a Nematode Model for the Developmental Regulation of Alternative Phenotypes

Polyphenisms, the capability of organisms to form two or more alternative phenotypes in response to environmental variation, are prevalent in nature. However, associated molecular mechanisms and potential general principles of polyphenisms among major organismal groups remain currently unknown. This review focuses on an emerging model system for developmental plasticity and polyphenism research, the nematode Pristionchus pacificus and explores mechanistic insight obtained through unbiased genetic, experimental and natural variation studies. Resulting findings identify a central role for epigenetic switches in the environmental control of alternative phenotypes and their micro–and macroevolution. Several features observed in P. pacificus are shared with insects and plants and might become general principles for the control of polyphenisms during development.

Developmental transcriptomics in Pristionchus reveals the logic of a plasticity gene regulatory network.

Developmental plasticity enables the production of alternative phenotypes in response to different environmental conditions. While significant advances in understanding the ecological and evolutionary implications of plasticity have been made, understanding its genetic basis has lagged. However, a decade of genetic screens in the model nematode Pristionchus pacificus has culminated in 30 genes which affect mouth-form plasticity. We also recently reported the critical window of environmental sensitivity, and therefore have clear expectations for when differential gene expression should matter. Here, we collated previous data into a gene-regulatory network (GRN), and performed developmental transcriptomics across different environmental conditions, genetic backgrounds, and mouth-form mutants to assess the regulatory logic of plasticity. We found that only two genes in the GRN (eud-1 and seud-1/sult-1) are sensitive to the environment during the critical window. Interestingly, the time points of their sensitivity differ, suggesting that they act as sequential checkpoints. We also observed temporal constraint upon the transcriptional effects of mutating the GRN and revealed unexpected feedback between mouth-form genes. Surprisingly, expression of seud-1/sult-1, but not eud-1, correlated with mouth form biases across different strains and species. Finally, a comprehensive analysis of all samples identified metabolism as a shared pathway for regulating mouth-form plasticity. These data are presented in a Shiny app to facilitate gene-expression comparisons across development in up to 14 different conditions. Collectively, our results suggest that mouth-form plasticity evolved a constrained, two-tiered logic to integrate environmental information leading up to the final developmental decision.

Genetic regulators of a resource polyphenism interact to couple predatory morphology and behaviour.

Phenotypic plasticity often requires the coordinated response of multiple traits observed individually as morphological, physiological or behavioural. The integration, and hence functionality, of this response may be influenced by whether and how these component traits share a genetic basis. In the case of polyphenism, or discrete plasticity, at least part of the environmental response is categorical, offering a simple readout for determining whether and to what degree individual components of a plastic response can be decoupled. Here, we use the nematode Pristionchus pacificus, which has a resource polyphenism allowing it to be a facultative predator of other nematodes, to understand the genetic integration of polyphenism. The behavioural and morphological consequences of perturbations to the polyphenism's genetic regulatory network show that both predatory activity and ability are strongly influenced by morphology, different axes of morphological variation are associated with different aspects of predatory behaviour, and rearing environment can decouple predatory morphology from behaviour. Further, we found that interactions between some polyphenism-modifying genes synergistically affect predatory behaviour. Our results show that the component traits of an integrated polyphenic response can be decoupled and, in principle, selected upon individually, and they suggest that multiple routes to functionally comparable phenotypes are possible.

Characterization of the Pristionchus pacificus "epigenetic toolkit" reveals the evolutionary loss of the histone methyltransferase complex PRC2.

Comparative approaches have revealed both divergent and convergent paths to achieving shared developmental outcomes. Thus, only through assembling multiple case studies can we understand biological principles. Yet, despite appreciating the conservation-or lack thereof-of developmental networks, the conservation of epigenetic mechanisms regulating these networks is poorly understood. The nematode Pristionchus pacificus has emerged as a model system of plasticity and epigenetic regulation as it exhibits a bacterivorous or omnivorous morph depending on its environment. Here, we determined the "epigenetic toolkit" available to P. pacificus as a resource for future functional work on plasticity, and as a comparison with Caenorhabditis elegans to investigate the conservation of epigenetic mechanisms. Broadly, we observed a similar cast of genes with putative epigenetic function between C. elegans and P. pacificus. However, we also found striking differences. Most notably, the histone methyltransferase complex PRC2 appears to be missing in P. pacificus. We described the deletion/pseudogenization of the PRC2 genes mes-2 and mes-6 and concluded that both were lost in the last common ancestor of P. pacificus and a related species P. arcanus. Interestingly, we observed the enzymatic product of PRC2 (H3K27me3) by mass spectrometry and immunofluorescence, suggesting that a currently unknown methyltransferase has been co-opted for heterochromatin silencing. Altogether, we have provided an inventory of epigenetic genes in P. pacificus to compare with C. elegans. This inventory will enable reverse-genetic experiments related to plasticity and has revealed the first loss of PRC2 in a multicellular organism.

Starvation resistance in the nematode Pristionchus pacificus requires a conserved supplementary nuclear receptor

Nuclear hormone receptors (NHRs) are a deeply-conserved superfamily of metazoan transcription factors, which fine-tune the expression of their regulatory target genes in response to a plethora of sensory inputs. In nematodes, NHRs underwent an explosive expansion and many species have hundreds of nhr genes, most of which remain functionally uncharacterized. However, recent studies have reported that two sister receptors, Ppa-NHR-1 and Ppa-NHR-40, are crucial regulators of feeding-structure morphogenesis in the diplogastrid model nematode Pristionchus pacificus. In the present study, we functionally characterize Ppa-NHR-10, the sister paralog of Ppa-NHR-1 and Ppa-NHR-40, aiming to reveal whether it too regulates aspects of feeding-structure development. We used CRISPR/CAS9-mediated mutagenesis to create small frameshift mutations of this nuclear receptor gene and applied a combination of geometric morphometrics and unsupervised clustering to characterize potential mutant phenotypes. However, we found that Ppa-nhr-10 mutants do not show aberrant feeding-structure morphologies. Instead, multiple RNA-seq experiments revealed that many of the target genes of this receptor are involved in lipid catabolic processes. We hypothesized that their mis-regulation could affect the survival of mutant worms during starvation, where lipid catabolism is often essential. Indeed, using novel survival assays, we found that mutant worms show drastically decreased starvation resistance, both as young adults and as dauer larvae. We also characterized genome-wide changes to the transcriptional landscape in P. pacificus when exposed to 24 h of acute starvation, and found that Ppa-NHR-10 partially regulates some of these responses. Taken together, these results demonstrate that Ppa-NHR-10 is broadly required for starvation resistance and regulates different biological processes than its closest paralogs Ppa-NHR-1 and Ppa-NHR-40.

Kin-recognition and predation shape collective behaviors in the cannibalistic nematode Pristionchus pacificus.

Kin-recognition is observed across diverse species forming an important behavioral adaptation influencing organismal interactions. In many species, the molecular mechanisms involved are difficult to characterize, but in the nematode Pristionchus pacificus molecular components regulating its kin-recognition system have been identified. These determine its predatory behaviors towards other con-specifics which prevents the killing and cannibalization of kin. Importantly, their impact on other interactions including collective behaviors is unknown. Here, we explored a high altitude adapted clade of this species which aggregates abundantly under laboratory conditions, to investigate the influence of the kin-recognition system on their group behaviours. By utilizing pairwise aggregation assays between distinct strains of P. pacificus with differing degrees of genetic relatedness, we observe aggregation between kin but not distantly related strains. In assays between distantly related strains, the aggregation ratio is frequently reduced. Furthermore, abolishing predation behaviors through CRISPR/Cas9 induced mutations in Ppa-nhr-40 result in rival strains successfully aggregating together. Finally, as Caenorhabditis elegans are found naturally occurring with P. pacificus, we also explored aggregation events between these species. Here, aggregates were dominated by P. pacificus with the presence of only a small number of predators proving sufficient to disrupt C. elegans aggregation dynamics. Thus, aggregating strains of P. pacificus preferentially group with kin, revealing competition and nepotism as previously unknown components influencing collective behaviors in nematodes.

Species-specific microRNA discovery and target prediction in the soybean cyst nematode

The soybean cyst nematode (SCN) is a devastating pathogen for economic and food security considerations. Although the SCN genome has recently been sequenced, the presence of any miRNA has not been systematically explored and reported. This paper describes the development of a species-specific SCN miRNA discovery pipeline and its application to the SCN genome. Experiments on well-documented model nematodes (Caenorhabditis elegans and Pristionchus pacificus) are used to tune the pipeline’s hyperparameters and confirm its recall and precision. Application to the SCN genome identifies 3342 high-confidence putative SCN miRNA. Prediction specificity within SCN is confirmed by applying the pipeline to RNA hairpins from known exonic regions of the SCN genome (i.e., sequences known to not be miRNA). Prediction recall is confirmed by building a positive control set of SCN miRNA, based on a limited deep sequencing experiment. Interestingly, a number of novel miRNA are predicted to be encoded within the intronic regions of effector genes, known to be involved in SCN parasitism, suggesting that these miRNA may also be involved in the infection process or virulence. Beyond miRNA discovery, gene targets within SCN are predicted for all high-confidence novel miRNA using a miRNA:mRNA target prediction system. Lastly, cross-kingdom miRNA targeting is investigated, where putative soybean mRNA targets are identified for novel SCN miRNA. All predicted miRNA and gene targets are made available in appendix and through a Borealis DataVerse open repository (https://borealisdata.ca/dataset.xhtml?persistentId=doi:10.5683/SP3/30DEXA).

Divergent combinations of cis-regulatory elements control the evolution of phenotypic plasticity.

The widespread occurrence of phenotypic plasticity across all domains of life demonstrates its evolutionary significance. However, how plasticity itself evolves and how it contributes to evolution is poorly understood. Here, we investigate the predatory nematode Pristionchus pacificus with its feeding structure plasticity using recombinant-inbred-line and quantitative-trait-locus (QTL) analyses between natural isolates. We show that a single QTL at a core developmental gene controls the expression of the cannibalistic morph. This QTL is composed of several cis-regulatory elements. Through CRISPR/Cas-9 engineering, we identify copy number variation of potential transcription factor binding sites that interacts with a single intronic nucleotide polymorphism. Another intronic element eliminates gene expression altogether, mimicking knockouts of the locus. Comparisons of additional isolates further support the rapid evolution of these cis-regulatory elements. Finally, an independent QTL study reveals evidence for parallel evolution at the same locus. Thus, combinations of cis-regulatory elements shape plastic trait expression and control nematode cannibalism.

Food choice of bacterial-feeding nematodes: The human pathogen Salmonella enterica compared to common soil bacteria

Free-living nematodes act as reservoirs and vectors of the food-borne pathogen Salmonella enterica, one of the leading causes of gastrointestinal human infections worldwide. Although the transport of this human pathogen through soil by nematodes has major concerns for food safety, little is known about the food preference for S. enterica in nematodes. To disentangle the trophic links between nematodes and bacteria, the food choice of the nematode species Acrobeloides buetschlii, Caenorhabditis elegans and Pristionchus pacificus was examined under semi-natural conditions using a modular soil system. S. enterica serovar Typhimurium LT2 was tested against five common soil bacteria, i.e. Arthrobacter alpinus, Bacillus subtilis, Escherichia coli OP50, Granulicella arctica and Pseudomonas putida IsoF in binary food choice assays. The tested nematodes showed a general preference for bacterial strains with a low C/N ratio (E. coli), and avoidance of strains with a high C/N ratio (G. arctica and A. alpinus), indicating that food choice is rather linked to nutritional value than to bacterial pathogenicity. Moreover, the enrichment opportunists C. elegans and P. pacificus showed a stronger attraction to a suitable bacterial diet compared to the general opportunist A. buetschlii, which also thrives food-depleted soils. Interestingly, there was no evidence regarding a differentiation between common soil bacteria and the pathogen S. enterica. Overall, S. enterica was used as resource by the tested bacterial-feeding soil nematodes, highlighting their vector potential for food-borne pathogens in agricultural soils.

Developmental genetics: The structural basis of malleable teeth

A chitin synthase is required for tooth development in the nematode Pristionchus pacificus, revealing the structural basis of phenotypically plastic feeding structures.

Experimental and theoretical support for costs of plasticity and phenotype in a nematode cannibalistic trait.

Developmental plasticity is the ability of a genotype to express multiple phenotypes under different environmental conditions and has been shown to facilitate the evolution of novel traits. However, while the associated cost of plasticity, i.e., the loss in fitness due to the ability to express plasticity in response to environmental change, and the cost of phenotype, i.e., the loss of fitness due to expressing a fixed phenotype across environments, have been theoretically predicted, empirically such costs remain poorly documented and little understood. Here, we use a plasticity model system, hermaphroditic nematode Pristionchus pacificus, to experimentally measure these costs in wild isolates under controlled laboratory conditions. P. pacificus can develop either a bacterial feeding or predatory mouth morph in response to different external stimuli, with natural variation of mouth-morph ratios between strains. We first demonstrated the cost of phenotype by analyzing fecundity and developmental speed in relation to mouth morphs across the P. pacificus phylogenetic tree. Then, we exposed P. pacificus strains to two distinct microbial diets that induce strain-specific mouth-form ratios. Our results indicate that the plastic strain does shoulder a cost of plasticity, i.e., the diet-induced predatory mouth morph is associated with reduced fecundity and slower developmental speed. In contrast, the non-plastic strain suffers from the cost of phenotype since its phenotype does not change to match the unfavorable bacterial diet but shows increased fitness and higher developmental speed on the favorable diet. Furthermore, using a stage-structured population model based on empirically derived life history parameters, we show how population structure can alleviate the cost of plasticity in P. pacificus. The results of the model illustrate the extent to which the costs associated with plasticity and its effect on competition depend on ecological factors. This study provides support for costs of plasticity and phenotype based on empirical and modeling approaches.

Application of ALFA-Tagging in the Nematode Model Organisms Caenorhabditis elegans and Pristionchus pacificus.

The detection, manipulation and purification of proteins is key in modern life sciences studies. To achieve this goal, a plethora of epitope tags have been employed in model organisms from bacteria to humans. Recently, the introduction of the rationally designed ALFA-tag resulted in a highly versatile tool with a very broad spectrum of potential applications. ALFA-tagged proteins can be detected by nanobodies, the single-domain antibodies of camelids, allowing for super-resolution microscopy and immunoprecipitation in biochemical applications. Here, we introduce ALFA-tagging into the two nematode model organisms Caenorhabditis elegans and Pristionchus pacificus. We show that the introduction of the DNA sequence, corresponding to the 13 amino acid sequence of the ALFA-tag, can easily be accommodated by CRISPR engineering. We provide examples of high-resolution protein expression in both nematodes. Finally, we use the GW182 ortholog Ppa-ain-1 to show successful pulldowns in P. pacificus. Thus, the ALFA-tag represents a novel epitope tag for nematode research with a broad spectrum of applications.

Molecular characterization of necromenic nematode Pristionchus pacificus and associated bacteria: A new record

The vast diversity of nematodes has adapted to endure in different trophic groups as free living and parasites of plants and animals. In search of potential entomopathogenic nematodes (EPNs), soil samples were collected from, the rhizosphere of Apple orchard in Darima, Nainital, Uttarakhand, India north-western Himalaya in August 2019. The whole experimental activity carried out at ICAR-Vivekanada Parvatiya Krishi Anusandhan Sansthan, Almora during 2019–2021. In present study, Pristionchus pacificus was isolated using Corcyra bait trap, identified based on morphological and molecular approaches, studied its host range, evaluated the phylogenetic tree of P. pacificus and isolated and characterized the nematode associated bacteria. The reproduction potential and frequency of emergence of P. pacificus differs significantly among the tested hosts on Corcyra, Spodoptera and Holotrichia larvae. The molecular diagnostics of associated bacteria based on 16S gene displayed the highest homology with Myroides marinus strain. The discovery of P. pacificus VLPP01 represents the first record from India and Himalayan region.

Multidimensional competition of nematodes affects plastic traits in a beetle ecosystem.

Resource competition has driven the evolution of novel polyphenisms in numerous organisms, enhancing fitness in constantly changing environmental conditions. In natural communities, the myriad interactions among diverse species are difficult to disentangle, but the multidimensional microscopic environment of a decaying insect teeming with bacteria and fighting nematodes provides pliable systems to investigate. Necromenic nematodes of the family Diplogastridae live on beetles worldwide, innocuously waiting for their hosts’ deaths to feast on the blooming bacteria. Often, more than one worm species either affiliates with the insect or joins the microbial meal; thus, competition over limited food ensues, and phenotypic plasticity provides perks for species capable of employing polyphenisms. The recently established system of cockchafer Gymnogaster bupthalma and its occasional co-infestation of Pristionchus mayeri and Acrostichus spp. has revealed that these worms will simultaneously utilize two polyphenisms to thrive in a competitive environment. While both genera maintain plastic capacities in mouth form (strictly bacterial-feeding and omnivorous predation) and developmental pathway (direct and arrested development, dauer), P. mayeri employs both when faced with competition from Acrostichus. Here, we took advantage of the malleable system and added a third competitor, model nematode Pristionchus pacificus. Intriguingly, with a third competitor, P. mayeri is quicker to exit dauer and devour available food, while Acrostichus hides in dauer, waiting for the two Pristionchus species to leave the immediate environment before resuming development. Thus, experimental manipulation of short-lived ecosystems can be used to study the roles of polyphenisms in organismal interactions and their potential significance for evolution.

Adaptation to environmental temperature in divergent clades of the nematode Pristionchus pacificus.

Because of ongoing climate change, populations of organisms are being subjected to stressful temperatures more often. This is especially problematic for ectothermic organisms, which are likely to be more sensitive to changes in temperature. Therefore, we need to know if ectotherms have adapted to environmental temperature and, if so, what are the evolutionary mechanisms behind such adaptation. Here, we use the nematode Pristionchus pacificus as a case study to investigate thermal adaptation on the Indian Ocean island of La Réunion, which experiences a range of temperatures from coast to summit. We study the evolution of high-temperature tolerance by constructing a phylogenetic tree of strains collected from many different thermal niches. We show that populations of P. pacificus at low altitudes have higher fertility at warmer temperatures. Most likely, this phenotype has arisen recently and at least twice independently, consistent with parallel evolution. We also studied low-temperature tolerance and showed that populations from high altitudes have increased their fertility at cooler temperatures. Together, these data indicate that P. pacificus strains on La Réunion are subject to divergent selection, adapting to hot and cold niches at the coast and summit of the volcano. Precisely defining these thermal niches provides essential information for models that predict the impact of future climate change on these populations.

Microbiomes: How a gut bacterium promotes healthier living in a nematode

A new study finds that a bacterium from the microbiome of the nematode Pristionchus pacificus can promote rapid growth, increased body size, and increased fecundity by inducing neuronal expression of TGF-β ligands. This is an intriguing example of how the microbiota induces systemic effects in the host by stimulating neuroendocrine signaling.

Synergistic interaction of gut microbiota enhances the growth of nematode through neuroendocrine signaling

Animals are associated with a diverse bacterial community that impacts host physiology. It is well known that nutrients and enzymes synthesized by bacteria largely expand host metabolic capacity. Bacteria also impact a wide range of animal physiology that solely depends on host genetics through direct interaction. However, studying the synergistic effects of the bacterial community remains challenging due to its complexity. The omnivorous nematode Pristionchus pacificus has limited digestive efficiency on bacteria. Therefore, we established a bacterial collection that represents the natural gut microbiota that are resistant to digestion. Using this collection, we show that the bacterium Lysinibacillus xylanilyticus by itself provides limited nutritional value, but in combination with Escherichia coli, it significantly promotes life-history traits of P.pacificus by regulating the neuroendocrine peptide in sensory neurons. This gut-to-brain communication depends on undigested L.xylanilyticus providing Pristionchus nematodes a specific fitness advantage to compete with nematodes that rupture bacteria efficiently. Using RNA-seq and CRISPR-induced mutants, we show that 1-h exposure to L.xylanilyticus is sufficient to stimulate the expression of daf-7-type TGF-β signaling ligands, which induce a global transcriptome change. In addition, several effects of L.xylanilyticus depend on TGF-β signaling, including olfaction, body size regulation, and a switch of energy allocation from lipid storage to reproduction. Our results reveal the beneficial effects of a gut bacterium to modify life-history traits and maximize nematode survival in natural habitats.

Predator–prey interactions: Strategic biting

The nematode Pristionchus pacificus occasionally encounters other nematode species that compete for similar resources. A new study shows that P. pacificus perform an aggressive patrolling and biting behavior to expel adult Caenorhabditis elegans nematodes from food patches.

Flexible reprogramming of Pristionchus pacificus motivation for attacking Caenorhabditis elegans in predator-prey competition

Animals with diverse diets must adapt their food priorities to a wide variety of environmental conditions. This diet optimization problem is especially complex for predators that compete with prey for food. Although predator-prey competition is widespread and ecologically critical, it remains difficult to disentangle predatory and competitive motivations for attacking competing prey. Here, we dissect the foraging decisions of the omnivorous nematode Pristionchus pacificus to reveal that its seemingly failed predatory attempts against Caenorhabditis elegans are actually motivated acts of efficacious territorial aggression. While P.pacificus easily kills and eats larval C.elegans with a single bite, adult C.elegans typically survives and escapes bites. However, non-fatal biting can provide competitive benefits by reducing access of adult C.elegans and its progeny to bacterial food that P.pacificus also eats. We show that the costs and benefits of both predatory and territorial outcomes influence how P.pacificus decides which food goal, prey or bacteria, should guide its motivation for biting. These predatory and territorial motivations impose different sets of rules for adjusting willingness to bite in response to changes in bacterial abundance. In addition to biting, predatory and territorial motivations also influence which search tactic P.pacificus uses to increase encounters with C.elegans. When treated with an octopamine receptor antagonist, P.pacificus switches from territorial to predatory motivation for both biting and search. Overall, we demonstrate that P.pacificus assesses alternate outcomes of attacking C.elegans and flexibly reprograms its foraging strategy to prioritize either prey or bacterial food.

The Role of Sulfation in Nematode Development and Phenotypic Plasticity.

Sulfation is poorly understood in most invertebrates and a potential role of sulfation in the regulation of developmental and physiological processes of these organisms remains unclear. Also, animal model system approaches did not identify many sulfation-associated mechanisms, whereas phosphorylation and ubiquitination are regularly found in unbiased genetic and pharmacological studies. However, recent work in the two nematodes Caenorhabditis elegans and Pristionchus pacificus found a role of sulfatases and sulfotransferases in the regulation of development and phenotypic plasticity. Here, we summarize the current knowledge about the role of sulfation in nematodes and highlight future research opportunities made possible by the advanced experimental toolkit available in these organisms.