Conserved switch genes that arose via whole-genome duplication regulate a cannibalistic nematode morph.

Experimental genetics in a nematode reveals a key role for developmental plasticity in the evolution of nutritional diversity.

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.

Feeding-structure morphogenesis in "rhabditid" and diplogastrid nematodes is not controlled by a conserved genetic module.

Disentangling the evolution of the molecular processes and genetic networks that facilitate the emergence of morphological novelties is one of the main objectives in evolutionary developmental biology. Here, we investigated the evolutionary history of a gene regulatory network controlling the development of novel tooth-like feeding structures in diplogastrid nematodes. Focusing on NHR-1 and NHR-40, the two transcription factors that regulate the morphogenesis of these feeding structures in Pristionchus pacificus, we sought to determine whether they have a similar function in Caenorhabditis elegans, an outgroup species to the Diplogastridae which has typical "rhabditid" flaps instead of teeth. Contrary to our initial expectations, we found that they do not have a similar function. While both receptors are co-expressed in the tissues that produce the feeding structures in the two nematodes, genetic inactivation of either receptor had no impact on feeding-structure morphogenesis in C. elegans. Transcriptomic experiments revealed that NHR-1 and NHR-40 have highly species-specific regulatory targets. These results suggest two possible evolutionary scenarios: either the genetic module responsible for feeding-structure morphogenesis in Diplogastridae already existed in the last common ancestor of C. elegans and P. pacificus, and subsequently disintegrated in the former as NHR-1 and NHR-40 acquired new targets, or it evolved in conjunction with teeth in Diplogastridae. These findings indicate that feeding-structure morphogenesis is regulated by different genetic programs in P. pacificus and C. elegans, hinting at developmental systems drift during the flap-to-tooth transformation. Further research in other "rhabditid" species is needed to fully reconstruct the developmental genetic changes which facilitated the evolution of novel feeding structures in Diplogastridae.

The role of plasticity and stochasticity in coexistence.

Species coexistence in ecological communities is a central feature of biodiversity. Different concepts, i.e., contemporary niche theory, modern coexistence theory, and the unified neutral theory, have identified many building blocks of such ecological assemblies. However, other factors, such as phenotypic plasticity and stochastic inter-individual variation, have received little attention, in particular in animals. For example, how resource polyphenisms resulting in predator-prey interactions affect coexistence is currently unknown. Here, we present an integrative theoretical-experimental framework using the nematode plasticity model Pristionchus pacificus with its well-studied mouth-form dimorphism resulting in cannibalism. We develop an individual-based model that relies upon synthetic data based on our empirical measurements of fecundity and polyphenism to preserve demographic heterogeneity. We demonstrate how the interplay between plasticity and individual stochasticity result in all-or-nothing outcomes at the local level. Coexistence is made possible when spatial structure is introduced.

Spatial and temporal heterogeneity alter the cost of plasticity in Pristionchus pacificus.

Phenotypic plasticity, the ability of a single genotype to produce distinct phenotypes under different environmental conditions, has become a leading concept in ecology and evolutionary biology, with the most extreme examples being the formation of alternative phenotypes (polyphenisms). However, several aspects associated with phenotypic plasticity remain controversial, such as the existence of associated costs. While already predicted by some of the pioneers of plasticity research, i.e. Schmalhausen and Bradshaw, experimental and theoretical approaches have provided limited support for the costs of plasticity. In experimental studies, one common restriction is the measurement of all relevant parameters over long time periods. Similarly, theoretical studies rarely use modelling approaches that incorporate specific experimentally-derived fitness parameters. Therefore, the existence of the costs of plasticity remains disputed. Here, we provide an integrative approach to understand the cost of adaptive plasticity and its ecological ramifications, by combining laboratory data from the nematode plasticity model system Pristionchus pacificus with a stage-structured population model. Taking advantage of measurements of two isogenic strains grown on two distinct diets, we illustrate how spatial and temporal heterogeneity with regard to the distribution of resources on a metapopulation can alter the outcome of the competition and alleviate the realized cost of plasticity.

A safety mechanism enables tissue-specific resistance to protein aggregation during aging in C. elegans.

During aging, proteostasis capacity declines and distinct proteins become unstable and can accumulate as protein aggregates inside and outside of cells. Both in disease and during aging, proteins selectively aggregate in certain tissues and not others. Yet, tissue-specific regulation of cytoplasmic protein aggregation remains poorly understood. Surprisingly, we found that the inhibition of 3 core protein quality control systems, namely chaperones, the proteasome, and macroautophagy, leads to lower levels of age-dependent protein aggregation in Caenorhabditis elegans pharyngeal muscles, but higher levels in body-wall muscles. We describe a novel safety mechanism that selectively targets newly synthesized proteins to suppress their aggregation and associated proteotoxicity. The safety mechanism relies on macroautophagy-independent lysosomal degradation and involves several previously uncharacterized components of the intracellular pathogen response (IPR). We propose that this protective mechanism engages an anti-aggregation machinery targeting aggregating proteins for lysosomal degradation.

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.

Histone 4 lysine 5/12 acetylation enables developmental plasticity of Pristionchus mouth form.

Development can be altered to match phenotypes with the environment, and the genetic mechanisms that direct such alternative phenotypes are beginning to be elucidated. Yet, the rules that govern environmental sensitivity vs. invariant development, and potential epigenetic memory, remain unknown. Here, we show that plasticity of nematode mouth forms is determined by histone 4 lysine 5 and 12 acetylation (H4K5/12ac). Acetylation in early larval stages provides a permissive chromatin state, which is susceptible to induction during the critical window of environmental sensitivity. As development proceeds deacetylation shuts off switch gene expression to end the critical period. Inhibiting deacetylase enzymes leads to fixation of prior developmental trajectories, demonstrating that histone modifications in juveniles can carry environmental information to adults. Finally, we provide evidence that this regulation was derived from an ancient mechanism of licensing developmental speed. Altogether, our results show that H4K5/12ac enables epigenetic regulation of developmental plasticity that can be stored and erased by acetylation and deacetylation, respectively.

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.

Chromosome fusions repatterned recombination rate and facilitated reproductive isolation during Pristionchus nematode speciation.

Large-scale genome-structural evolution is common in various organisms. Recent developments in speciation genomics revealed the importance of inversions, whereas the role of other genome-structural rearrangements, including chromosome fusions, have not been well characterized. We study genomic divergence and reproductive isolation of closely related nematodes: the androdioecious (hermaphroditic) model Pristionchus pacificus and its dioecious sister species Pristionchus exspectatus. A chromosome-level genome assembly of P. exspectatus using single-molecule and Hi-C sequencing revealed a chromosome-wide rearrangement relative to P. pacificus. Strikingly, genomic characterization and cytogenetic studies including outgroup species Pristionchus occultus indicated two independent fusions involving the same chromosome, ChrIR, between these related species. Genetic linkage analysis indicated that these fusions altered the chromosome-wide pattern of recombination, resulting in large low-recombination regions that probably facilitated the coevolution between some of the ~14.8% of genes across the entire genomes. Quantitative trait locus analyses for hybrid sterility in all three sexes revealed that major quantitative trait loci mapped to the fused chromosome ChrIR. While abnormal chromosome segregations of the fused chromosome partially explain hybrid female sterility, hybrid-specific recombination that breaks linkage of genes in the low-recombination region was associated with hybrid male sterility. Thus, recent chromosome fusions repatterned recombination rate and drove reproductive isolation during Pristionchus speciation.

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 developmental (phenotypic) plasticity. In particular, the identification of developmental switch genes that can sense environmental stimuli and reprogram developmental processes has confirmed long-standing evolutionary theory. 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. We find that environmental temperature does influence mouth-form plasticity in most of the 10 wild isolates of P. pacificus tested in this study. We used one of these strains, P. pacificus RSA635, for detailed molecular analysis. 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.

Chitin contributes to the formation of a feeding structure in a predatory nematode.

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.

Evolution and Diversity of TGF-ß Pathways are Linked with Novel Developmental and Behavioral Traits.

Transforming growth factor-ß (TGF-ß) signaling is essential for numerous biologic functions. It is a highly conserved pathway found in all metazoans including the nematode Caenorhabditis elegans, which has also been pivotal in identifying many components. Utilizing a comparative evolutionary approach, we explored TGF-ß signaling in nine nematode species and revealed striking variability in TGF-ß gene frequency across the lineage. Of the species analyzed, gene duplications in the DAF-7 pathway appear common with the greatest disparity observed in Pristionchus pacificus. Specifically, multiple paralogues of daf-3, daf-4 and daf-7 were detected. To investigate this additional diversity, we induced mutations in 22 TGF-ß components and generated corresponding double, triple, and quadruple mutants revealing both conservation and diversification in function. Although the DBL-1 pathway regulating body morphology appears highly conserved, the DAF-7 pathway exhibits functional divergence, notably in some aspects of dauer formation. Furthermore, the formation of the phenotypically plastic mouth in P. pacificus is partially influenced through TGF-ß with the strongest effect in Ppa-tag-68. This appears important for numerous processes in P. pacificus but has no known function in C. elegans. Finally, we observe behavioral differences in TGF-ß mutants including in chemosensation and the establishment of the P. pacificus kin-recognition signal. Thus, TGF-ß signaling in nematodes represents a stochastic genetic network capable of generating novel functions through the duplication and deletion of associated genes.

A New Hope: A Hermaphroditic Nematode Enables Analysis of a Recent Whole Genome Duplication Event.

Whole genome duplication (WGD) is often considered a major driver of evolution that leads to phenotypic novelties. However, the importance of WGD for evolution is still controversial because most documented WGD events occurred anciently and few experimental systems amenable to genetic analysis are available. Here, we report a recent WGD event in the hermaphroditic nematode Allodiplogaster sudhausi and present a comparison with a gonochoristic (male/female) sister species that did not undergo WGD. Self-fertilizing reproduction of A. sudhausi makes it amenable to functional analysis and an ideal system to study WGD events. We document WGD in A. sudhausi through karyotype analysis and whole genome sequencing, the latter of which allowed us to 1) identify functional bias in retention of protein domains and metabolic pathways, 2) show most duplicate genes are under evolutionary constraint, 3) show a link between sequence and expression divergence, and 4) characterize differentially expressed duplicates. We additionally show WGD is associated with increased body size and an abundance of repeat elements (36% of the genome), including a recent expansion of the DNA-hAT/Ac transposon family. Finally, we demonstrate the use of CRISPR/Cas9 to generate mutant knockouts, whereby two WGD-derived duplicate genes display functional redundancy in that they both need to be knocked out to generate a phenotype. Together, we present a novel experimental system that is convenient for examining and characterizing WGD-derived genes both computationally and functionally.

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.

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.

The improved genome of the nematode Parapristionchus giblindavisi provides insights into lineage-specific gene family evolution.

Nematodes such as Caenorhabditis elegans and Pristionchus pacificus are extremely successful model organisms for comparative biology. Several studies have shown that phenotypic novelty but also conserved processes are controlled by taxon-restricted genes. To trace back the evolution of such new or rapidly evolving genes, a robust phylogenomic framework is indispensable. Here, we present an improved version of the genome of Parapristionchus giblindavisi which is the only known member of the sister group of Pristionchus. Relative to the previous short-read assembly, the new genome is based on long reads and displays higher levels of contiguity, completeness, and correctness. Specifically, the number of contigs dropped from over 7,303 to 735 resulting in an N50 increase from 112 to 791 kb. We made use of the new genome to revisit the evolution of multiple gene families. This revealed Pristionchus-specific expansions of several environmentally responsive gene families and a Pristionchus-specific loss of the de novo purine biosynthesis pathway. Focusing on the evolution of sulfatases and sulfotransferases, which control the mouth form plasticity in P. pacificus, reveals differences in copy number and genomic configurations between the genera Pristionchus and Parapristionchus. Altogether, this demonstrates the utility of the P. giblindavisi genome to date and polarizes lineage-specific patterns.

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.

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-ß signaling ligands, which induce a global transcriptome change. In addition, several effects of L. xylanilyticus depend on TGF-ß 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.