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.

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.

Nematode biphasic 'boom and bust' dynamics are dependent on host bacterial load while linking dauer and mouth-form polyphenisms.

Cross-kingdom interactions involve dynamic processes that shape terrestrial ecosystems and represent striking examples of co-evolution. The multifaceted relationships of entomopathogenic nematodes with their insect hosts and symbiotic bacteria are well-studied cases of co-evolution and pathogenicity. In contrast, microbial interactions in soil after the natural death of insects and other invertebrates are minimally understood. In particular, the turnover and succession of nematodes and bacteria during insect decay have not been well documented - although it represents a rich ecological niche with multiple species interactions. Here, we utilize developmentally plastic nematode Pristionchus pacificus and its associated scarab beetles as models. On La Réunion Island, we collected rhinoceros beetle Oryctes borbonicus, induced death, and placed carcasses in cages both on the island and in a mock-natural environment in the laboratory controlling for high spatial and temporal resolution. Investigating nematode population density and dispersal dynamics, we were able to connect two imperative plasticities, dauer and mouth form. We observed a biphasic 'boom and bust' dispersal dynamic of dauer larvae that corresponds to bacterial load on carcasses but not bacterial type. Strikingly, all post-dauer adults have the predatory mouth form, demonstrating novel intricate interactions on decaying insect hosts. Thus, ecologically relevant survival strategies incorporate critical plastic traits.

Rhabditophanes diutinus a parthenogenetic clade IV nematode with dauer larvae.

Comparative studies using non-parasitic model species such as Caenorhabditis elegans, have been very helpful in investigating the basic biology and evolution of parasitic nematodes. However, as phylogenetic distance increases, these comparisons become more difficult, particularly when outside of the nematode clade to which C. elegans belongs (V). One of the reasons C. elegans has nevertheless been used for these comparisons, is that closely related well characterized free-living species that can serve as models for parasites of interest are frequently not available. The Clade IV parasitic nematodes Strongyloides are of great research interest due to their life cycle and other unique biological features, as well as their medical and veterinary importance. Rhabditophanes, a closely related free-living genus, forms part of the Strongyloidoidea nematode superfamily. Rhabditophanes diutinus (= R. sp. KR3021) was included in the recent comparative genomic analysis of the Strongyloididae, providing some insight into the genomic nature of parasitism. However, very little is known about this species, limiting its usefulness as a research model. Here we provide a species description, name the species as R. diutinus and investigate its life cycle and subsequently gene expression in multiple life stages. We identified two previously unreported starvation induced life stages: dauer larvae and arrested J2 (J2A) larvae. The dauer larvae are morphologically similar to and are the same developmental stage as dauers in C. elegans and infective larvae in Strongyloides. As in C. elegans and Strongyloides, dauer formation is inhibited by treatment with dafachronic acid, indicating some genetic control mechanisms are conserved. Similarly, the expression patterns of putative dauer/infective larva control genes resemble each other, in particular between R. diutinus and Strongyloides spp. These findings illustrate and increase the usefulness of R. diutinus as a non-parasitic, easy to work with model species for the Strongyloididae for studying the evolution of parasitism as well as many aspects of the biology of Strongyloides spp, in particular the formation of infective larvae.

Adult Influence on Juvenile Phenotypes by Stage-Specific Pheromone Production.

Many animal and plant species respond to population density by phenotypic plasticity. To investigate if specific age classes and/or cross-generational signaling affect density-dependent plasticity, we developed a dye-based method to differentiate co-existing nematode populations. We applied this method to Pristionchus pacificus, which develops a predatory mouth form to exploit alternative resources and kill competitors in response to high population densities. Remarkably, adult, but not juvenile, crowding induces the predatory morph in other juveniles. High-performance liquid chromatography-mass spectrometry of secreted metabolites combined with genetic mutants traced this result to the production of stage-specific pheromones. In particular, the P. pacificus-specific di-ascaroside#1 that induces the predatory morph is induced in the last juvenile stage and young adults, even though mouth forms are no longer plastic in adults. Cross-generational signaling between adults and juveniles may serve as an indication of rapidly increasing population size, arguing that age classes are an important component of phenotypic plasticity.

Environmental influence on Pristionchus pacificus mouth form through different culture methods.

Environmental cues can impact development to elicit distinct phenotypes in the adult. The consequences of phenotypic plasticity can have profound effects on morphology, life cycle, and behavior to increase the fitness of the organism. The molecular mechanisms governing these interactions are beginning to be elucidated in a few cases, such as social insects. Nevertheless, there is a paucity of systems that are amenable to rigorous experimentation, preventing both detailed mechanistic insight and the establishment of a generalizable conceptual framework. The mouth dimorphism of the model nematode Pristionchus pacificus offers the rare opportunity to examine the genetics, genomics, and epigenetics of environmental influence on developmental plasticity. Yet there are currently no easily tunable environmental factors that affect mouth-form ratios and are scalable to large cultures required for molecular biology. Here we present a suite of culture conditions to toggle the mouth-form phenotype of P. pacificus. The effects are reversible, do not require the costly or labor-intensive synthesis of chemicals, and proceed through the same pathways previously examined from forward genetic screens. Different species of Pristionchus exhibit different responses to culture conditions, demonstrating unique gene-environment interactions, and providing an opportunity to study environmental influence on a macroevolutionary scale.

A species-specific nematocide that results in terminal embryogenesis.

Nematode-insect interactions are ubiquitous, complex and constantly changing as the host and nematode coevolve. The entomophilic nematode Pristionchus pacificus is found on a myriad beetle species worldwide, although the molecular dynamics of this relationship are largely unknown. To better understand how host cues affect P. pacificus embryogenesis, we characterized the threshold of sensitivity to the pheromone (Z)-7-tetradecen-2-one (ZTDO) by determining the minimum exposure duration and developmental window that results in P. pacificus embryonic lethality. We found early-stage embryos exposed to volatile ZTDO for as few as 4 h all display terminal embryogenesis, characterized by punctuated development up to 48 h later, with abnormal morphology and limited cavity formation. To determine if the pheromone arrests pre-hatching development by suffocating or permeabilizing the eggshells, we raised embryos under anoxic conditions and also examined eggshell permeability using the lipophilic dye FM4-64. We found that asphyxiating the embryos arrested embryogenesis in a reversible manner but did not phenocopy the effects of ZTDO exposure, whereas the ZTDO-induced disruption of embryogenesis did correlate with increased eggshell permeability. The effects of ZTDO are also highly specific, as other lipid insect compounds do not produce any detectable embryocidal effect. The high specificity and unusual teratogenic effect of ZTDO may be important in mediating the host-nematode relationship by regulating P. pacificus development.

The genetics of phenotypic plasticity in nematode feeding structures.

Phenotypic plasticity has been proposed as an ecological and evolutionary concept. Ecologically, it can help study how genes and the environment interact to produce robust phenotypes. Evolutionarily, as a facilitator it might contribute to phenotypic novelty and diversification. However, the discussion of phenotypic plasticity remains contentious in parts due to the absence of model systems and rigorous genetic studies. Here, we summarize recent work on the nematode Pristionchus pacificus, which exhibits a feeding plasticity allowing predatory or bacteriovorous feeding. We show feeding plasticity to be controlled by developmental switch genes that are themselves under epigenetic control. Phylogenetic and comparative studies support phenotypic plasticity and its role as a facilitator of morphological novelty and diversity.