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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 plastic response to environment, 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 of competition depend on ecological factors. This study provides comprehensive support for the costs of plasticity and phenotype based on empirical and modeling approaches.Impact SummaryA genotype able to express a range of phenotypes in response to environmental conditions, that is to demonstrate developmental plasticity, would be a Darwinian demon, able to infinitely adapt and outcompete those genotypes that require genetic change to express a phenotype fit to an environment. It has been suggested that the absence of such demons in nature is due to the cost of plasticity, i.e., developmental plasticity results in a reduction of biological fitness compared to a genotype that facultatively expresses a phenotype matching the environment. While conceptually simple, measuring the cost of plasticity in nature has proven a major challenge. We use the nematode P. pacificus to measure the cost of plasticity. During its development, P. pacificus can assume one of two possible mouth forms: predatory or non-predatory. The likelihood developing any of these two mouth forms is determined by a gene regulatory network, which itself is affected by a wide range on environmental conditions, including diet. We used two strains of P. pacificus and grew them on two different bacterial diets. The plastic strain was capable of switching from non-predatory to predatory mouth form depending on the diet, while the non-plastic strain could only express the predatory mouth form on either of the diets. By measuring the number eggs laid in both strain on each diet, we show that the plastic response is associated with a reduction in fecundity, thus providing a clear example of the cost of plasticity. We then use a stage-structured model to simulate the population dynamics of the plastic and the non-plastic strains. Our simulation show that the cost of plasticity is highly context dependent and its ecological ramifications can be greatly influenced by biotic and abiotic factors.

Transforming growth factor-β (TGF-β) 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-β signaling in nine nematode species and revealed striking variability in TGF-β 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-β 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-β 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-β mutants including in chemosensation and the establishment of the P. pacificus kin-recognition signal. Thus, TGF-β signaling in nematodes represents a stochastic genetic network capable of generating novel functions through the duplication and deletion of associated genes.

Developmental plasticity influences cooperative versus competitive social action strategies in animals., Resource polyphenisms, where single genotypes produce alternative feeding strategies in response to changing environments, are thought to be facilitators of evolutionary novelty. However, understanding the interplay between environment, morphology, and behavior and its significance is complex. We explore a radiation of Pristionchus nematodes with discrete polyphenic mouth forms and associated microbivorous versus cannibalistic traits. Notably, comparing 29 Pristionchus species reveals that reproductive mode strongly correlates with mouth-form plasticity. Male-female species exhibit the microbivorous morph and avoid parent-offspring conflict as indicated by genetic hybrids. In contrast, hermaphroditic species display cannibalistic morphs encouraging competition. Testing predation between 36 co-occurring strains of the hermaphrodite P. pacificus showed that killing inversely correlates with genomic relatedness. These empirical data together with theory reveal that polyphenism (plasticity), kin recognition, and relatedness are three major factors that shape cannibalistic behaviors. Thus, developmental plasticity influences cooperative versus competitive social action strategies in diverse animals.

Summary 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., Graphical Abstract, Highlights • Novel vital dye method for tracking mixed nematode populations • Adult, but not juvenile, crowding induces the omnivorous morph in P. pacificus • Omnivorous morph-inducing pheromones are produced late in development • Age class is an important component of density-dependent phenotypic plasticity, Physiology; Genetics; Cell Biology; Developmental Biology

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.

Nematodes such as Caenorhabditis elegans are powerful systems to study basically all aspects of biology. Their species richness together with tremendous genetic knowledge from C. elegans facilitate the evolutionary study of biological functions using reverse genetics. However, the ability to identify orthologs of candidate genes in other species can be hampered by erroneous gene annotations. To improve gene annotation in the nematode model organism Pristionchus pacificus, we performed a genome-wide screen for C. elegans genes with potentially incorrectly annotated P. pacificus orthologs. We initiated a community-based project to manually inspect more than two thousand candidate loci and to propose new gene models based on recently generated Iso-seq and RNA-seq data. In most cases, misannotation of C. elegans orthologs was due to artificially fused gene predictions and completely missing gene models. The community-based curation raised the gene count from 25,517 to 28,036 and increased the single copy ortholog completeness level from 86% to 97%. This pilot study demonstrates how even small-scale crowdsourcing can drastically improve gene annotations. In future, similar approaches can be used for other species, gene sets, and even larger communities thus making manual annotation of large parts of the genome feasible.

Summary Switching between alternative complex phenotypes is often regulated by "supergenes," polymorphic clusters of linked genes such as in butterfly mimicry. In contrast, phenotypic plasticity results in alternative complex phenotypes controlled by environmental influences rather than polymorphisms. Here, we show that the developmental switch gene regulating predatory versus non-predatory mouth-form plasticity in the nematode Pristionchus pacificus is part of a multi-gene locus containing two sulfatases and two α-N-acetylglucosaminidases ( nag ). We provide functional characterization of all four genes, using CRISPR-Cas9-based reverse genetics, and show that nag genes and the previously identified eud-1 /sulfatase have opposing influences. Members of the multi-gene locus show non-overlapping neuronal expression and epistatic relationships. The locus architecture is conserved in the entire genus Pristionchus . Interestingly, divergence between paralogs is counteracted by gene conversion, as inferred from phylogenies and genotypes of CRISPR-Cas9-induced mutants. Thus, we found that physical linkage accompanies regulatory linkage between switch genes controlling plasticity in P. pacificus .