Metazoan operons accelerate recovery from growth-arrested states.

Existing theories explain why operons are advantageous in prokaryotes, but their occurrence in metazoans is an enigma. Nematode operon genes, typically consisting of growth genes, are significantly upregulated during recovery from growth-arrested states. This expression pattern is anticorrelated to nonoperon genes, consistent with a competition for transcriptional resources. We find that transcriptional resources are initially limiting during recovery and that recovering animals are highly sensitive to any additional decrease in transcriptional resources. We provide evidence that operons become advantageous because, by clustering growth genes into operons, fewer promoters compete for the limited transcriptional machinery, effectively increasing the concentration of transcriptional resources and accelerating recovery. Mathematical modeling reveals how a moderate increase in transcriptional resources can substantially enhance transcription rate and recovery. This design principle occurs in different nematodes and the chordate C. intestinalis. As transition from arrest to rapid growth is shared by many metazoans, operons could have evolved to facilitate these processes.

DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence.

Quiescence, an actively-maintained reversible state of cell cycle arrest, is not well understood. PTEN is one of the most frequently lost tumor suppressors in human cancers and regulates quiescence of stem cells and cancer cells. The sole PTEN ortholog in Caenorhabditis elegans is daf-18. In a C. elegans loss-of-function mutant for daf-18, primordial germ cells (PGCs) divide inappropriately in L1 larvae hatched into starvation conditions, in a TOR-dependent manner. Here, we further investigated the role of daf-18 in maintaining PGC quiescence in L1 starvation. We found that maternal or zygotic daf-18 is sufficient to maintain cell cycle quiescence, that daf-18 acts in the germ line and soma, and that daf-18 affects timing of PGC divisions in fed animals. Importantly, our results also implicate daf-18 in repression of germline zygotic gene activation, though not in germline fate specification. However, TOR is less important to germline zygotic gene expression, suggesting that in the absence of food, daf-18/PTEN prevents inappropriate germline zygotic gene activation and cell division by distinct mechanisms.

Correction: Maternal Diet and Insulin-Like Signaling Control Intergenerational Plasticity of Progeny Size and Starvation Resistance.

[This corrects the article DOI: 10.1371/journal.pgen.1006396.].

Maternal Diet and Insulin-Like Signaling Control Intergenerational Plasticity of Progeny Size and Starvation Resistance.

Maternal effects of environmental conditions produce intergenerational phenotypic plasticity. Adaptive value of these effects depends on appropriate anticipation of environmental conditions in the next generation, and mismatch between conditions may contribute to disease. However, regulation of intergenerational plasticity is poorly understood. Dietary restriction (DR) delays aging but maternal effects have not been investigated. We demonstrate maternal effects of DR in the roundworm C. elegans. Worms cultured in DR produce fewer but larger progeny. Nutrient availability is assessed in late larvae and young adults, rather than affecting a set point in young larvae, and maternal age independently affects progeny size. Reduced signaling through the insulin-like receptor daf-2/InsR in the maternal soma causes constitutively large progeny, and its effector daf-16/FoxO is required for this effect. nhr-49/Hnf4, pha-4/FoxA, and skn-1/Nrf also regulate progeny-size plasticity. Genetic analysis suggests that insulin-like signaling controls progeny size in part through regulation of nhr-49/Hnf4, and that pha-4/FoxA and skn-1/Nrf function in parallel to insulin-like signaling and nhr-49/Hnf4. Furthermore, progeny of DR worms are buffered from adverse consequences of early-larval starvation, growing faster and producing more offspring than progeny of worms fed ad libitum. These results suggest a fitness advantage when mothers and their progeny experience nutrient stress, compared to an environmental mismatch where only progeny are stressed. This work reveals maternal provisioning as an organismal response to DR, demonstrates potentially adaptive intergenerational phenotypic plasticity, and identifies conserved pathways mediating these effects.

Food perception without ingestion leads to metabolic changes and irreversible developmental arrest in C. elegans.

BACKGROUND: Developmental physiology is very sensitive to nutrient availability. For instance, in the nematode Caenorhabditis elegans, newly hatched L1-stage larvae require food to initiate postembryonic development. In addition, larvae arrested in the dauer diapause, a non-feeding state of developmental arrest that occurs during the L3 stage, initiate recovery when exposed to food. Despite the essential role of food in C. elegans development, the contribution of food perception versus ingestion on physiology has not been delineated. RESULTS: We used a pharmacological approach to uncouple the effects of food (bacteria) perception and ingestion in C. elegans. Perception was not sufficient to promote postembryonic development in L1-stage larvae. However, L1 larvae exposed to food without ingestion failed to develop upon return to normal culture conditions, instead displaying an irreversible arrest phenotype. Inhibition of gene expression during perception rescued subsequent development, demonstrating that the response to perception without feeding is deleterious. Perception altered DAF-16/FOXO subcellular localization, reflecting activation of insulin/IGF signaling (IIS). The insulin-like peptide daf-28 was specifically required, suggesting perception in chemosensory neurons, where it is expressed, regulates peptide synthesis and possibly secretion. However, genetic manipulation of IIS did not modify the irreversible arrest phenotype caused by food perception, revealing that wild-type function of the IIS pathway is not required to produce this phenotype and that other pathways affected by perception of food in the absence of its ingestion are likely to be involved. Gene expression and Nile red staining showed that food perception could alter lipid metabolism and storage. We found that starved larvae sense environmental polypeptides, with similar molecular and developmental effects as perception of bacteria. Environmental polypeptides also promoted recovery from dauer diapause, suggesting that perception of polypeptides plays an important role in the life history of free-living nematodes. CONCLUSIONS: We conclude that actual ingestion of food is required to initiate postembryonic development in C. elegans. We also conclude that polypeptides are perceived as a food-associated cue in this and likely other animals, initiating a signaling and gene regulatory cascade that alters metabolism in anticipation of feeding and development, but that this response is detrimental if feeding does not occur.

Nonselective autophagy reduces mitochondrial content during starvation in Caenorhabditis elegans.

Starvation significantly alters cellular physiology, and signs of aging have been reported to occur during starvation. Mitochondria are essential to the regulation of cellular energetics and aging. We sought to determine whether mitochondria exhibit signs of aging during starvation and whether quality control mechanisms regulate mitochondrial physiology during starvation. We describe effects of starvation on mitochondria in the first and third larval stages of the nematode Caenorhabditis elegans. When starved, C. elegans larvae enter developmental arrest. We observed fragmentation of the mitochondrial network, a reduction in mitochondrial DNA (mtDNA) copy number, and accumulation of DNA damage during starvation-induced developmental arrest. Mitochondrial function was also compromised by starvation. Starved worms had lower basal, maximal, and ATP-linked respiration. These observations are consistent with reduced mitochondrial quality, similar to mitochondrial phenotypes during aging. Using pharmacological and genetic approaches, we found that worms deficient for autophagy were short-lived during starvation and recovered poorly from extended starvation, indicating sensitivity to nutrient stress. Autophagy mutants unc-51/Atg1 and atg-18/Atg18 maintained greater mtDNA content than wild-type worms during starvation, suggesting that autophagy promotes mitochondrial degradation during starvation. unc-51 mutants also had a proportionally smaller reduction in oxygen consumption rate during starvation, suggesting that autophagy also contributes to reduced mitochondrial function. Surprisingly, mutations in genes involved in mitochondrial fission and fusion as well as selective mitophagy of damaged mitochondria did not affect mitochondrial content during starvation. Our results demonstrate the profound influence of starvation on mitochondrial physiology with organismal consequences, and they show that these physiological effects are influenced by autophagy.

dbl-1/TGF-ß and daf-12/NHR Signaling Mediate Cell-Nonautonomous Effects of daf-16/FOXO on Starvation-Induced Developmental Arrest.

Nutrient availability has profound influence on development. In the nematode C. elegans, nutrient availability governs post-embryonic development. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This "L1 arrest" (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-ß, a ligand for the Sma/Mab pathway, daf-12/NHR and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-ß and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-ß, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-ß and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-ß and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.

Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways.

Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint.

Ins-4 and daf-28 function redundantly to regulate C. elegans L1 arrest.

Caenorhabditis elegans larvae reversibly arrest development in the first larval stage in response to starvation (L1 arrest or L1 diapause). Insulin-like signaling is a critical regulator of L1 arrest. However, the C. elegans genome encodes 40 insulin-like peptides, and it is unknown which peptides participate in nutritional control of L1 development. Work in other contexts has revealed that insulin-like genes can promote development ("agonists") or developmental arrest ("antagonists"), suggesting that such agonists promote L1 development in response to feeding. We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified thirteen candidate agonists and eight candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists is largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in control of L1 development. Transcriptional regulation of candidate agonists is most significant in the intestine, as if internal nutrient status is a more important influence on transcription than sensory perception. Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 developmental dynamics, though simultaneous disruption of ins-4 and daf-28 increases survival of L1 arrest. Furthermore, overexpression of ins-4, ins-6 or daf-28 alone decreases survival and promotes cell division during starvation. These results suggest extensive functional overlap among insulin-like genes in nutritional control of L1 development while highlighting the role of ins-4, daf-28 and to a lesser extent ins-6.

Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.

Succinylated octopamine ascarosides and a new pathway of biogenic amine metabolism in Caenorhabditis elegans.

The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in Caenorhabditis elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLC-MS/MS-based targeted metabolomics, we identified novel ascarosides incorporating a side chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine-derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.

Developmental and conditional regulation of DAF-2/INSR ubiquitination in Caenorhabditis elegans.

Insulin/IGF signaling (IIS) regulates developmental and metabolic plasticity. Conditional regulation of insulin-like peptide expression and secretion promotes different phenotypes in different environments. However, IIS can also be regulated by other, less-understood mechanisms. For example, stability of the only known insulin/IGF receptor in C. elegans, DAF-2/INSR, is regulated by CHIP-dependent ubiquitination. Disruption of chn-1/CHIP reduces longevity in C. elegans by increasing DAF-2/INSR abundance and IIS activity in adults. Likewise, mutation of a ubiquitination site causes daf-2(gk390525) to display gain-of-function phenotypes in adults. However, we show that this allele displays loss-of-function phenotypes in larvae, and that its effect on IIS activity transitions from negative to positive during development. In contrast, the allele acts like a gain-of-function in larvae cultured at high temperature, inhibiting temperature-dependent dauer formation. Disruption of chn-1/CHIP causes an increase in IIS activity in starved L1 larvae, unlike daf-2(gk390525). CHN-1/CHIP ubiquitinates DAF-2/INSR at multiple sites. These results suggest that the sites that are functionally relevant to negative regulation of IIS vary in larvae and adults, at different temperatures, and in nutrient-dependent fashion, revealing additional layers of IIS regulation.

Structure-specific variation in per- and polyfluoroalkyl substances toxicity among genetically diverse Caenorhabditis elegans strains.

Background: There are >14,500 structurally diverse per- and polyfluoroalkyl substances (PFAS). Despite knowledge that these "forever chemicals" are in 99% of humans, mechanisms of toxicity and adverse health effects are incompletely known. Furthermore, the contribution of genetic variation to PFAS susceptibility and health consequences is unknown. Objectives: We determined the toxicity of a structurally distinct set of PFAS in twelve genetically diverse strains of the genetic model system Caenorhabditis elegans. Methods: Dose-response curves for four perfluoroalkyl carboxylic acids (PFNA, PFOA, PFPeA, and PFBA), two perfluoroalkyl sulfonic acids (PFOS and PFBS), two perfluoroalkyl sulfonamides (PFOSA and PFBSA), two fluoroether carboxylic acids (GenX and PFMOAA), one fluoroether sulfonic acid (PFEESA), and two fluorotelomers (6:2 FCA and 6:2 FTS) were determined in the C. elegans laboratory reference strain, N2, and eleven genetically diverse wild strains. Body length was quantified by image analysis at each dose after 48 hr of developmental exposure of L1 arrest-synchronized larvae to estimate effective concentration values (EC50). Results: There was a significant range in toxicity among PFAS: PFOSA > PFBSA ≈ PFOS ≈ PFNA > PFOA > GenX ≈ PFEESA > PFBS ≈ PFPeA ≈ PFBA. Long-chain PFAS had greater toxicity than short-chain, and fluorosulfonamides were more toxic than carboxylic and sulfonic acids. Genetic variation explained variation in susceptibility to PFBSA, PFOS, PFBA, PFOA, GenX, PFEESA, PFPeA, and PFBA. There was significant variation in toxicity among C. elegans strains due to chain length, functional group, and between legacy and emerging PFAS. Conclusion: C. elegans respond to legacy and emerging PFAS of diverse structures, and this depends on specific structures and genetic variation. Harnessing the natural genetic diversity of C. elegans and the structural complexity of PFAS is a powerful New Approach Methodology (NAM) to investigate structure-activity relationships and mechanisms of toxicity which may inform regulation of other PFAS to improve human and environmental health.

Alternative somatic and germline gene-regulatory strategies during starvation-induced developmental arrest.

Nutrient availability governs growth and quiescence, and many animals arrest development when starved. Using C. elegans L1 arrest as a model, we show that gene expression changes deep into starvation. Surprisingly, relative expression of germline-enriched genes increases for days. We conditionally degrade the large subunit of RNA polymerase II using the auxin-inducible degron system and analyze absolute expression levels. We find that somatic transcription is required for survival, but the germline maintains transcriptional quiescence. Thousands of genes are continuously transcribed in the soma, though their absolute abundance declines, such that relative expression of germline transcripts increases given extreme transcript stability. Aberrantly activating transcription in starved germ cells compromises reproduction, demonstrating important physiological function of transcriptional quiescence. This work reveals alternative somatic and germline gene-regulatory strategies during starvation, with the soma maintaining a robust transcriptional response to support survival and the germline maintaining transcriptional quiescence to support future reproductive success.

Genetic analysis of daf-18/PTEN missense mutants for starvation resistance and developmental regulation during Caenorhabditis elegans L1 arrest.

Mutations in the well-known tumor suppressor PTEN are observed in many cancers. PTEN is a dual-specificity phosphatase that harbors lipid and protein-phosphatase activities. The Caenorhabditis elegans PTEN ortholog is daf-18, which has pleiotropic effects on dauer formation, aging, starvation resistance, and development. Function of 3 daf-18 point-mutants, G174E, D137A, and C169S, had previously been investigated using high-copy transgenes in a daf-18 null background. These alleles were generated based on their mammalian counterparts and were treated as though they specifically disrupt lipid or protein-phosphatase activity, or both, respectively. Here, we investigated these alleles using genome editing of endogenous daf-18. We assayed 3 traits relevant to L1 starvation resistance, and we show that each point mutant is essentially as starvation-sensitive as a daf-18 null mutant. Furthermore, we show that G174E and D137A do not complement each other, suggesting overlapping effects on lipid and protein-phosphatase activity. We also show that each allele has strong effects on nucleocytoplasmic localization of DAF-16/FoxO and dauer formation, both of which are regulated by PI3K signaling, similar to a daf-18 null allele. In addition, each allele also disrupts M-cell quiescence during L1 starvation, though D137A has a weaker effect than the other alleles, including the null. Our results confirm that daf-18/PTEN is important for promoting starvation resistance and developmental arrest and that it is a potent regulator of PI3K signaling, and they highlight challenges of using genetic analysis to link specific DAF-18/PTEN enzymatic activities to particular phenotypes.

Using population selection and sequencing to characterize natural variation of starvation resistance in Caenorhabditis elegans.

Starvation resistance is important to disease and fitness, but the genetic basis of its natural variation is unknown. Uncovering the genetic basis of complex, quantitative traits such as starvation resistance is technically challenging. We developed a synthetic-population (re)sequencing approach using molecular inversion probes (MIP-seq) to measure relative fitness during and after larval starvation in Caenorhabditis elegans. We applied this competitive assay to 100 genetically diverse, sequenced, wild strains, revealing natural variation in starvation resistance. We confirmed that the most starvation-resistant strains survive and recover from starvation better than the most starvation-sensitive strains using standard assays. We performed genome-wide association (GWA) with the MIP-seq trait data and identified three quantitative trait loci (QTL) for starvation resistance, and we created near isogenic lines (NILs) to validate the effect of these QTL on the trait. These QTL contain numerous candidate genes including several members of the Insulin/EGF Receptor-L Domain (irld) family. We used genome editing to show that four different irld genes have modest effects on starvation resistance. Natural variants of irld-39 and irld-52 affect starvation resistance, and increased resistance of the irld-39; irld-52 double mutant depends on daf-16/FoxO. DAF-16/FoxO is a widely conserved transcriptional effector of insulin/IGF signaling (IIS), and these results suggest that IRLD proteins modify IIS, although they may act through other mechanisms as well. This work demonstrates efficacy of using MIP-seq to dissect a complex trait and it suggests that irld genes are natural modifiers of starvation resistance in C. elegans.

Chromosome-Level Reference Genomes for Two Strains of Caenorhabditis briggsae: An Improved Platform for Comparative Genomics.

The publication of the Caenorhabditis briggsae reference genome in 2003 enabled the first comparative genomics studies between C. elegans and C. briggsae, shedding light on the evolution of genome content and structure in the Caenorhabditis genus. However, despite being widely used, the currently available C. briggsae reference genome is substantially less complete and structurally accurate than the C. elegans reference genome. Here, we used high-coverage Oxford Nanopore long-read and chromosome-conformation capture data to generate chromosome-level reference genomes for two C. briggsae strains: QX1410, a new reference strain closely related to the laboratory AF16 strain, and VX34, a highly divergent strain isolated in China. We also sequenced 99 recombinant inbred lines generated from reciprocal crosses between QX1410 and VX34 to create a recombination map and identify chromosomal domains. Additionally, we used both short- and long-read RNA sequencing data to generate high-quality gene annotations. By comparing these new reference genomes to the current reference, we reveal that hyper-divergent haplotypes cover large portions of the C. briggsae genome, similar to recent reports in C. elegans and C. tropicalis. We also show that the genomes of selfing Caenorhabditis species have undergone more rearrangement than their outcrossing relatives, which has biased previous estimates of rearrangement rate in Caenorhabditis. These new genomes provide a substantially improved platform for comparative genomics in Caenorhabditis and narrow the gap between the quality of genomic resources available for C. elegans and C. briggsae.

Liquid-culture protocols for synchronous starvation, growth, dauer formation, and dietary restriction of Caenorhabditis elegans.

Standard laboratory culture of Caenorhabditis elegans utilizes solid growth media with a bacterial food source. However, this culture method limits control of food availability and worm population density, factors that impact many life-history traits. Here, we describe liquid-culture protocols for precisely modulating bacterial food availability and population density, facilitating reliable production of arrested L1 larvae, dauer larvae, dietarily restricted worms, or well-fed worms. Worms can be grown in small quantities for standard assays or in the millions for other applications. For complete details on the use and execution of these protocols, please refer to Hibshman et al. (2016), Webster et al. (2018), and Jordan et al. (2019).

Intergenerational adaptations to stress are evolutionarily conserved, stress-specific, and have deleterious trade-offs.

Despite reports of parental exposure to stress promoting physiological adaptations in progeny in diverse organisms, there remains considerable debate over the significance and evolutionary conservation of such multigenerational effects. Here, we investigate four independent models of intergenerational adaptations to stress in Caenorhabditis elegans - bacterial infection, eukaryotic infection, osmotic stress, and nutrient stress - across multiple species. We found that all four intergenerational physiological adaptations are conserved in at least one other species, that they are stress -specific, and that they have deleterious tradeoffs in mismatched environments. By profiling the effects of parental bacterial infection and osmotic stress exposure on progeny gene expression across species, we established a core set of 587 genes that exhibited a greater than twofold intergenerational change in expression in response to stress in C. elegans and at least one other species, as well as a set of 37 highly conserved genes that exhibited a greater than twofold intergenerational change in expression in all four species tested. Furthermore, we provide evidence suggesting that presumed adaptive and deleterious intergenerational effects are molecularly related at the gene expression level. Lastly, we found that none of the effects we detected of these stresses on C. elegans F1 progeny gene expression persisted transgenerationally three generations after stress exposure. We conclude that intergenerational responses to stress play a substantial and evolutionarily conserved role in regulating animal physiology and that the vast majority of the effects of parental stress on progeny gene expression are reversible and not maintained transgenerationally.

Selfing is the safest sex for Caenorhabditis tropicalis.

Mating systems have profound effects on genetic diversity and compatibility. The convergent evolution of self-fertilization in three Caenorhabditis species provides a powerful lens to examine causes and consequences of mating system transitions. Among the selfers, Caenorhabditis tropicalis is the least genetically diverse and most afflicted by outbreeding depression. We generated a chromosomal-scale genome for C. tropicalis and surveyed global diversity. Population structure is very strong, and islands of extreme divergence punctuate a genomic background that is highly homogeneous around the globe. Outbreeding depression in the laboratory is caused largely by multiple Medea-like elements, genetically consistent with maternal toxin/zygotic antidote systems. Loci with Medea activity harbor novel and duplicated genes, and their activity is modified by mito-nuclear background. Segregating Medea elements dramatically reduce fitness, and simulations show that selfing limits their spread. Frequent selfing in C. tropicalis may therefore be a strategy to avoid Medea-mediated outbreeding depression.