Natural variation in infection specificity of Caenorhabditis briggsae isolates by two RNA viruses.

Antagonistic relationships such as host-virus interactions potentially lead to rapid evolution and specificity in interactions. The Orsay virus is so far the only horizontal virus naturally infecting the nematode C. elegans. In contrast, several related RNA viruses infect its congener C. briggsae, including Santeuil (SANTV) and Le Blanc (LEBV) viruses. Here we focus on the host's intraspecific variation in sensitivity to these two intestinal viruses. Many temperate-origin C. briggsae strains, including JU1264 and JU1498, are sensitive to both, while many tropical strains, such as AF16, are resistant to both. Interestingly, some C. briggsae strains exhibit a specific resistance, such as the HK104 strain, specifically resistant to LEBV. The viral sensitivity pattern matches the strains' geographic and genomic relationships. The heavily infected strains mount a seemingly normal small RNA response that is insufficient to suppress viral infection, while the resistant strains show no small RNA response, suggesting an early block in viral entry or replication. We use a genetic approach from the host side to map genomic regions participating in viral resistance polymorphisms. Using Advanced Intercrossed Recombinant Inbred Lines (RILs) between virus-resistant AF16 and SANTV-sensitive HK104, we detect Quantitative Trait Loci (QTLs) on chromosomes IV and III. Building RILs between virus-sensitive JU1498 and LEBV-resistant HK104 followed by bulk segregant analysis, we identify a chromosome II QTL. In both cases, further introgressions of the regions confirmed the QTLs. This diversity provides an avenue for studying virus entry, replication, and exit mechanisms, as well as host-virus specificity and the host response to a specific virus infection.

Experimental evolution of an RNA virus in Caenorhabditis elegans.

The discovery of Orsay virus (OrV), the first virus infecting wild populations of Caenorhabditis elegans, has boosted studies of viral immunity pathways in this nematode. Considering the many advantages that C. elegans offers for fundamental research in host-pathogen interactions, this pathosystem has high potential to become a model system for experimental virus evolution studies. However, the evolutionary constraints - i.e, the balance between genetic variation, selection, drift and historical contingency- operating in this pathosystem have barely been explored. Here we describe for the first time an evolution experiment of two different OrV strains in C. elegans. Comparison of the two ancestral strains showed differences in infectivity and sequence, and highlighted the importance of consistently normalize viral inocula for meaningful comparisons among strains. After 10 serial passages of evolution, we report slight changes in infectivity and non-synonymous mutations fixed in the evolved viral populations. In addition, we observed numerous minor variants emerging in the viral population. These minor variants were not randomly distributed along the genome but concentrated in polymorphic genomic regions. Overall, our work established the grounds for future experimental virus evolution studies using Caenorhabditis nematodes.

Caenorhabditis elegans defective-pharynx and constipated mutants are resistant to Orsay virus infection.

C. elegans animals with a compromised pharynx accumulate bacteria in their intestinal lumen and activate a transcriptional response that includes anti-bacterial response genes. In this study, we demonstrate that animals with defective pharynxes are resistant to Orsay virus (OrV) infection. This resistance is observed for animals grown on Escherichia coli OP50 and on Comamonas BIGb0172, a bacterium naturally associated with C. elegans . The viral resistance observed in defective-pharynx mutants does not seem to result from constitutive transcriptional immune responses against viruses. OrV resistance is also observed in mutants with defective defecation, which share with the pharynx-defective perturbations in the regulation of their intestinal contents and altered lipid metabolism. The underlying mechanisms of viral resistance in pharynx- and defecation-defective mutants remain elusive.

Caenorhabditis elegans immune responses to microsporidia and viruses.

The model organism Caenorhabditis elegans is susceptible to infection by obligate intracellular pathogens, specifically microsporidia and viruses. These intracellular pathogens infect intestinal cells, or, for some microsporidia, epidermal cells. Strikingly, intestinal cell infections by viruses or microsporidia trigger a common transcriptional response, activated in part by the ZIP-1 transcription factor. Among the strongest activated genes in this response are ubiquitin-pathway members and members of the pals family, an intriguing gene family with cross-regulations of different members of genomic clusters. Some of the induced genes participate in host defense against the pathogens, for example through ubiquitin-mediated inhibition. Other mechanisms defend the host specifically against viral infections, including antiviral RNA interference and uridylation. These various immune responses are altered by environmental factors and by intraspecific genetic variation of the host. These pathogens were first isolated 15 years ago and much remains to be discovered using C. elegans genetics; also, other intracellular pathogens of C. elegans may yet to be discovered.

Naturally-associated bacteria modulate Orsay virus infection of Caenorhabditis elegans.

Microbes associated with an organism can significantly modulate its susceptibility to viral infections, but our understanding of the influence of individual microbes remains limited. The nematode Caenorhabditis elegans is a model organism that in nature inhabits environments rich in bacteria. Here, we examine the impact of 71 naturally associated bacteria on C. elegans susceptibility to its only known natural virus, the Orsay virus. Our findings reveal that viral infection of C. elegans is significantly influenced by monobacterial environments. Compared to an Escherichia coli environmental reference, the majority of tested bacteria reduced C. elegans susceptibility to viral infection. This reduction is not caused by virion degradation or poor animal nutrition by the bacteria. The repression of viral infection by the bacterial strains Chryseobacterium JUb44 and Sphingobacterium BIGb0172 does not require the RIG-I homolog DRH-1, which is known to activate antiviral responses such as RNA interference and transcriptional regulation. Our research highlights the necessity of considering natural biotic environments in viral infection studies and opens the way future research on host-microbe-virus interactions.

Genome-wide association and environmental suppression of the mortal germline phenotype of wild C. elegans.

The animal germline lineage needs to be maintained along generations. However, some Caenorhabditis elegans wild isolates display a mortal germline phenotype, leading to sterility after several generations at 25°C. Using a genome-wide association approach, we detect a significant peak on chromosome III around 5 Mb, confirmed by introgressions. Thus, a seemingly deleterious genotype is maintained at intermediate frequency in the species. Environmental rescue is a likely explanation, and indeed associated bacteria and microsporidia suppress the phenotype of wild isolates as well as mutants in small RNA inheritance (nrde-2) and histone modifications (set-2). Escherichia coli strains of the K-12 lineage suppress the phenotype compared to B strains. By shifting a wild strain from E. coli K-12 to E. coli B, we find that memory of the suppressing condition is maintained over several generations. Thus, the mortal germline phenotype of wild C. elegans is in part revealed by laboratory conditions and may represent variation in epigenetic inheritance and environmental interactions. This study also points to the importance of non-genetic memory in the face of environmental variation.

A QTL on chromosome IV explains a natural variation of QR.pap final position in Caenorhabditis elegans.

In Caenorhabditis elegans , the QR neuroblast and its progeny migrate from the posterior to the anterior part of the animal during the L1 stage. We previously showed that the final position of QR.pa daughters varies among C. elegans wild isolates, with CB4932 displaying a particularly anterior QR.pap position (Dubois et al., 2021). Here, we study the genetic basis of the variation between isolates CB4932 and JU1242. We show that JU1242 alleles behave in a mostly dominant fashion. Using a Bulk Segregant Analysis, we detect a quantitative trait locus (QTL) region on chromosome IV. This QTL was confirmed using reciprocal chromosome IV introgressions.

Precise temporal control of neuroblast migration through combined regulation and feedback of a Wnt receptor.

Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that controls neuroblast migration in Caenorhabditis elegans as a tractable system to study a robust, cell-intrinsic timing mechanism in vivo. Single-molecule mRNA quantification showed that the expression of the receptor increases non-linearly, a dynamic that is predicted to enhance timing precision over an unregulated, linear increase in timekeeper abundance. We show that this upregulation depends on transcriptional activation, providing in vivo evidence for a model in which the timing of receptor expression is regulated through an accumulating activator that triggers expression when a specific threshold is reached. This timing mechanism acts across a cell division that occurs in the neuroblast lineage and is influenced by the asymmetry of the division. Finally, we show that positive feedback of receptor expression through the canonical Wnt pathway enhances temporal precision. We conclude that robust cell-intrinsic timing can be achieved by combining regulation and feedback of the timekeeper gene.

Bacterial filamentation as a mechanism for cell-to-cell spread within an animal host.

Intracellular pathogens are challenged with limited space and resources while replicating in a single host cell. Mechanisms for direct invasion of neighboring host cells have been discovered in cell culture, but we lack an understanding of how bacteria directly spread between host cells in vivo. Here, we describe the discovery of intracellular bacteria that use filamentation for spreading between the intestinal epithelial cells of a natural host, the rhabditid nematode Oscheius tipulae. The bacteria, which belong to the new species Bordetella atropi, can infect the nematodes following a fecal-oral route, and reduce host life span and fecundity. Filamentation requires UDP-glucose biosynthesis and sensing, a highly conserved pathway that is used by other bacteria to detect rich conditions and inhibit cell division. Our results indicate that B. atropi uses a pathway that normally regulates bacterial cell size to trigger filamentation inside host cells, thus facilitating cell-to-cell dissemination.

De Novo Genome Assembly of Auanema Melissensis, a Trioecious Free-Living Nematode.

Nematodes of the genus Auanema are interesting models for studying sex determination mechanisms because their populations consist of three sexual morphs (males, females, and hermaphrodites) and produce skewed sex ratios. Here, we introduce a new undescribed species of this genus, Auanema melissensis n. sp., together with its draft nuclear genome. This species is also trioecious and does not cross with the other described species A. rhodensis or A. freiburgensis. Similar to A. freiburgensis, A. melissensis' maternal environment influences the hermaphrodite versus female sex determination of the offspring. The genome of A. melissensis is ~60 Mb, containing 11,040 protein-coding genes and 8.07% of repeat sequences. Using the estimated ancestral chromosomal gene content (Nigon elements), it was possible to identify putative X chromosome scaffolds.

Infection of C. elegans by Haptoglossa Species Reveals Shared Features in the Host Response to Oomycete Detection.

Oomycetes are a group of eukaryotic organisms that includes many important pathogens of animals and plants. Within this group, the Haptoglossa genus is characterised by the presence of specialised gun cells carrying a harpoon-like infection apparatus. While several Haptoglossa pathogens have been morphologically described, there are currently no host systems developed to study the infection process or host responses in the lab. In this study, we report that Haptoglossa species are potent natural pathogens of Caenorhabditis nematodes. Using electron microscopy, we characterise the infection process in C. elegans and demonstrate that the oomycete causes excessive tissue degradation upon entry in the body cavity, whilst leaving the host cuticle intact. We also report that the host transcriptional response to Haptoglossa infection shares similarities with the response against the oomycete Myzocytiopsis humicola, a key example of which is the induction of chitinase-like (chil) genes in the hypodermis. We demonstrate that this shared feature of the host response can be mounted by pathogen detection without any infection, as previously shown for M. humicola. These results highlight similarities in the nematode immune response to natural infection by phylogenetically distinct oomycetes.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria.

Caenorhabditis elegans (C. elegans) has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Natural genetic variation drives microbiome selection in the Caenorhabditis elegans gut.

Host genetic landscapes can shape microbiome assembly in the animal gut by contributing to the establishment of distinct physiological environments. However, the genetic determinants contributing to the stability and variation of these microbiome types remain largely undefined. Here, we use the free-living nematode Caenorhabditis elegans to identify natural genetic variation among wild strains of C. elegans that drives assembly of distinct microbiomes. To achieve this, we first established a diverse model microbiome that represents the strain-level phylogenetic diversity naturally encountered by C. elegans in the wild. Using this community, we show that C. elegans utilizes immune, xenobiotic, and metabolic signaling pathways to favor the assembly of different microbiome types. Variations in these pathways were associated with enrichment for specific commensals, including the Alphaproteobacteria Ochrobactrum. Using RNAi and mutant strains, we showed that host selection for Ochrobactrum is mediated specifically by host insulin signaling pathways. Ochrobactrum recruitment is blunted in the absence of DAF-2/IGFR and modulated by the competitive action of insulin signaling transcription factors DAF-16/FOXO and PQM-1/SALL2. Further, the ability of C. elegans to enrich for Ochrobactrum as adults is correlated with faster animal growth rates and larger body size at the end of development. These results highlight a new role for the highly conserved insulin signaling pathways in the regulation of gut microbiome composition in C. elegans.

Balancing selection maintains hyper-divergent haplotypes in Caenorhabditis elegans.

Across diverse taxa, selfing species have evolved independently from outcrossing species thousands of times. The transition from outcrossing to selfing decreases the effective population size, effective recombination rate and heterozygosity within a species. These changes lead to a reduction in genetic diversity, and therefore adaptive potential, by intensifying the effects of random genetic drift and linked selection. Within the nematode genus Caenorhabditis, selfing has evolved at least three times, and all three species, including the model organism Caenorhabditis elegans, show substantially reduced genetic diversity relative to outcrossing species. Selfing and outcrossing Caenorhabditis species are often found in the same niches, but we still do not know how selfing species with limited genetic diversity can adapt to these environments. Here, we examine the whole-genome sequences from 609 wild C. elegans strains isolated worldwide and show that genetic variation is concentrated in punctuated hyper-divergent regions that cover 20% of the C. elegans reference genome. These regions are enriched in environmental response genes that mediate sensory perception, pathogen response and xenobiotic stress response. Population genomic evidence suggests that genetic diversity in these regions has been maintained by long-term balancing selection. Using long-read genome assemblies for 15 wild strains, we show that hyper-divergent haplotypes contain unique sets of genes and show levels of divergence comparable to levels found between Caenorhabditis species that diverged millions of years ago. These results provide an example of how species can avoid the evolutionary dead end associated with selfing.

Evolution: Drivers in a Traffic Jam.

Genetic loci coding for a toxin and its antidote behave like selfish elements. Two new studies find an accumulation of such elements in one species.

Temporally regulated cell migration is sensitive to variation in body size.

Few studies have measured the robustness to perturbations of the final position of a long-range migrating cell. In the nematode Caenorhabditis elegans, the QR neuroblast migrates anteriorly, while undergoing three division rounds. We study the final position of two of its great-granddaughters, the end of migration of which was previously shown to depend on a timing mechanism. We find that the variance in their final position is similar to that of other long-range migrating neurons. As expected from the timing mechanism, the position of QR descendants depends on body size, which we varied by changing maternal age or using body size mutants. Using a mathematical model, we show that body size variation is partially compensated for. Applying environmental perturbations, we find that the variance in final position increased following starvation at hatching. The mean position is displaced upon a temperature shift. Finally, highly significant variation was found among C. elegans wild isolates. Overall, this study reveals that the final position of these neurons is quite robust to stochastic variation, shows some sensitivity to body size and to external perturbations, and varies in the species.This article has an associated 'The people behind the papers' interview.

Diversification and hybrid incompatibility in auto-pseudogamous species of Mesorhabditis nematodes.

BACKGROUND: Pseudogamy is a reproductive system in which females rely on the sperm of males to activate their oocytes, generally parasitizing males of other species, but do not use the sperm DNA. The nematode Mesorhabditis belari uses a specific form of pseudogamy, where females produce their own males as a source of sperm. Males develop from rare eggs with true fertilization, while females arise by gynogenesis. Males thus do not contribute their genome to the female offspring. Here, we explored the diversity of reproductive mode within the Mesorhabditis genus and addressed species barriers in pseudogamous species. RESULTS: To this end, we established a collection of over 60 Mesorhabditis strains from soil and rotting vegetal matter. We found that males from pseudogamous species displayed a reduced size of their body, male tail and sperm cells compared to males of sexual Mesorhabditis species, as expected for males that face little competition. Using rDNA sequences and crosses, we could define 11 auto-pseudogamous biological species, with closely related species pairs and a possible single origin of pseudogamy in the Mesorhabditis genus. Most crosses between males and females of different species did not even produce female progeny. This surprising species barrier in pseudogamous egg activation was pre or postcopulatory depending on the species pair. In the latter case, when hybrid embryos were produced, most arrested before the first embryonic cell division. Hybrid incompatibility between auto-pseudogamous species was due to defective interaction between sperm and oocyte as well as defective reconstitution of zygotic centrosomes. CONCLUSIONS: We established a collection of sexual and pseudo-sexual species which offer an ideal framework to explore the origin and consequences of transition to asexuality. Our results demonstrate that speciation occurs in the pseudogamous state. Whereas genomic conflicts are responsible for hybrid incompatibility in sexual species, we here reveal that centrosomes constitute key organelles in the establishment of species barrier.

A broad mutational target explains a fast rate of phenotypic evolution.

The rapid evolution of a trait in a clade of organisms can be explained by the sustained action of natural selection or by a high mutational variance, that is the propensity to change under spontaneous mutation. The causes for a high mutational variance are still elusive. In some cases, fast evolution depends on the high mutation rate of one or few loci with short tandem repeats. Here, we report on the fastest evolving cell fate among vulva precursor cells in Caenorhabditis nematodes, that of P3.p. We identify and validate causal mutations underlying P3.p's high mutational variance. We find that these positions do not present any characteristics of a high mutation rate, are scattered across the genome and the corresponding genes belong to distinct biological pathways. Our data indicate that a broad mutational target size is the cause of the high mutational variance and of the corresponding fast phenotypic evolutionary rate.

Heritable characteristics or traits of a group of organisms, for example the large brain size of primates or the hooves of a horse, are determined by genes, the environment, and by the interactions between them. Traits can change over time and generations when enough mutations in these genes have spread in a species to result in visible differences. However, some traits, such as the large brain of primates, evolve faster than others, but why this is the case has been unclear. It could be that a few specific genes important for that trait in question mutate at a high rate, or, that many genes affect the trait, creating a lot of variation for natural selection to choose from. Here, Besnard, Picao-Osorio et al. studied the roundworm Caenorhabditis elegans to better understand the causes underlying the different rates of trait evolution. These worms have a short life cycle and evolve quickly over many generations, making them an ideal candidate for studying mutation rates in different traits. Previous studies have shown that one of C. elegans' six cells of the reproductive system evolves faster than the others. To investigate this further, Besnard, Picao-Osorio et al. analysed the genetic mutations driving change in this cell in 250 worm generations. The results showed that five mutations in five different genes ­ all responsible for different processes in the cells ­ were behind the supercharged evolution of this particular cell. This suggests that fast evolution results from natural selection acting upon a collection of genes, rather than one gene, and that many genes and pathways shape this trait. In conclusion, these results demonstrate that how traits are coded at the molecular level, in one gene or many, can influence the rate at which they evolve.

CeMbio - The Caenorhabditis elegans Microbiome Resource.

The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g., human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural Caenorhabditis elegans microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the C. elegans model system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions in C. elegans.

Natural Variation in a Dendritic Scaffold Protein Remodels Experience-Dependent Plasticity by Altering Neuropeptide Expression.

The extent to which behavior is shaped by experience varies between individuals. Genetic differences contribute to this variation, but the neural mechanisms are not understood. Here, we dissect natural variation in the behavioral flexibility of two Caenorhabditis elegans wild strains. In one strain, a memory of exposure to 21% O2 suppresses CO2-evoked locomotory arousal; in the other, CO2 evokes arousal regardless of previous O2 experience. We map that variation to a polymorphic dendritic scaffold protein, ARCP-1, expressed in sensory neurons. ARCP-1 binds the Ca2+-dependent phosphodiesterase PDE-1 and co-localizes PDE-1 with molecular sensors for CO2 at dendritic ends. Reducing ARCP-1 or PDE-1 activity promotes CO2 escape by altering neuropeptide expression in the BAG CO2 sensors. Variation in ARCP-1 alters behavioral plasticity in multiple paradigms. Our findings are reminiscent of genetic accommodation, an evolutionary process by which phenotypic flexibility in response to environmental variation is reset by genetic change.