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1.  A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral RNA dicing and antiviral immunity 
eLife  2013;2:e00994.
RNA interference defends against viral infection in plant and animal cells. The nematode Caenorhabditis elegans and its natural pathogen, the positive-strand RNA virus Orsay, have recently emerged as a new animal model of host-virus interaction. Using a genome-wide association study in C. elegans wild populations and quantitative trait locus mapping, we identify a 159 base-pair deletion in the conserved drh-1 gene (encoding a RIG-I-like helicase) as a major determinant of viral sensitivity. We show that DRH-1 is required for the initiation of an antiviral RNAi pathway and the generation of virus-derived siRNAs (viRNAs). In mammals, RIG-I-domain containing proteins trigger an interferon-based innate immunity pathway in response to RNA virus infection. Our work in C. elegans demonstrates that the RIG-I domain has an ancient role in viral recognition. We propose that RIG-I acts as modular viral recognition factor that couples viral recognition to different effector pathways including RNAi and interferon responses.
DOI: http://dx.doi.org/10.7554/eLife.00994.001
eLife digest
Most organisms—from bacteria to mammals—have at least a rudimentary immune system that can detect and defend against pathogens, particularly viruses. This defense mechanism, which is known as the innate immune system, uses sensor proteins to recognize viral RNA, and then mobilizes other immune components to attack the invaders.
The specific mechanisms used to destroy viruses differ between species. In mammals, a protein called RIG-1 binds to viral RNA and activates a signaling pathway that leads to the production of interferons: immune proteins named after their ability to ‘interfere’ with viral replication. Plants and insects do not use interferons, but instead use a mechanism called RNA interference, in which long double-stranded RNAs are cleaved into shorter fragments.
The nematode worm C. elegans also deploys RNA interference against viruses but, in contrast to insects and plants, worms do not possess a specific set of RNA interference enzymes that participate solely in the antiviral response. They do, however, express a protein called DRH-1 that is related to the RIG-I protein found in mammals.
To investigate whether DRH-1 contributes to innate immunity in C. elegans, Ashe et al. infected 97 strains of C. elegans from around the world with a virus, and showed that some strains were more sensitive to the virus than others, with certain strains showing complete resistance. By comparing a sensitive strain with a resistant one, Ashe et al. revealed that viral sensitivity was caused by a mutation in the gene encoding DRH-1.
Further experiments showed that DRH-1 is required for the first step in RNA interference. Ashe et al. have thus identified a conserved role for RIG-1 in initiating antiviral responses, and propose that the protein couples virus recognition to distinct defense mechanisms in different evolutionary groups.
DOI: http://dx.doi.org/10.7554/eLife.00994.002
doi:10.7554/eLife.00994
PMCID: PMC3793227  PMID: 24137537
RNA interference; immunity; virus infection; C. elegans
2.  A Streamlined System for Species Diagnosis in Caenorhabditis (Nematoda: Rhabditidae) with Name Designations for 15 Distinct Biological Species 
PLoS ONE  2014;9(4):e94723.
The rapid pace of species discovery outstrips the rate of species description in many taxa. This problem is especially acute for Caenorhabditis nematodes, where the naming of distinct species would greatly improve their visibility and usage for biological research, given the thousands of scientists studying Caenorhabditis. Species description and naming has been hampered in Caenorhabditis, in part due to the presence of morphologically cryptic species despite complete biological reproductive isolation and often enormous molecular divergence. With the aim of expediting species designations, here we propose and apply a revised framework for species diagnosis and description in this group. Our solution prioritizes reproductive isolation over traditional morphological characters as the key feature in delineating and diagnosing new species, reflecting both practical considerations and conceptual justifications. DNA sequence divergence criteria help prioritize crosses for establishing patterns of reproductive isolation among the many species of Caenorhabditis known to science, such as with the ribosomal internal transcribed spacer-2 (ITS2) DNA barcode. By adopting this approach, we provide new species name designations for 15 distinct biological species, thus increasing the number of named Caenorhabditis species in laboratory culture by nearly 3-fold. We anticipate that the improved accessibility of these species to the research community will expand the opportunities for study and accelerate our understanding of diverse biological phenomena.
doi:10.1371/journal.pone.0094723
PMCID: PMC3984244  PMID: 24727800
3.  Two Leucobacter Strains Exert Complementary Virulence on Caenorhabditis Including Death by Worm-Star Formation 
Current Biology  2013;23(21):2157-2161.
Summary
The nematode Caenorhabditis elegans has been much studied as a host for microbial infection. Some pathogens can infect its intestine [1, 2], while others attack via its external surface [1, 3–6]. Cultures of Caenorhabditis isolated from natural environments have yielded new nematode pathogens, such as microsporidia and viruses [7, 8]. We report here a novel mechanism for bacterial attack on worms, discovered during investigation of a diseased and coinfected natural isolate of Caenorhabditis from Cape Verde. Two related coryneform pathogens (genus Leucobacter) were obtained from this isolate, which had complementary effects on C. elegans and related nematodes. One pathogen, Verde1, was able to cause swimming worms to stick together irreversibly by their tails, leading to the rapid formation of aggregated “worm-stars.” Adult worms trapped in these aggregates were immobilized and subsequently died, with concomitant growth of bacteria. Trapped larval worms were sometimes able to escape from worm-stars by undergoing autotomy, separating their bodies into two parts. The other pathogen, Verde2, killed worms after rectal invasion, in a more virulent version of a previously studied infection [6]. Resistance to killing by Verde2, by means of alterations in host surface glycosylation, resulted in hypersensitivity to Verde1, revealing a trade-off in bacterial susceptibility. Conversely, a sublethal surface infection of worms with Verde1 conferred partial protection against Verde2. The formation of worm-stars by Verde1 occurred only when worms were swimming in liquid but provides a striking example of asymmetric warfare as well as a bacterial equivalent to the trapping strategies used by nematophagous fungi [4].
Highlights
•Verde1 bacteria can trap swimming worms by tail adhesion and worm-star formation•Nematodes trapped in worm-stars are immobilized, killed, and used for nutrition•Trapped larval worms can escape from worm-stars by whole-body autotomy•Resistance to virulent Verde2 bacteria results in lethal hypersensitivity to Verde1
doi:10.1016/j.cub.2013.08.060
PMCID: PMC3898767  PMID: 24206844
4.  Complete Genome Sequence of Le Blanc Virus, a Third Caenorhabditis Nematode-Infecting Virus 
Journal of Virology  2012;86(21):11940.
Orsay virus and Santeuil virus, the first known viruses capable of naturally infecting the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, respectively, were recently identified by high-throughput sequencing of wild Caenorhabditis strains. By similar analysis of another wild C. briggsae isolate, we have now discovered and sequenced the complete genome of a third novel virus, Le Blanc virus, that is distantly related to Orsay and Santeuil viruses. All three viruses are positive-sense RNA viruses with bipartite genomes that are most closely related to nodaviruses. Identification of a third virus capable of infecting Caenorhabditis nematodes enables comparative analysis of this clade of viruses and strengthens this model for investigating virus-host interactions.
doi:10.1128/JVI.02025-12
PMCID: PMC3486331  PMID: 23043172
5.  Species richness, distribution and genetic diversity of Caenorhabditis nematodes in a remote tropical rainforest 
Background
In stark contrast to the wealth of detail about C. elegans developmental biology and molecular genetics, biologists lack basic data for understanding the abundance and distribution of Caenorhabditis species in natural areas that are unperturbed by human influence.
Methods
Here we report the analysis of dense sampling from a small, remote site in the Amazonian rain forest of the Nouragues Natural Reserve in French Guiana.
Results
Sampling of rotting fruits and flowers revealed proliferating populations of Caenorhabditis, with up to three different species co-occurring within a single substrate sample, indicating remarkable overlap of local microhabitats. We isolated six species, representing the highest local species richness for Caenorhabditis encountered to date, including both tropically cosmopolitan and geographically restricted species not previously isolated elsewhere. We also documented the structure of within-species molecular diversity at multiple spatial scales, focusing on 57 C. briggsae isolates from French Guiana. Two distinct genetic subgroups co-occur even within a single fruit. However, the structure of C. briggsae population genetic diversity in French Guiana does not result from strong local patterning but instead presents a microcosm of global patterns of differentiation. We further integrate our observations with new data from nearly 50 additional recently collected C. briggsae isolates from both tropical and temperate regions of the world to re-evaluate local and global patterns of intraspecific diversity, providing the most comprehensive analysis to date for C. briggsae population structure across multiple spatial scales.
Conclusions
The abundance and species richness of Caenorhabditis nematodes is high in a Neotropical rainforest habitat that is subject to minimal human interference. Microhabitat preferences overlap for different local species, although global distributions include both cosmopolitan and geographically restricted groups. Local samples for the cosmopolitan C. briggsae mirror its pan-tropical patterns of intraspecific polymorphism. It remains an important challenge to decipher what drives Caenorhabditis distributions and diversity within and between species.
doi:10.1186/1471-2148-13-10
PMCID: PMC3556333  PMID: 23311925
Caenorhabditis; Species richness; Population structure; C. briggsae; Nucleotide diversity
6.  Chromosome-scale selective sweeps shape Caenorhabditis elegans genomic diversity 
Nature genetics  2012;44(3):285-290.
The nematode Caenorhabditis elegans is central to research in molecular, cell, and developmental biology, but nearly all of this research has been conducted on a single strain. Comparatively little is known about the population genomic and evolutionary history of this species. We characterized C. elegans genetic variation by high-throughput selective sequencing of a worldwide collection of 200 wild strains, identifying 41,188 single nucleotide polymorphisms. Unexpectedly, C. elegans genome variation is dominated by a set of commonly shared haplotypes on four of the six chromosomes, each spanning many megabases. Population-genetic modeling shows that this pattern was generated by chromosome-scale selective sweeps that have reduced variation worldwide; at least one of these sweeps likely occurred in the past few hundred years. These sweeps, which we hypothesize to be a result of human activity, have dramatically reshaped the global C. elegans population in the recent past.
doi:10.1038/ng.1050
PMCID: PMC3365839  PMID: 22286215
7.  Population dynamics and habitat sharing of natural populations of Caenorhabditis elegans and C. briggsae 
BMC Biology  2012;10:59.
Background
The nematode Caenorhabditis elegans is a major model organism in laboratory biology. Very little is known, however, about its ecology, including where it proliferates. In the past, C. elegans was mainly isolated from human-made compost heaps, where it was overwhelmingly found in the non-feeding dauer diapause stage.
Results
C. elegans and C. briggsae were found in large, proliferating populations in rotting plant material (fruits and stems) in several locations in mainland France. Both species were found to co-occur in samples isolated from a given plant species. Population counts spanned a range from one to more than 10,000 Caenorhabditis individuals on a single fruit or stem. Some populations with an intermediate census size (10 to 1,000) contained no dauer larvae at all, whereas larger populations always included some larvae in the pre-dauer or dauer stages. We report on associated micro-organisms, including pathogens. We systematically sampled a spatio-temporally structured set of rotting apples in an apple orchard in Orsay over four years. C. elegans and C. briggsae were abundantly found every year, but their temporal distributions did not coincide. C. briggsae was found alone in summer, whereas both species co-occurred in early fall and C. elegans was found alone in late fall. Competition experiments in the laboratory at different temperatures show that C. briggsae out-competes C. elegans at high temperatures, whereas C. elegans out-competes C. briggsae at lower temperatures.
Conclusions
C. elegans and C. briggsae proliferate in the same rotting vegetal substrates. In contrast to previous surveys of populations in compost heaps, we found fully proliferating populations with no dauer larvae. The temporal sharing of the habitat by the two species coincides with their temperature preference in the laboratory, with C. briggsae populations growing faster than C. elegans at higher temperatures, and vice at lower temperatures.
doi:10.1186/1741-7007-10-59
PMCID: PMC3414772  PMID: 22731941
8.  Quantitative variation in autocrine signaling and pathway crosstalk in the Caenorhabditis vulva network 
Current biology : CB  2011;21(7):527-538.
SUMMARY
Background
Biological networks experience quantitative change in response to environmental and evolutionary variation. Computational modeling allows exploration of network parameter space, corresponding to such variations. The intercellular signaling network underlying Caenorhabditis vulva development specifies three fates in a row of six precursor cells, yielding a quasi-invariant 3°-3°-2°-1°-2°-3° cell fate pattern. Two seemingly conflicting verbal models of vulval precursor cell fate specification have been proposed: sequential induction by the EGF-MAP kinase and Notch pathways, or morphogen-based induction by the former.
Results
To study the mechanistic and evolutionary system properties of this network, we combine experimental studies with computational modeling, using a model that keeps the network architecture constant but varies parameters. We first show that the Delta autocrine loop can play an essential role in 2° fate specification. With this autocrine loop, the same network topology can be quantitatively tuned to use in the six-cell row morphogen-based or sequential patterning mechanisms, which may act singly, cooperatively or redundantly. Moreover, different quantitative tunings of this same network can explain vulval patterning observed experimentally in C. elegans, C. briggsae, C. remanei and C. brenneri. We experimentally validate model predictions, such as interspecific differences in isolated vulva precursor cell behavior and in spatial regulation of Notch activity.
Conclusions
Our study illustrates how quantitative variation in the same network comprises developmental patterning modes that were considered qualitatively distinct and also accounts for evolution among closely related species.
doi:10.1016/j.cub.2011.02.040
PMCID: PMC3084603  PMID: 21458263
9.  Evolution of Susceptibility to Ingested Double-Stranded RNAs in Caenorhabditis Nematodes 
PLoS ONE  2012;7(1):e29811.
Background
The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs) and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus.
Principal Findings
We study the sensitivity of many new species of Caenorhabditis to ingested dsRNAs matching a conserved actin gene sequence from the nematode Oscheius tipulae. We find ample variation in the Caenorhabditis genus in the ability to mount an RNAi response. We map this sensitivity onto a phylogenetic tree, and show that sensitivity or insensitivity have evolved convergently several times. We uncover several evolutionary losses in sensitivity, which may have occurred through distinct mechanisms. We could render C. remanei and C. briggsae sensitive to ingested dsRNAs by transgenesis of the Cel-sid-2 gene. We thus provide tools for RNA interference studies in these species. We also show that transgenesis by injection is possible in many Caenorhabditis species.
Conclusions
The ability of animals to take up dsRNAs or to respond to them by gene inactivation is under rapid evolution in the Caenorhabditis genus. This study provides a framework and tools to use RNA interference and transgenesis in various Caenorhabditis species for further comparative and evolutionary studies.
doi:10.1371/journal.pone.0029811
PMCID: PMC3256175  PMID: 22253787
10.  Role of Pleiotropy in the Evolution of a Cryptic Developmental Variation in Caenorhabditis elegans 
PLoS Biology  2012;10(1):e1001230.
Using vulval phenotypes in Caenorhabditis elegans, the authors show that cryptic genetic variation can evolve through selection for pleiotropic effects that alter fitness, and identify a cryptic variant that has conferred enhanced fitness on domesticated worms under laboratory conditions.
Robust biological systems are expected to accumulate cryptic genetic variation that does not affect the system output in standard conditions yet may play an evolutionary role once phenotypically expressed under a strong perturbation. Genetic variation that is cryptic relative to a robust trait may accumulate neutrally as it does not change the phenotype, yet it could also evolve under selection if it affects traits related to fitness in addition to its cryptic effect. Cryptic variation affecting the vulval intercellular signaling network was previously uncovered among wild isolates of Caenorhabditis elegans. Using a quantitative genetic approach, we identify a non-synonymous polymorphism of the previously uncharacterized nath-10 gene that affects the vulval phenotype when the system is sensitized with different mutations, but not in wild-type strains. nath-10 is an essential protein acetyltransferase gene and the homolog of human NAT10. The nath-10 polymorphism also presents non-cryptic effects on life history traits. The nath-10 allele carried by the N2 reference strain leads to a subtle increase in the egg laying rate and in the total number of sperm, a trait affecting the trade-off between fertility and minimal generation time in hermaphrodite individuals. We show that this allele appeared during early laboratory culture of N2, which allowed us to test whether it may have evolved under selection in this novel environment. The derived allele indeed strongly outcompetes the ancestral allele in laboratory conditions. In conclusion, we identified the molecular nature of a cryptic genetic variation and characterized its evolutionary history. These results show that cryptic genetic variation does not necessarily accumulate neutrally at the whole-organism level, but may evolve through selection for pleiotropic effects that alter fitness. In addition, cultivation in the laboratory has led to adaptive evolution of the reference strain N2 to the laboratory environment, which may modify other phenotypes of interest.
Author Summary
Robustness is a property of biological systems that ensures the production of reproducible phenotypes in spite of underlying environmental, stochastic, and genetic variability. A consequence of robustness is that potentially functional genetic variation is free to accumulate in natural populations because it is buffered at the phenotypic level. Even if this so-called “cryptic” genetic variation has no obvious effects under standard conditions, it may become phenotypically expressed upon major genetic or environmental perturbations. Here we used the model organism Caenorhabditis elegans to identify genetic variations involved in the cryptic evolution of vulval cell fate induction between wild strains. We found that a mutation in the essential nath-10 gene not only contributes to cryptic genetic variation in the vulval system, but also affects key life history traits that are expected to be under a strong selective pressure (brood size, age at sexual maturity, sperm number and rate of progeny production). Indeed, an allele of nath-10 that emerged during the laboratory domestication of C. elegans about 50 years ago confers a strong competitive advantage over the ancestral allele under laboratory conditions. A genetic variation that is cryptic for a robust trait can therefore affect more sensitive phenotypes and thus evolve under selection.
doi:10.1371/journal.pbio.1001230
PMCID: PMC3250502  PMID: 22235190
11.  A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits 
Background
The nematode Caenorhabditis elegans is a major laboratory model in biology. Only ten Caenorhabditis species were available in culture at the onset of this study. Many of them, like C. elegans, were mostly isolated from artificial compost heaps, and their more natural habitat was unknown.
Results
Caenorhabditis nematodes were found to be proliferating in rotten fruits, flowers and stems. By collecting a large worldwide set of such samples, 16 new Caenorhabditis species were discovered. We performed mating tests to establish biological species status and found some instances of semi-fertile or sterile hybrid progeny. We established barcodes for all species using ITS2 rDNA sequences. By obtaining sequence data for two rRNA and nine protein-coding genes, we determined the likely phylogenetic relationships among the 26 species in culture. The new species are part of two well-resolved sister clades that we call the Elegans super-group and the Drosophilae super-group. We further scored phenotypic characters such as reproductive mode, mating behavior and male tail morphology, and discuss their congruence with the phylogeny. A small space between rays 2 and 3 evolved once in the stem species of the Elegans super-group; a narrow fan and spiral copulation evolved once in the stem species of C. angaria, C. sp. 8 and C. sp. 12. Several other character changes occurred convergently. For example, hermaphroditism evolved three times independently in C. elegans, C. briggsae and C. sp. 11. Several species can co-occur in the same location or even the same fruit. At the global level, some species have a cosmopolitan distribution: C. briggsae is particularly widespread, while C. elegans and C. remanei are found mostly or exclusively in temperate regions, and C. brenneri and C. sp. 11 exclusively in tropical zones. Other species have limited distributions, for example C. sp. 5 appears to be restricted to China, C. sp. 7 to West Africa and C. sp. 8 to the Eastern United States.
Conclusions
Caenorhabditis are "fruit worms", not soil nematodes. The 16 new species provide a resource and their phylogeny offers a framework for further studies into the evolution of genomic and phenotypic characters.
doi:10.1186/1471-2148-11-339
PMCID: PMC3277298  PMID: 22103856
12.  Evolution of Caenorhabditis Mitochondrial Genome Pseudogenes and Caenorhabditis briggsae Natural Isolates 
Molecular Biology and Evolution  2009;27(5):1087-1096.
Although most metazoan mitochondrial genomes are highly streamlined and encode little noncoding DNA outside of the “AT” region, the accumulation of mitochondrial pseudogenes and other types of noncoding DNA has been observed in a growing number of animal groups. The nematode species Caenorhabditis briggsae harbors two mitochondrial DNA (mtDNA) pseudogenes, named Ψnad5-1 and Ψnad5-2, presumably derived from the nad5 protein-coding gene. Here, we provide an in-depth analysis of mtDNA pseudogene evolution in C. briggsae natural isolates and related Caenorhabditis species. Mapping the observed presence and absence of the pseudogenes onto phylogenies suggests that Ψnad5-1 originated in the ancestor to C. briggsae and its recently discovered outcrossing relative species Caenorhabditis sp. 5 and Caenorhabditis sp. 9. However, Ψnad5-1 was not detected in Caenorhabditis sp. 9 natural isolates, suggesting a lineage-specific loss of this pseudogene in this species. Our results corroborated the previous finding that Ψnad5-2 originated within C. briggsae. The observed pattern of mitochondrial pseudogene gain and loss in Caenorhabditis was inconsistent with predictions of the tandem duplication–random loss model of mitochondrial genome evolution and suggests that intralineage recombination–like mechanisms might play a major role in Caenorhabditis mtDNA evolution. Natural variation was analyzed at the pseudogenes and flanking mtDNA sequences in 141 geographically diverse C. briggsae natural isolates. Although phylogenetic analysis placed the majority of isolates into the three previously established major intraspecific clades of C. briggsae, two new and unexpected haplotypes fell outside of these conventional groupings. Ψnad5-2 copy number variation was observed among C. briggsae isolates collected from the same geographic site. Patterns of nucleotide diversity were analyzed in Ψnad5-1 and Ψnad5-2, and confidence intervals were found to overlap values from synonymous sites in protein-coding genes, consistent with neutral expectations. Our findings provide new insights into the mode and tempo of mitochondrial genome and pseudogene evolution both within and between Caenorhabditis nematode species.
doi:10.1093/molbev/msp318
PMCID: PMC2857805  PMID: 20026478
Caenorhabditis; mitochondrial genome; nucleotide diversity; phylogenetics; pseudogene
13.  Natural and Experimental Infection of Caenorhabditis Nematodes by Novel Viruses Related to Nodaviruses 
PLoS Biology  2011;9(1):e1000586.
Novel viruses have been discovered in wild Caenorahbditis nematode isolates and can now be used to explore host antiviral pathways, nematode ecology, and host-pathogen co-evolution.
An ideal model system to study antiviral immunity and host-pathogen co-evolution would combine a genetically tractable small animal with a virus capable of naturally infecting the host organism. The use of C. elegans as a model to define host-viral interactions has been limited by the lack of viruses known to infect nematodes. From wild isolates of C. elegans and C. briggsae with unusual morphological phenotypes in intestinal cells, we identified two novel RNA viruses distantly related to known nodaviruses, one infecting specifically C. elegans (Orsay virus), the other C. briggsae (Santeuil virus). Bleaching of embryos cured infected cultures demonstrating that the viruses are neither stably integrated in the host genome nor transmitted vertically. 0.2 µm filtrates of the infected cultures could infect cured animals. Infected animals continuously maintained viral infection for 6 mo (∼50 generations), demonstrating that natural cycles of horizontal virus transmission were faithfully recapitulated in laboratory culture. In addition to infecting the natural C. elegans isolate, Orsay virus readily infected laboratory C. elegans mutants defective in RNAi and yielded higher levels of viral RNA and infection symptoms as compared to infection of the corresponding wild-type N2 strain. These results demonstrated a clear role for RNAi in the defense against this virus. Furthermore, different wild C. elegans isolates displayed differential susceptibility to infection by Orsay virus, thereby affording genetic approaches to defining antiviral loci. This discovery establishes a bona fide viral infection system to explore the natural ecology of nematodes, host-pathogen co-evolution, the evolution of small RNA responses, and innate antiviral mechanisms.
Author Summary
The nematode C. elegans is a robust model organism that is broadly used in biology. It also has great potential for the study of host-microbe interactions, as it is possible to systematically knockout almost every gene in high-throughput fashion to examine the potential role of each gene in infection. While C. elegans has been successfully applied to the study of bacterial infections, only limited studies of antiviral responses have been possible since no virus capable of infecting any Caenorhabditis nematode in laboratory culture has previously been described. Here we report the discovery of natural viruses infecting wild isolates of C. elegans and its relative C. briggsae. These novel viruses are most closely related to the ssRNA nodaviruses, but have larger genomes than other described nodaviruses and clearly represent a new taxon of virus. We were able to use these viruses to infect a variety of laboratory nematode strains. We show that mutant worms defective in the RNA interference pathway, an antiviral system known to operate in a number of organisms, accumulate more viral RNA than wild type strains. The discovery of these viruses will enable further studies of host-virus interactions in C. elegans and the identification of other host mechanisms that counter viral infection.
doi:10.1371/journal.pbio.1000586
PMCID: PMC3026760  PMID: 21283608
14.  Bias and Evolution of the Mutationally Accessible Phenotypic Space in a Developmental System 
PLoS Genetics  2010;6(3):e1000877.
Genetic and developmental architecture may bias the mutationally available phenotypic spectrum. Although such asymmetries in the introduction of variation may influence possible evolutionary trajectories, we lack quantitative characterization of biases in mutationally inducible phenotypic variation, their genotype-dependence, and their underlying molecular and developmental causes. Here we quantify the mutationally accessible phenotypic spectrum of the vulval developmental system using mutation accumulation (MA) lines derived from four wild isolates of the nematodes Caenorhabditis elegans and C. briggsae. The results confirm that on average, spontaneous mutations degrade developmental precision, with MA lines showing a low, yet consistently increased, proportion of developmental defects and variants. This result indicates strong purifying selection acting to maintain an invariant vulval phenotype. Both developmental system and genotype significantly bias the spectrum of mutationally inducible phenotypic variants. First, irrespective of genotype, there is a developmental bias, such that certain phenotypic variants are commonly induced by MA, while others are very rarely or never induced. Second, we found that both the degree and spectrum of mutationally accessible phenotypic variation are genotype-dependent. Overall, C. briggsae MA lines exhibited a two-fold higher decline in precision than the C. elegans MA lines. Moreover, the propensity to generate specific developmental variants depended on the genetic background. We show that such genotype-specific developmental biases are likely due to cryptic quantitative variation in activities of underlying molecular cascades. This analysis allowed us to identify the mutationally most sensitive elements of the vulval developmental system, which may indicate axes of potential evolutionary variation. Consistent with this scenario, we found that evolutionary trends in the vulval system concern the phenotypic characters that are most easily affected by mutation. This study provides an empirical assessment of developmental bias and the evolution of mutationally accessible phenotypes and supports the notion that such bias may influence the directions of evolutionary change.
Author Summary
Random mutation does not generate random phenotypic variation because genetic and developmental architecture may constrain and bias the mutationally inducible phenotypic spectrum. Understanding such biases in the introduction of phenotypic variation is thus essential to reveal which phenotypes can ultimately be explored and selected through evolution. Here we used lines which had accumulated spontaneous random mutation over 250 generations starting from four distinct wild isolates of the nematode species C. briggsae and C. elegans, to study how a developmental system—vulval cell fate patterning—responds to mutational perturbations. We show that developmental defects and variants increase upon mutation accumulation in lines derived from all four isolates. However, some mutationally induced phenotypic variants occur more frequently than others, and the degree and spectrum of developmental variation further differed between isolates. These results illustrate how the phenotypic spectrum induced by random mutation can be biased due to both developmental system features and variation in the genetic background. Moreover, the mutationally most sensitive phenotypic characters are the ones that show most evolutionary variation among closely related species. These observations show how random mutation translates into a biased, limited range of phenotypes—a phenomenon likely impacting possible trajectories of phenotypic evolution.
doi:10.1371/journal.pgen.1000877
PMCID: PMC2837400  PMID: 20300655
15.  Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans 
PLoS Biology  2008;6(12):e309.
For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.
Author Summary
The small roundworm Caenorhabditis elegans is an important model system for many areas of biology, but little is known about its natural ecology. We have identified an intracellular parasite from C. elegans in its natural habitat isolated near Paris and have named it Nematocida parisii, or nematode-killer from Paris. N. parisii defines a new genus and species of microsporidia. Microsporidia are ubiquitous eukaryotic pathogens that are thought to be highly reduced fungi and are emerging pathogens of humans. The microsporidian N. parisii invades and resides in C. elegans intestinal cells where it goes through a multistep life cycle and eventually escapes out of intestinal cells, leaving holes in the terminal web, an important cellular structure. We have found N. parisii and a related Nematocida species in several wild-caught roundworms, indicating that microsporidian infections may be relatively common for C. elegans in the wild. The C. elegans/N. parisii interaction provides a valuable system in which to study microsporidian infections in a whole animal, and a convenient and inexpensive system in which to screen for anti-microsporidian drugs.
A newly identified intracellular pathogen of wild-caughtCaenorhabditis elegans represents a new microsporidian species that will provide a model to study this class of pathogen in humans.
doi:10.1371/journal.pbio.0060309
PMCID: PMC2596862  PMID: 19071962
16.  RNA interference in nematodes and the chance that favored Sydney Brenner 
Journal of Biology  2008;7(9):34.
The efficiency of RNA interference varies between different organisms, even among nematodes. A recent report of successful RNA interference in the nematode Panagrolaimus superbus in BMC Molecular Biology has implications for the comparative study of the functional genomics of nematode species, and prompts reflections on the choice of Caenorhabditis elegans as a model organism.
doi:10.1186/jbiol97
PMCID: PMC2776389  PMID: 19014674
17.  Three Biological Species Closely Related to Rhabditis (Oscheius) pseudodolichura Körner in Osche, 1952 
Journal of Nematology  2001;33(2-3):104-109.
The Oscheius subgenus (Nematoda: Rhabditidae) comprises several common free-living hermaphroditic species. Morphological identification is difficult due to a lack of reliable characters to discriminate species. We studied 32 strains that are closely related to Rhabditis (Oscheius) pseudodolichura and R. (O.) tipulae. We present results from mating experiments between the strains and sequence data from the internal transcribed spacer region of ribosomal RNA, allowing discrimination of three closely related biological species.
PMCID: PMC2638136  PMID: 19266004
ITS; nematode; Oscheius; PCR-RFLP; systematics

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