Although the nematode Caenorhabditis elegans is a major model organism in diverse biological areas and well studied under laboratory conditions, little is known about its ecology. Therefore, characterization of the species’ natural habitats should provide a new perspective on its otherwise well-studied biology. The currently best characterized populations are in France, demonstrating that C. elegans prefers nutrient- and microorganism-rich substrates such as rotting fruits and decomposing plant matter. In order to extend these findings, we sampled C. elegans continuously across 1.5 years from rotting apples and compost heaps in three North German locations.
C. elegans was found throughout summer and autumn in both years. It shares its habitat with the related nematode species C. remanei, which could thus represent an important competitor for a similar ecological niche. The two species were isolated from the same site, but rarely the same substrate sample. In fact, C. elegans was mainly found on compost and C. remanei on rotten apples, possibly suggesting niche separation. The occurrence of C. elegans itself was related to environmental humidity and rain, although the correlation was significant for only one sampling site each. Additional associations between nematode prevalence and abiotic parameters could not be established.
Taken together, our findings vary from the previous results for French C. elegans populations in that the considered German populations always coexisted with the congeneric species C. remanei (rather than C. briggsae as in France) and that C. elegans prevalence can associate with humidity and rain (rather than temperature, as suggested for French populations). Consideration of additional locations and time points is thus essential for full appreciation of the nematode's natural ecology.
Caenorhabditis elegans; Caenorhabditis remanei; Niche separation; Competition
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.
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.
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.
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.
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.
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.
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.
Caenorhabditis; Species richness; Population structure; C. briggsae; Nucleotide diversity
The Caenorhabditis elegans dauer larva is a facultative state of diapause. Mutations affecting dauer signal transduction and morphogenesis have been reported. Of these, most that result in constitutive formation of dauer larvae are temperature-sensitive (ts). The daf-31 mutant was isolated in genetic screens looking for novel and underrepresented classes of mutants that form dauer and dauer-like larvae non-conditionally. Dauer-like larvae are arrested in development and have some, but not all, of the normal dauer characteristics. We show here that daf-31 mutants form dauer-like larvae under starvation conditions but are sensitive to SDS treatment. Moreover, metabolism is shifted to fat accumulation in daf-31 mutants. We cloned the daf-31 gene and it encodes an ortholog of the arrest-defective-1 protein (ARD1) that is the catalytic subunit of the major N alpha-acetyltransferase (NatA). A daf-31 promoter::GFP reporter gene indicates daf-31 is expressed in multiple tissues including neurons, pharynx, intestine and hypodermal cells. Interestingly, overexpression of daf-31 enhances the longevity phenotype of daf-2 mutants, which is dependent on the forkhead transcription factor (FOXO) DAF-16. We demonstrate that overexpression of daf-31 stimulates the transcriptional activity of DAF-16 without influencing its subcellular localization. These data reveal an essential role of NatA in controlling C. elegans life history and also a novel interaction between ARD1 and FOXO transcription factors, which may contribute to understanding the function of ARD1 in mammals.
The development of a living organism is influenced by the environmental conditions such as nutrient availability. Under starvation conditions, the C. elegans larvae will enter a special developmental stage called dauer larva. An insulin-like signaling pathway controls dauer formation as well as adult lifespan by inhibiting the activity of FOXO transcription factor DAF-16 that regulates expression of stress-resistant genes. Here we isolate a new gene called daf-31; this gene encodes a protein that regulates C. elegans larval development, metabolism and adult lifespan. This protein has been found in other species to be part of an enzyme that functions to modify other proteins. We show that overexpression of our newly discovered protein stimulates the transcriptional activity of DAF-16. Interestingly, abnormal regulation of human proteins similar to DAF-31 results in tumor formation. It is known that human FOXO proteins prevent tumorigenesis. Therefore, it is possible that abnormal DAF-31 activity may lead to tumor growth by reducing DAF-16 activity. Thus, the present study may not only contribute to understanding the role of a universal enzyme in controlling development, metabolism and lifespan in other organisms besides worms but may also shed light on the mechanisms of tumorigenesis in humans.
The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.
With the Caenorhabditis briggsae genome now in hand, C. elegans biologists have a powerful new research tool to refine their knowledge of gene function in C. elegans and to study the path of genome evolution
The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.
The nematode Caenorhabditis briggsae is increasingly used for comparisons with its more famous relative, C. elegans. To improve genomic resources for C. briggsae, we created two sets of inbred lines derived from crosses between diverged C. briggsae strains. High-throughput genotyping of these has improved the resolution of the recombination map and genome assembly. It also allows detailed comparisons of recombination both within and between species. Unexpectedly, we found that alleles from one parental strain were much more likely to be fixed on three of the six chromosomes in one of the sets of lines. One of these biases is caused by a pronounced developmental delay in F2 progeny that is seen in both reciprocal crosses, whereas the other two manifest in only one of the two cross directions. This indicates that the parental strains have diverged in both nuclear and nuclear-cytoplasmic interactions, either because of local adaptation or restricted gene flow across much of the genome.
The ascarosides form a family of small molecules that have been isolated from cultures of the nematode Caenorhabditis elegans. They are often referred to as “dauer pheromones” because most of them induce formation of long-lived and highly stress resistant dauer larvae. More recent studies have shown that ascarosides serve additional functions as social signals and mating pheromones. Thus, ascarosides have multiple functions. Until now, it has been generally assumed that ascarosides are constitutively expressed during nematode development.
Cultures of C. elegans were developmentally synchronized on controlled diets. Ascarosides released into the media, as well as stored internally, were quantified by LC/MS. We found that ascaroside biosynthesis and release were strongly dependent on developmental stage and diet. The male attracting pheromone was verified to be a blend of at least four ascarosides, and peak production of the two most potent mating pheromone components, ascr#3 and asc#8 immediately preceded or coincided with the temporal window for mating. The concentration of ascr#2 increased under starvation conditions and peaked during dauer formation, strongly supporting ascr#2 as the main population density signal (dauer pheromone). After dauer formation, ascaroside production largely ceased and dauer larvae did not release any ascarosides. These findings show that both total ascaroside production and the relative proportions of individual ascarosides strongly correlate with these compounds' stage-specific biological functions.
Ascaroside expression changes with development and environmental conditions. This is consistent with multiple functions of these signaling molecules. Knowledge of such differential regulation will make it possible to associate ascaroside production to gene expression profiles (transcript, protein or enzyme activity) and help to determine genetic pathways that control ascaroside biosynthesis. In conjunction with findings from previous studies, our results show that the pheromone system of C. elegans mimics that of insects in many ways, suggesting that pheromone signaling in C. elegans may exhibit functional homology also at the sensory level. In addition, our results provide a strong foundation for future behavioral modeling studies.
Mitochondrial DNA (mtDNA) mutations underlie a variety of human genetic disorders and are associated with the aging process. mtDNA polymorphisms are widely used in a variety of evolutionary applications. Although mtDNA mutation spectra are known to differ between distantly related model organisms, the extent to which mtDNA mutation processes vary between more closely related species and within species remains enigmatic. We analyzed mtDNA divergence in two sets of 250-generation Caenorhabditis briggsae mutation-accumulation (MA) lines, each derived from a different natural isolate progenitor: strain HK104 from Okayama, Japan, and strain PB800 from Ohio, United States. Both sets of C. briggsae MA lines accumulated numerous large heteroplasmic mtDNA deletions, whereas only one similar event was observed in a previous analysis of Caenorhabditis elegans MA line mtDNA. Homopolymer length change mutations were frequent in both sets of C. briggsae MA lines and occurred in both intergenic and protein-coding gene regions. The spectrum of C. briggsae mtDNA base substitution mutations differed from the spectrum previously observed in C. elegans. In C. briggsae, the HK104 MA lines experienced many different base substitution types, whereas the PB800 lines displayed only C:G → T:A transitions, although the difference was not significant. Over half of the mtDNA base substitutions detected in the C. briggsae MA lines were in a heteroplasmic state, whereas all those previously characterized in C. elegans MA line mtDNA were fixed changes, indicating a narrower mtDNA bottleneck in C. elegans as compared with C. briggsae. Our results show that C. briggsae mtDNA is highly susceptible to large deletions and that the mitochondrial mutation process varies between Caenorhabditis nematode species.
bottleneck; heteroplasmy; mutation-accumulation line; nematode
The infectious form of many parasitic nematodes, which afflict over one billion people globally, is a developmentally arrested third-stage larva (L3i). The parasitic nematode Strongyloides stercoralis differs from other nematode species that infect humans, in that its life cycle includes both parasitic and free-living forms, which can be leveraged to investigate the mechanisms of L3i arrest and activation. The free-living nematode Caenorhabditis elegans has a similar developmentally arrested larval form, the dauer, whose formation is controlled by four pathways: cyclic GMP (cGMP) signaling, insulin/IGF-1-like signaling (IIS), transforming growth factor β (TGFβ) signaling, and biosynthesis of dafachronic acid (DA) ligands that regulate a nuclear hormone receptor. We hypothesized that homologous pathways are present in S. stercoralis, have similar developmental regulation, and are involved in L3i arrest and activation. To test this, we undertook a deep-sequencing study of the polyadenylated transcriptome, generating over 2.3 billion paired-end reads from seven developmental stages. We constructed developmental expression profiles for S. stercoralis homologs of C. elegans dauer genes identified by BLAST searches of the S. stercoralis genome as well as de novo assembled transcripts. Intriguingly, genes encoding cGMP pathway components were coordinately up-regulated in L3i. In comparison to C. elegans, S. stercoralis has a paucity of genes encoding IIS ligands, several of which have abundance profiles suggesting involvement in L3i development. We also identified seven S. stercoralis genes encoding homologs of the single C. elegans dauer regulatory TGFβ ligand, three of which are only expressed in L3i. Putative DA biosynthetic genes did not appear to be coordinately regulated in L3i development. Our data suggest that while dauer pathway genes are present in S. stercoralis and may play a role in L3i development, there are significant differences between the two species. Understanding the mechanisms governing L3i development may lead to novel treatment and control strategies.
Parasitic nematodes infect over one billion people worldwide and cause many diseases, including strongyloidiasis, filariasis, and hookworm disease. For many of these parasites, including Strongyloides stercoralis, the infectious form is a developmentally arrested and long-lived thirdstage larva (L3i). Upon encountering a host, L3i quickly resume development and mature into parasitic adults. In the free-living nematode Caenorhabditis elegans, a similar developmentally arrested third-stage larva, known as the dauer, is regulated by four key cellular mechanisms. We hypothesized that similar cellular mechanisms control L3i arrest and activation. Therefore, we used deep-sequencing technology to characterize the S. stercoralis transcriptome (RNAseq), which allowed us to identify S. stercoralis homologs of components of these four mechanisms and examine their temporal regulation. We found similar temporal regulation between S. stercoralis and C. elegans for components of two mechanisms, but dissimilar temporal regulation for two others, suggesting conserved as well as novel modes of developmental regulation for L3i. Understanding L3i development may lead to novel control strategies as well as new treatments for strongyloidiasis and other diseases caused by parasitic nematodes.
Accurate identification of synteny blocks is an important step in comparative genomics towards the understanding of genome architecture and expression. Most computer programs developed in the last decade for identifying synteny blocks have limitations. To address these limitations, we recently developed a robust program called OrthoCluster, and an online database OrthoClusterDB. In this work, we have demonstrated the application of OrthoCluster in identifying synteny blocks between the genomes of Caenorhabditis elegans and Caenorhabditis briggsae, two closely related hermaphrodite nematodes.
Initial identification and analysis of synteny blocks using OrthoCluster enabled us to systematically improve the genome annotation of C. elegans and C. briggsae, identifying 52 potential novel genes in C. elegans, 582 in C. briggsae, and 949 novel orthologous relationships between these two species. Using the improved annotation, we have detected 3,058 perfect synteny blocks that contain no mismatches between C. elegans and C. briggsae. Among these synteny blocks, the majority are mapped to homologous chromosomes, as previously reported. The largest perfect synteny block contains 42 genes, which spans 201.2 kb in Chromosome V of C. elegans. On average, perfect synteny blocks span 18.8 kb in length. When some mismatches (interruptions) are allowed, synteny blocks ("imperfect synteny blocks") that are much larger in size are identified. We have shown that the majority (80%) of the C. elegans and C. briggsae genomes are covered by imperfect synteny blocks. The largest imperfect synteny block spans 6.14 Mb in Chromosome X of C. elegans and there are 11 synteny blocks that are larger than 1 Mb in size. On average, imperfect synteny blocks span 63.6 kb in length, larger than previously reported.
We have demonstrated that OrthoCluster can be used to accurately identify synteny blocks and have found that synteny blocks between C. elegans and C. briggsae are almost three-folds larger than previously identified.
To determine whether the distinctive features of Caenorhabditis elegans chromosomal organization are shared with the C. briggsae genome, we constructed a single nucleotide polymorphism–based genetic map to order and orient the whole genome shotgun assembly along the six C. briggsae chromosomes. Although these species are of the same genus, their most recent common ancestor existed 80–110 million years ago, and thus they are more evolutionarily distant than, for example, human and mouse. We found that, like C. elegans chromosomes, C. briggsae chromosomes exhibit high levels of recombination on the arms along with higher repeat density, a higher fraction of intronic sequence, and a lower fraction of exonic sequence compared with chromosome centers. Despite extensive intrachromosomal rearrangements, 1:1 orthologs tend to remain in the same region of the chromosome, and colinear blocks of orthologs tend to be longer in chromosome centers compared with arms. More strikingly, the two species show an almost complete conservation of synteny, with 1:1 orthologs present on a single chromosome in one species also found on a single chromosome in the other. The conservation of both chromosomal organization and synteny between these two distantly related species suggests roles for chromosome organization in the fitness of an organism that are only poorly understood presently.
The importance of chromosomal organization in the fitness of a species is only poorly understood. The publication of the C. elegans genome sequence in 1998 revealed features of higher level organization that suggested its chromosomes were organized into distinct domains. Chromosome arms were accumulating changes more rapidly than the centers of chromosomes. In this paper, we have compared the organization of the nematode C. briggsae genome with that of C. elegans. By building a genetic map based on DNA variations between two strains of C. briggsae, and by using that map to organize the draft genome sequence of C. briggsae published in 2003, we found the following: (1) Intrachromosomal rearrangements are frequent within and even between arms but are less common within central regions and between arms and centers. (2) Genes have remained overwhelmingly on the same chromosomes. (3) The distinctive features that distinguish C. elegans arms from centers also are seen in C. briggsae chromosomes. The conservation of these features between these two species, despite the approximately 100 million years since their most recent common ancestor, provides clear evidence of the selective advantages of the domain architecture of chromosomes. The continuing association of genes on the same chromosomes suggests that this may also be advantageous.
The conservation of both chromosomal organization and synteny between two distantly related species suggests roles for chromosome organization in the fitness of an organism.
Organisms live in environments that vary. For life-history traits that vary across environments, fitness will be maximised when the phenotype is appropriately matched to the environmental conditions. For the free-living nematode Caenorhabditis elegans, we have investigated how two major life-history traits, (i) the development of environmentally resistant dauer larvae and (ii) reproduction, respond to environmental stress (high population density and low food availability), and how these traits vary between lines and the genetic basis of this variation.
We found that lines of C. elegans vary in their phenotypic plasticity of dauer larva development, i.e. there is variation in the likelihood of developing into a dauer larva for the same environmental change. There was also variation in how lifetime fecundity and the rate of reproduction changed under conditions of environmental stress. These traits were related, such that lines that are highly plastic for dauer larva development also maintain a high population growth rate when stressed. We identified quantitative trait loci (QTL) on two chromosomes that control the dauer larva development and population size phenotypes. The QTLs affecting the dauer larva development and population size phenotypes on chromosome II are closely linked, but are genetically separable. This chromosome II QTL controlling dauer larva development does not encompass any loci previously identified to control dauer larva development. This chromosome II region contains many predicted 7-transmembrane receptors. Such proteins are often involved in information transduction, which is clearly relevant to the control of dauer larva development.
C. elegans alters both its larval development and adult reproductive strategy in response to environmental stress. Together the phenotypic and genotypic data suggest that these two major life-history traits are co-ordinated responses to environmental stress and that they are, at least in part, controlled by the same genomic regions.
Third-stage larvae (L3) of the canine hookworm, Ancylostoma caninum, undergo arrested development preceding transmission to a host. Many of the mRNAs up-regulated at this stage are likely to encode proteins that facilitate the transition from a free-living to a parasitic larva. The initial phase of mammalian host invasion by A. caninum L3 (herein termed “activation”) can be mimicked in vitro by culturing L3 in serum-containing medium.
The mRNAs differentially transcribed between activated and non-activated L3 were identified by suppression subtractive hybridisation (SSH). The analysis of these mRNAs on a custom oligonucleotide microarray printed with the SSH expressed sequence tags (ESTs) and publicly available A. caninum ESTs (non-subtracted) yielded 602 differentially expressed mRNAs, of which the most highly represented sequences encoded members of the pathogenesis-related protein (PRP) superfamily and proteases. Comparison of these A. caninum mRNAs with those of Caenorhabditis elegans larvae exiting from developmental (dauer) arrest demonstrated unexpectedly large differences in gene ontology profiles. C. elegans dauer exiting L3 up-regulated expression of mostly intracellular molecules involved in growth and development. Such mRNAs are virtually absent from activated hookworm larvae, and instead are over-represented by mRNAs encoding extracellular proteins with putative roles in host-parasite interactions.
Although this should not invalidate C. elegans dauer exit as a model for hookworm activation, it highlights the limitations of this free-living nematode as a model organism for the transition of nematode larvae from a free-living to a parasitic state.
Hookworms are soil-transmitted nematodes that parasitize hundreds of millions of people in developing countries. Here we describe the genes expressed when hookworm larvae make the transition from a developmentally arrested free-living form to a tissue-penetrating parasitic stage. Ancylostoma caninum can be “tricked” into thinking it has penetrated host skin by incubating free-living larvae in host serum – this is called “activation”. To comprehensively identify genes involved in activation, we used suppressive subtractive hybridization to clone genes that were up- or down-regulated in activated larvae, with a particular focus on up-regulated genes. The subtracted genes, as well as randomly sequenced (non-subtracted) genes from public databases were then printed on a microarray to further explore differential expression. We compared predicted gene functions between activated hookworms and the free-living nematode, Caenorhabditis elegans, exiting developmental arrest (dauer), and found enormous differences in the types of genes expressed. Genes encoding secreted proteins involved in parasitism were over-represented in activated hookworms whereas genes involved in growth and development dominated in C. elegans exiting dauer. Our data implies that C. elegans dauer exit is not a reliable model for exit from developmental arrest of hookworm larvae. Many of these genes likely play critical roles in host-parasite interactions, and are therefore worthy of pursuit for vaccine and drug development.
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.
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.
Infective third-stage larvae (L3i) of the human parasite Strongyloides stercoralis share many morphological, developmental, and behavioral attributes with Caenorhabditis elegans dauer larvae. The ‘dauer hypothesis’ predicts that the same molecular genetic mechanisms control both dauer larval development in C. elegans and L3i morphogenesis in S. stercoralis. In C. elegans, the phosphatidylinositol-3 (PI3) kinase catalytic subunit AGE-1 functions in the insulin/IGF-1 signaling (IIS) pathway to regulate formation of dauer larvae. Here we identify and characterize Ss-age-1, the S. stercoralis homolog of the gene encoding C. elegans AGE-1. Our analysis of the Ss-age-1 genomic region revealed three exons encoding a predicted protein of 1,209 amino acids, which clustered with C. elegans AGE-1 in phylogenetic analysis. We examined temporal patterns of expression in the S. stercoralis life cycle by reverse transcription quantitative PCR and observed low levels of Ss-age-1 transcripts in all stages. To compare anatomical patterns of expression between the two species, we used Ss-age-1 or Ce-age-1 promoter::enhanced green fluorescent protein reporter constructs expressed in transgenic animals for each species. We observed conservation of expression in amphidial neurons, which play a critical role in developmental regulation of both dauer larvae and L3i. Application of the PI3 kinase inhibitor LY294002 suppressed L3i in vitro activation in a dose-dependent fashion, with 100 µM resulting in a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08–0.13) in the odds of resumption of feeding for treated L3i in comparison to the control. Together, these data support the hypothesis that Ss-age-1 regulates the development of S. stercoralis L3i via an IIS pathway in a manner similar to that observed in C. elegans dauer larvae. Understanding the mechanisms by which infective larvae are formed and activated may lead to novel control measures and treatments for strongyloidiasis and other soil-transmitted helminthiases.
Animals use environmental information to make developmental decisions to maximise their fitness. The nematode Caenorhabditis elegans measures its environment to decide between arresting development as dauer larvae or continuing to grow and reproduce. Worms are thought to use ascarosides as signals of population density and this signalling is thought to be a species-wide honest signal. We compared recently wild C. elegans lines’ dauer larva arrest when presented with the same ascaroside signals and in different food environments.
We find that the hitherto canonical dauer larva response does not hold among these lines. Ascaroside molecules can, depending on the food environment, both promote and repress dauer larva formation. Further, these recently wild C. elegans lines also produce ascaroside mixtures that induce a wide diversity of dauer larva formation responses. We further find that the lines differ in the quantity and ratios of ascaroside molecules that they release. Some of the dauer larva formation responses are consistent with dishonest signalling.
Together, the results suggest that the idea that dauer larva formation is an honestly-signalled C. elegans-wide effect does not hold. Rather, the results suggest that ascaroside-based signalling is a public broadcast information system, but where the correct interpretation of that information depends on the worms’ context, and is a system open to dishonest signalling.
C. elegans; Dauer; Arrest; Ascaroside; Signalling
Evolution can follow predictable genetic trajectories1, indicating that discrete environmental shifts can select for reproducible genetic changes2-4. Conspecific individuals are an important feature of an animal's environment, and a potential source of selective pressures. We show here that adaptation of two Caenorhabditis species to growth at high density, a feature common to domestic environments, occurs by reproducible genetic changes to pheromone receptor genes. Chemical communication through pheromones that accumulate during high-density growth causes young nematode larvae to enter the long-lived but non-reproductive dauer stage. Two strains of Caenorhabditis elegans grown at high density have independently acquired multigenic resistance to pheromone-induced dauer formation. In each strain, resistance to the pheromone ascaroside C3 results from a deletion that disrupts the adjacent chemoreceptor genes serpentine receptor class g (srg)-36 and -37. Through misexpression experiments, we show that these genes encode redundant G protein-coupled receptors for ascaroside C3. Multigenic resistance to dauer formation has also arisen in high-density cultures of a different nematode species, Caenorhabditis briggsae, resulting in part from deletion of an srg gene paralogous to srg-36 and srg-37. These results demonstrate rapid remodeling of the chemoreceptor repertoire as an adaptation to specific environments, and indicate that parallel changes to a common genetic substrate can affect life history traits across species.
A dual mechanism regulates the developmental fate choice in C. elegans in response to population density: variation of the threshold of DA hormone required to commit to a certain fate and a positive feedback loop that amplifies this hormonal signal to ensure an organism-wide developmental fate choice.
Many animals can choose between different developmental fates to maximize fitness. Despite the complexity of environmental cues and life history, different developmental fates are executed in a robust fashion. The nematode Caenorhabditis elegans serves as a powerful model to examine this phenomenon because it can adopt one of two developmental fates (adulthood or diapause) depending on environmental conditions. The steroid hormone dafachronic acid (DA) directs development to adulthood by regulating the transcriptional activity of the nuclear hormone receptor DAF-12. The known role of DA suggests that it may be the molecular mediator of environmental condition effects on the developmental fate decision, although the mechanism is yet unknown. We used a combination of physiological and molecular biology techniques to demonstrate that commitment to reproductive adult development occurs when DA levels, produced in the neuroendocrine XXX cells, exceed a threshold. Furthermore, imaging and cell ablation experiments demonstrate that the XXX cells act as a source of DA, which, upon commitment to adult development, is amplified and propagated in the epidermis in a DAF-12 dependent manner. This positive feedback loop increases DA levels and drives adult programs in the gonad and epidermis, thus conferring the irreversibility of the decision. We show that the positive feedback loop canalizes development by ensuring that sufficient amounts of DA are dispersed throughout the body and serves as a robust fate-locking mechanism to enforce an organism-wide binary decision, despite noisy and complex environmental cues. These mechanisms are not only relevant to C. elegans but may be extended to other hormonal-based decision-making mechanisms in insects and mammals.
During development, many animals choose between mutually exclusive fates, such as workers, soldiers, or queens in bees or ants. The choice between states is uniform throughout the animal since mixtures of these fates are not observed in the wild. The nematode Caenorhabditis elegans larvae integrate environmental conditions and have two choices: mature into reproductive adults or arrest development as dauer larvae—a latent form that can survive harsh conditions. The decision between both fates is governed by the hormone dafachronic acid (DA), however its regulation during development in response to environmental conditions has been unclear. In this study we show how two mechanisms are responsible for the integration of environmental conditions and the coordination of the decision between many tissues. We first show that a threshold mechanism integrates population density with the internal amount of DA made in the head. A normal population density has a low threshold of DA needed for worms to become adults, whereas a high population density increases this threshold and leads worms to develop into dauer larvae. We then show that the low levels of DA released from the head are amplified in the hypodermis (the main body syncytial epithelium) via a positive feedback loop, coordinating the decision over the animal. Disruption of this positive feedback yields abnormal adults. We propose that the positive feedback serves as a fate-locking mechanism enforcing an organismal binary decision—either adult or dauer—despite noisy and uncertain environmental conditions.
In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-β and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation) were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis), but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia), implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT) (required to build cilia) is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.
C. elegans mutants that either fail to form or arrest development as dauer larvae, a stress-resistant lifestage, usually have defects in genes involved in evolutionarily conserved signaling pathways. In this study, we identified the gene mutated in daf-25 mutant strains, which inappropriately arrest as dauer larvae and are also defective in the sense of smell. The mammalian counterpart of DAF-25 is Ankmy2, a protein of unknown function that contains three ankyrin repeats and a zinc finger MYND domain, both of which are predicted to bind other protein(s). We show that DAF-25/Ankmy2 is required for the proper localization of a membrane-bound guanylyl cyclase—a class of protein that functions in cyclic GMP signaling—to cilia, which are conserved sensory organelles. We further demonstrate that mammalian Ankmy2 binds the retinal guanylyl cyclase GC1, suggesting a role for Ankmy2 in vision—which critically depends on cyclic GMP signal transduction—suggesting the potential involvement of Ankmy2 in human retinal disease, as well as other cilia-related diseases such as obesity.
Mutation rate often increases with environmental temperature, but establishing causality is complicated. Asymmetry between physiological stress and deviation from the optimal temperature means that temperature and stress are often confounded. We allowed mutations to accumulate in two species of Caenorhabditis for approximately 100 generations at 18°C and for approximately 165 generations at 26°C; 26°C is stressful for Caenorhabditis elegans but not for Caenorhabditis briggsae. We report mutation rates at a set of microsatellite loci and estimates of the per-generation decay of fitness (ΔMw), the genomic mutation rate for fitness (U) and the average effect of a new mutation (E[a]), assayed at both temperatures. In C. elegans, the microsatellite mutation rate is significantly greater at 26°C than at 18°C whereas in C. briggsae there is only a slight, non-significant increase in mutation rate at 26°C, consistent with stress-dependent mutation in C. elegans. The fitness data from both species qualitatively reinforce the microsatellite results. The fitness results of C. elegans are potentially complicated by selection but also suggest temperature-dependent mutation; the difference between the two species suggests that physiological stress plays a significant role in the mutational process.
mutation accumulation; fitness; microsatellite; metabolic rate
Caenorhabditis elegans is a preeminent model organism, but the natural ecology of this nematode has been elusive. A four-year survey of French orchards published in BMC Biology reveals thriving populations of C. elegans (and Caenorhabditis briggsae) in rotting fruit and plant stems. Rather than being simply a 'soil nematode', C. elegans appears to be a 'plant-rot nematode'. These studies signal a growing interest in the integrated genomics and ecology of these tractable animals.
See research article http://www.biomedcentral.com/1741-7007/10/59
Parasitic hookworms and the free-living model nematode Caenorhabtidis elegans share a developmental arrested stage, called the dauer stage in C. elegans and the infective third-stage larva (L3) in hookworms. One of the key transcription factors that regulate entrance to and exit from developmental arrest is the forkhead transcription factor DAF-16/FoxO. During the dauer stage, DAF-16 is activated and localized in the nucleus. DAF-16 is negatively regulated by phosphorylation by the upstream kinase AKT, which causes DAF-16 to localize out of the nucleus and the worm to exit from dauer. DAF-16 is conserved in hookworms, and hypothesized to control recovery from L3 arrest during infection. Lacking reverse genetic techniques for use in hookworms, we used C. elegans complementation assays to investigate the function of Ancylostoma caninum DAF-16 during entrance and exit from L3 developmental arrest. We performed dauer switching assays and observed the restoration of the dauer phenotype when Ac-DAF-16 was expressed in temperature-sensitive dauer defective C. elegans daf-2(e1370);daf-16(mu86) mutants. AKT phosphorylation site mutants of Ac-DAF-16 were also able to restore the dauer phenotype, but surprisingly allowed dauer exit when temperatures were lowered. We used fluorescence microscopy to localize DAF-16 during dauer and exit from dauer in C. elegans DAF-16 mutant worms expressing Ac-DAF-16, and found that Ac-DAF-16 exited the nucleus during dauer exit. Surprisingly, Ac-DAF-16 with mutated AKT phosphorylation sites also exited the nucleus during dauer exit. Our results suggest that another mechanism may be involved in the regulation DAF-16 nuclear localization during recovery from developmental arrest.
In the nematode Caenorhabditis elegans, the appropriate induction of dauer larvae development within growing populations is likely to be a primary determinant of genotypic fitness. The underlying genetic architecture of natural genetic variation in dauer formation has, however, not been thoroughly investigated. Here, we report extensive natural genetic variation in dauer larvae development within growing populations across multiple wild isolates. Moreover, bin mapping of introgression lines (ILs) derived from the genetically divergent isolates N2 and CB4856 reveals 10 quantitative trait loci (QTLs) affecting dauer formation. Comparison of individual ILs to N2 identifies an additional eight QTLs, and sequential IL analysis reveals six more QTLs. Our results also show that a behavioural, laboratory-derived, mutation controlled by the neuropeptide Y receptor homolog npr-1 can affect dauer larvae development in growing populations. These findings illustrate the complex genetic architecture of variation in dauer larvae formation in C. elegans and may help to understand how the control of variation in dauer larvae development has evolved.
Caenorhabditis elegans; dauer larvae; npr-1; QTL mapping; complex traits; introgression lines
Somatic and germline sex determination pathways have diverged significantly in animals, making comparisons between taxa difficult. To overcome this difficulty, we compared the genes in the germline sex determination pathways of Caenorhabditis elegans and C. briggsae, two Caenorhabditis species with similar reproductive systems and sequenced genomes. We demonstrate that C. briggsae has orthologs of all known C. elegans sex determination genes with one exception: fog-2. Hermaphroditic nematodes are essentially females that produce sperm early in life, which they use for self fertilization. In C. elegans, this brief period of spermatogenesis requires FOG-2 and the RNA-binding protein GLD-1, which together repress translation of the tra-2 mRNA. FOG-2 is part of a large C. elegans FOG-2-related protein family defined by the presence of an F-box and Duf38/FOG-2 homogy domain. A fog-2-related gene family is also present in C. briggsae, however, the branch containing fog-2 appears to have arisen relatively recently in C. elegans, post-speciation. The C-terminus of FOG-2 is rapidly evolving, is required for GLD-1 interaction, and is likely critical for the role of FOG-2 in sex determination. In addition, C. briggsae gld-1 appears to play the opposite role in sex determination (promoting the female fate) while maintaining conserved roles in meiotic progression during oogenesis. Our data indicate that the regulation of the hermaphrodite germline sex determination pathway at the level of FOG-2/GLD-1/tra-2 mRNA is fundamentally different between C. elegans and C. briggsae, providing functional evidence in support of the independent evolution of self-fertile hermaphroditism. We speculate on the convergent evolution of hermaphroditism in Caenorhabditis based on the plasticity of the C. elegans germline sex determination cascade, in which multiple mutant paths yield self fertility.
A comparison of sex determination genes in C. elegans and C. briggsae provides evidence in support of the convergent evolution of self-fertile hermaphroditism in the Caenorhabditis clade
Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions, Caenorhabditis elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, Pristionchus pacificus, and Strongyloides ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs, and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer versus nondauer stages and infective larvae versus free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved “dauer-infective” expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest and long-term survival in free-living and parasitic nematodes.
microRNA; nematodes; dauer larvae; post-transcriptional regulation; parasites