The genomic basis of Red Queen dynamics during rapid reciprocal host-pathogen coevolution.

Red Queen dynamics, involving coevolutionary interactions between species, are ubiquitous, shaping the evolution of diverse biological systems. To date, information on the underlying selection dynamics and the involved genome regions is mainly available for bacteria-phage systems or only one of the antagonists of a eukaryotic host-pathogen interaction. We add to our understanding of these important coevolutionary interactions using an experimental host-pathogen model, which includes the nematode Caenorhabditis elegans and its pathogen Bacillus thuringiensis We combined experimental evolution with time-shift experiments, in which a focal host or pathogen is tested against a coevolved antagonist from the past, present, or future, followed by genomic analysis. We show that (i) coevolution occurs rapidly within few generations, (ii) temporal coadaptation at the phenotypic level is found in parallel across replicate populations, consistent with antagonistic frequency-dependent selection, (iii) genomic changes in the pathogen match the phenotypic pattern and include copy number variations of a toxin-encoding plasmid, and (iv) host genomic changes do not match the phenotypic pattern and likely involve selective responses at more than one locus. By exploring the dynamics of coevolution at the phenotypic and genomic level for both host and pathogen simultaneously, our findings demonstrate a more complex model of the Red Queen, consisting of distinct selective processes acting on the two antagonists during rapid and reciprocal coadaptation.

Cytoplasmic-Nuclear Incompatibility Between Wild Isolates of Caenorhabditis nouraguensis.

How species arise is a fundamental question in biology. Species can be defined as populations of interbreeding individuals that are reproductively isolated from other such populations. Therefore, understanding how reproductive barriers evolve between populations is essential for understanding the process of speciation. Hybrid incompatibility (for example, hybrid sterility or lethality) is a common and strong reproductive barrier in nature. Here we report a lethal incompatibility between two wild isolates of the nematode Caenorhabditis nouraguensis Hybrid inviability results from the incompatibility between a maternally inherited cytoplasmic factor from each strain and a recessive nuclear locus from the other. We have excluded the possibility that maternally inherited endosymbiotic bacteria cause the incompatibility by treating both strains with tetracycline and show that hybrid death is unaffected. Furthermore, cytoplasmic-nuclear incompatibility commonly occurs between other wild isolates, indicating that this is a significant reproductive barrier within C. nouraguensis We hypothesize that the maternally inherited cytoplasmic factor is the mitochondrial genome and that mitochondrial dysfunction underlies hybrid death. This system has the potential to shed light on the dynamics of divergent mitochondrial-nuclear coevolution and its role in promoting speciation.

Leucobacter musarum subsp. musarum sp. nov., subsp. nov., Leucobacter musarum subsp. japonicus subsp. nov., and Leucobacter celer subsp. astrifaciens subsp. nov., three nematopathogenic bacteria isolated from Caenorhabditis, with an emended description of Leucobacter celer.

Three Gram-stain-positive, irregular-rod-shaped, non-motile, non-spore-forming bacteria were isolated from nematodes collected from Santa Antao, Cabo Verde (CBX151T, CBX152T) and Kakegawa, Japan (CBX130T). Based on 16S rRNA gene sequence similarity, strains CBX130T, CBX151T and CBX152T were shown to belong to the genus Leucobacter. This affiliation was supported by chemotaxonomic data (2,4-diaminobutyric acid in the cell wall; major respiratory quinones MK-10 and MK-11; major polar lipids phosphatidylglycerol and diphosphatidylglycerol; major fatty acids anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0). Strains CBX130T and CBX152T were found to share salient characteristics. Based on morphological, physiological, chemotaxonomic and biochemical analysis, strain CBX152T represents a novel species of the genus Leucobacter, for which the name Leucobacter musarum sp. nov. (type strain CBX152T = DSM 27160T = CIP 110721T) is proposed. Two subspecies of Leucobacter musarum sp. nov. are proposed: Leucobacter musarum sp. nov. subsp. musarum subsp. nov. (type strain CBX152T = DSM 27160T = CIP 110721T) and Leucobacter musarum sp. nov. subsp. japonicus subsp. nov. (type strain CBX130T = DSM 27158T = CIP 110719T). The third novel strain, CBX151T, showed genetic similarities with Leucobacter celer NAL101T indicating that these strains belong to the same species. Based on morphological, physiological, chemotaxonomic and biochemical differences it is proposed to split the species Leucobacter celer into two novel subspecies, Leucobacter celer subsp. celer subsp. nov. (type strain NAL101T = KACC 14220T = JCM 16465T) and Leucobacter celer subsp. astrifaciens subsp. nov. (type strain CBX151T = DSM 27159T = CIP 110720T), and to emend the description of Leucobacter celerShin et al. 2011.

Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae.

BACKGROUND: Entomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity. RESULTS: We report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99% sequence identity in rDNA sequence and orthology across 85.6% of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8%) were present in Serratia while 33 (84.6%) and 35 (89%) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively. CONCLUSION: The majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are enriched in putative functions that are biologically relevant to an entomopathogenic lifestyle, including non-ribosomal peptide synthetases, bacteriocins, fimbrial biogenesis, ushering proteins, toxins, secondary metabolite secretion and multiple drug resistance/efflux systems. By revealing the early stages of adaptation to this lifestyle, the Serratia sp. SCBI genome underscores the fact that in EPN formation the composite end result - killing, bioconversion, cadaver protection and recolonization- can be achieved by dissimilar mechanisms. This genome sequence will enable further study of the evolution of entomopathogenic nematode-bacteria complexes.

Effect of the bacterium Serratia marcescens SCBI on the longevity and reproduction of the nematode Caenorhabditis briggsae KT0001.

BACKGROUND: Extensive research effort has advanced our understanding of Caenorhabditis as a model system, but its natural association with bacteria remains to be explored in an ecological context. Explored associations vary vastly from mutualistic to parasitic. Serratia marcescens has been shown to be pathogenic to Caenorhabditis with a fitness cost. The recent isolation of an entomopathogenic Caenorhabditis briggsae KT0001/S. marcescens SCBI association from the wild has allowed us to examine under laboratory conditions whether such an association poses a serious cost to Caenorhabditis as previously surmised for other Serratia. RESULTS: A fecundity table of Caenorhabditis briggsae KT0001 fed on S. marcescens SCBI and the control fed on E. coli OP50 is presented. We found no significant difference in survivorship or total fecundity between the S. marcescens SCBI fed and E. coli OP50 fed Caenorhabditis briggsae KT0001. Only the mean onset of reproduction was significantly different between the two groups with E. coli fed C. briggsae maturing earlier (2.12 days) than those fed on Serratia (2.42 days). CONCLUSION: S. marcescens SCBI is not highly pathogenic to C. briggsae KT0001 indicating that the entomopathogenicity reported for this association may be beneficial for both the nematode and bacteria. In light of the fact that hitherto conducted experimental tests conform to widely held view that Serratia are highly pathogenic to Caenorhabditis, the absence of a high fitness cost for C. briggsae we report here may indicate that this entomopathogenic association is non-transient suggesting nematode/bacterial associations in the wild may vary greatly. Consequently, broad generalizations about nematode/bacterial associations should be interpreted with care.

Microsporidian infection in a free-living marine nematode.

Microsporidia are unicellular fungi that are obligate endoparasites. Although nematodes are one of the most abundant and diverse animal groups, the only confirmed report of microsporidian infection was that of the "nematode killer" (Nematocida parisii). N. parisii was isolated from a wild Caenorhabditis sp. and causes an acute and lethal intestinal infection in a lab strain of Caenorhabditis elegans. We set out to characterize a microsporidian infection in a wild nematode to determine whether the infection pattern of N. parisii in the lab is typical of microsporidian infections in nematodes. We describe a novel microsporidian species named Sporanauta perivermis (marine spore of roundworms) and characterize its infection in its natural host, the free-living marine nematode Odontophora rectangula. S. perivermis is not closely related to N. parisii and differs strikingly in all aspects of infection. Examination by transmission electron microscopy (TEM) revealed that the infection was localized in the hypodermal and muscle tissues only and did not involve the intestines. Fluorescent in situ hybridization (FISH) confirmed infection in the muscle and hypodermis, and surprisingly, it also revealed that the parasite infects O. rectangula eggs, suggesting a vertical mode of transmission. Our observations highlight the importance of studying parasites in their natural hosts and indicate that not all nematode-infecting microsporidia are "nematode killers"; instead, microsporidiosis can be more versatile and chronic in the wild.

Influence of temperature on the physiology and virulence of the insect pathogen Serratia sp. Strain SCBI.

The physiology of a newly recognized Serratia species, termed South African Caenorhabditis briggsae Isolate (SCBI), which is both a nematode mutualist and an insect pathogen, was investigated and compared to that of Serratia marcescens Db11, a broad-host-range pathogen. The two Serratia strains had comparable levels of virulence for Manduca sexta and similar cytotoxic activity patterns, but motility and lipase and hemolytic activities differed significantly between them.

Population dynamics and habitat sharing of natural populations of Caenorhabditis elegans and C. briggsae.

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.

An insect pathogenic symbiosis between a Caenorhabditis and Serratia.

We described an association between a strain of the nematode Caenorhabditis briggsae, i.e. KT0001, and the bacteria Serratia sp. SCBI (South African Caenorhabditis briggsae isolate), which was able to kill the insect Galleria. Here we show that the Serratia sp. SCBI lines the gut of the nematode, similar to the Heterorhabditis-Photorhabdus complex, indicating that the association is possibly internal. We also expand on the relevance of this tripartite, i.e. insect-nematode-bacteria, interaction in the broader evolutionary context and Caenorhabditis natural history.

An entomopathogenic Caenorhabditis briggsae.

Caenorhabditis elegans is a premier model organism upon which considerable knowledge of basic cell and developmental biology has been built. Yet, as is true for many traditional model systems, we have limited knowledge of the ecological context in which these systems evolved, severely limiting our understanding of gene function. A better grasp of the ecology of model systems would help us immensely in understanding the functionality of genes and evolution of genomes in an environmental context. Consequently, there are ongoing efforts to uncover natural populations of this model system globally. Here, we describe the discovery of a Caenorhabditis briggsae strain and its bacterial associate (Serratia sp.) that form an entomopathogenic complex in the wild. Laboratory experiments confirm that this nematode and its natural bacterial associate can penetrate, kill and reproduce in an insect host and that the bacterial associate can induce this insect pathogenic life cycle in other Caenorhabditis species, including C. elegans. Our findings suggest that this life history may be widespread in nature and critical to the understanding of the biology of this important model organism. Caenorhabditis-insect interaction could be a key factor in our quest for a better grasp of gene functionality in this important model species. The discovered association, consequently, would provide an ecological framework for functional genomics of Caenorhabditis.

Caenorhabditis is a metazoan host for Legionella.

We investigated whether nematodes contribute to the persistence, differentiation and amplification of Legionella species in soil, an emerging source for Legionnaires' disease. Here we show that Legionella spp. colonize the intestinal tracts of Caenorhabditis nematodes leading to worm death. Susceptibility to Legionella is influenced by innate immune responses governed by the p38 mitogen-activated protein kinase and insulin/insulin growth factor-1 receptor signalling pathways. We also show that L. pneumophila colonizes the intestinal tract of nematodes cultivated in soil. To distinguish between transient infection and persistence, plate-fed and soil-extracted nematodes-fed fluorescent strains of L. pneumophila were analysed. Bacteria replicated within the nematode intestinal tract, did not invade surrounding tissue, and were excreted as differentiated forms that were transmitted to offspring. Interestingly, the ultrastructural features of the differentiated bacterial forms were similar to cyst-like forms observed within protozoa, amoeba and mammalian cell lines. While intestinal colonization of L. pneumophila dotA and icmT mutant strains did not alter the survival rate of nematodes in comparison to wild-type strains, nematodes colonized with the dot/icm mutant strains exhibited significantly increased levels of germline apoptosis. Taken together, these studies show that nematodes may serve as natural hosts for these organisms and thereby contribute to their dissemination in the environment and suggest that the remarkable ability of L. pneumophila to subvert host cell signalling and evade mammalian immune responses evolved through the natural selection associated with cycling between protozoan and metazoan hosts.

Sex-dependent resistance to the pathogenic fungus Cryptococcus neoformans.

Sex differences occur in most species and affect a variety of biological traits including morphology, behavior, and life history. The nematode Caenorhabditis elegans exists as a population of self-fertile hermaphrodites with occasional males, which differ anatomically and behaviorally from hermaphrodites. Here we show that male C. elegans also differ from hermaphrodites in their susceptibility to a fungal pathogen, Cryptococcus neoformans. Wild-type males show greater resistance than hermaphrodite animals to killing by this pathogen and this resistance can be induced in hermaphrodite animals by inappropriate activation of the male sex-determination pathway. Resistance is molecularly determined, rather than resulting from behavioral changes or reproductive differences, and requires the activity of the stress-response transcription factor DAF-16. Finally, we demonstrate that resistance to C. neoformans correlates broadly with longevity within the Caenorhabditis genus. Our results hint at an overlap between the pathways controlling immunity and longevity and raise the possibility that differential regulation of these pathways may contribute to sex-dependent and species-dependent variation.

Alterations of life span in the nematode Caenorhabditis elegans under monoxenic culture conditions.

The nematode Caenorhabditis elegans was cultured monoxenically with E. coli as a food source and the influence of the bacterial growth conditions on the life span was studied. When bacterial growth was restricted by reducing the concentration of bactopeptone, which was supplied as the energy source in nematode growth medium (NGM), the nematode's life span tended to be prolonged without a marked effect on postembryonic development. The effect of bactopeptone on the life span was clearly observed during the postreproductive period (that is, after the egg-laying stage of the wild-type C. elegans) rather than during the larval to young adult stage. Evidence is presented that this alteration of the life span was not brought about by any factor in the bactopeptone but by the concentration of bacteria.