The nematode Pristionchus pacificus (Nematoda: Diplogastridae) is associated with the oriental beetle Exomala orientalis (Coleoptera: Scarabaeidae) in Japan

Pristionchus pacificus has been developed as a nematode satellite organism in evolutionary developmental biology. Detailed studies of vulva development revealed multiple differences in genetic and molecular control in P. pacificus compared to the model organism Caenorhabditis elegans. To place evolutionary developmental biology in a comprehensive evolutionary context, such studies have to be complemented with ecology. In recent field studies in western Europe and eastern North America we found 11 Pristionchus species that are closely associated with scarab beetles and the Colorado potato beetle. However, P. pacificus was not commonly found in association with scarab beetles in these studies. Here, we describe the results of a similar survey of scarab beetles in Japan. Pristionchus pacificus was the most common Pristionchus species on scarab beetles in Japan, with 40 out of 43 (93%) isolates. The other Pristionchus isolates represent three novel species, which we refer to as Pristionchus sp. 11, Pristionchus sp. 14, and Pristionchus sp. 15. Thirty-seven of the established P. pacificus strains were found on the oriental beetle Exomala orientalis. Laboratory studies with the sex pheromone (Z)-7-tetradecen-2-one of the oriental beetle revealed that P. pacificus shows strong olfactory attraction to the beetle's sex pheromone, which provides a potential mechanism for the recognition and interaction of P. pacificus and E. orientalis. Together, this study identifies P. pacificus as the most common Pristionchus nematode in field studies in Japan, identifies E. orientalis as an important host species, and provides the basis for the ecology of P. pacificus.     

Mutation rates and intraspecific divergence of the mitochondrial genome of Pristionchus pacificus

Evolutionary reconstruction of the natural history of an organism ultimately requires knowledge about the development, population genetics, ecology, and phylogeny of the species. Such investigations would benefit from studies of mutational processes because mutations are the source of natural variation. The nematode Pristionchus pacificus has been developed as a model organism in evolutionary biology by comparing its development with Caenorhabditis elegans. Pristionchus pacificus and related species are associated with scarab beetles, and their ecology and phylogeny are well known. More than 200 P. pacificus isolates from all over the world are available for this cosmopolitan species. We generated mutation accumulation (MA) lines in P. pacificus to study spontaneous mutation rates in the mitochondrial genome and compared mutation rate estimates with natural variation between nine representative isolates of the species. The P. pacificus mitochondrial genome is 15,955 bp in length and is typical for nematodes. Pristionchus pacificus has all known mitochondrial genes and contains an unusual suppressor transfer RNA (tRNA) for the codon UAA. This has most likely influenced the spectrum of observable mutations because 6 of 12 mutations found in the 82 MA lines analyzed are nonsense mutations that can be suppressed by the suppressor tRNA. The overall mutation rate in P. pacificus is 7.6 × 10?? per site per generation and is less than one order of magnitude different from estimates in C. elegans and Drosophila. Using this mutation rate estimate in a comparison of the mitochondrial genome of nine P. pacificus isolates, we calculate the minimum time to the most recent common ancestor at 10?-10? generations. The combination of mutation rate analysis with intraspecific divergence provides a powerful tool for the reconstruction of the natural history of P. pacificus, and we discuss the ecological implication of these findings.     

Microevolutionary analysis of the nematode genus Pristionchus suggests a recent evolution of redundant developmental mechanisms during vulva formation

SUMMARY To identify the mechanisms by which molecular variation is introduced into developmental systems, microevolutionary approaches to evolutionary developmental biology have to be taken. Here, we describe the molecular and developmental characterization of laboratory strains of the nematode genus Pristionchus , which lays a foundation for a microevolutionary analysis of vulva development. We describe 13 laboratory strains of the Pristionchus genus that are derived from natural isolates from around the world. Mating experiments and ITS sequence analysis indicated that these 13 strains represent four different species: the gonochoristic species P. lheritieri and three hermaphroditic species, P. pacificus , P. maupasi , and an as yet undescribed species Pristionchus sp., respectively. P. pacificus is represented by five different strains isolated from California, Washington, Hawaii, Ontario, and Poland. Developmental differences during vulva formation are observed between strains from different species but also between strains of P. pacificus , like the strains from California and Poland. In particular, redundant developmental mechanisms present during vulva formation in P. pacificus var. California are absent in other strains. Amplified restriction fragment length polymorphism (AFLP) analyses of the P. pacificus strains revealed that the American strains are highly polymorphic. In contrast, the developmentally distinct strain from Poland is identical to the Californian strain, suggesting that the developmental differences rely on a small number of changes in developmental control genes rather than the accumulation of changes at multiple loci.     

Molecular phylogeny of a red-snow-crab species complex using mitochondrial and nuclear DNA markers

Nucleotide sequence variation of mitochondrial DNA COI and nuclear rRNA gene regions was used to reconstruct phylogenetic relationships for the red-snow-crab species complex, including the red snow crab, Chionoecetes japonicus, its nominal subspecies, C. japonicus pacificus, and the triangle tanner crab, C. angulatus. The topologies of the Bayesian and neighbor-joining (NJ) trees of the COI and of NJ trees of rRNA sequences placed C. japonicus and C. angulatus in a single clade. The net sequence divergence between these taxa was d(net) = 0.000 in COI, and strongly suggests that these taxa represent a single species. In contrast, haplotypes in C. j. pacificus clustered separately from the C. japonicus - C. angulatus clade. Net sequence divergence from C. japonicus - C. angulatus to C. j. pacificus was d(net) = 0.026 in COI, indicating that C. j. pacificus should be elevated to a separate species, C. pacificus. A 165 bp insert appeared in the rRNA gene of C. j. pacificus, but was absent in the remaining species of Chionoecetes. This autapomorphic condition in C. j. pacificus adds support for an independent evolution of this taxon. Evolutionary divergences between these taxa may reflect contrasting evolutionary process influenced by ocean bathymetry.     

Nematodes of the genus Pristionchus are closely associated with scarab beetles and the Colorado potato beetle in Western Europe

Evolutionary developmental biology examines how changes in developmental programmes give rise to developmental and, ultimately, morphological novelty. To this end, comparisons of related but distinct organisms have to be performed. The diplogastrid nematode Pristionchus pacificus has been developed as a satellite system for a detailed comparison of various developmental processes to the model organism Caenorhabditis elegans, a rhabditid nematode. In addition to developmental and genetic studies, a genomic platform has been established to analyse the biology of this organism. However, only little is known about where and how Pristionchus pacificus and its relatives live in the wild. Here we show that nematodes of the genus Pristionchus live in close association with scarabaeoid beetles and the Colorado potato beetle. In total, we generated 371 isogenic female lines from 4242 beetles collected at 25 sampling sites all over Europe. Isogenic female lines were subjected to sequence analysis and mating experiments for species determination. The 371 isolates fell into six species. Two hermaphroditic species account for about 60% of the collected nematodes. We found Pristionchus maupasi almost exclusively on cockchafers and Pristionchus entomophagus predominantly on dung beetles. Colorado potato beetles carried the gonochoristic species Pristionchus uniformis, which was only rarely observed on scarabaeoid beetles. We describe the initial evidence for the association of Pristionchus nematodes with beetles and provide a phylogeny based on sequence analysis of the small subunit ribosomal RNA gene.     

Species-specific recognition of beetle cues by the nematode Pristionchus maupasi

SUMMARY The environment has a strong effect on development as is best seen in the various examples of phenotypic plasticity. Besides abiotic factors, the interactions between organisms are part of the adaptive forces shaping the evolution of species. To study how ecology influences development, model organisms have to be investigated in their environmental context. We have recently shown that the nematode Pristionchus pacificus and its relatives are closely associated with scarab beetles with a high degree of species specificity. For example, P. pacificus is associated with the oriental beetle Exomala orientalis in Japan and the northeastern United States, whereas Pristionchus maupasi is primarily isolated from cockchafers of the genus Melolontha in Europe. Here, we investigate how Pristionchus nematodes identify their specific insect hosts by using chemotaxis studies originally established in Caenorhabditis elegans . We observed that P. maupasi is exclusively attracted to phenol, one of the sex attractants of Melolontha beetles, and that attraction was also observed when washes of adult beetles were used instead of pure compounds. Furthermore, P. maupasi chemoattraction to phenol synergizes with plant volatiles such as the green leaf alcohol and linalool, demonstrating that nematodes can integrate distinct chemical senses from multiple trophic levels. In contrast, another cockchafer-associated nematode, Diplogasteriodes magnus , was not strongly attracted to phenol. We conclude that interception of the insect communication system might be a recurring strategy of Pristionchus nematodes but that different nematodes use distinct chemical cues for finding their beetle hosts.     

Isolation of mutations with dumpy-like phenotypes and of collagen genes in the nematode Pristionchus pacificus

The nematode Pristionchus pacificus was developed as a satellite system in evolutionary developmental biology and forward and reverse genetic approaches allow a detailed comparison of various developmental processes between P. pacificus and Caenorhabditis elegans. To facilitate map-based cloning in P. pacificus, a genome map was generated including a genetic linkage map of approximately 300 molecular markers and a physical map of 10,000 BAC clones. Here, we describe the isolation and characterization of more than 40 morphological mutations that can be used as genetic markers. These mutations fall into 12 Dumpy genes and one Roller gene that represent morphological markers for all six P. pacificus chromosomes. Using an in silico approach, we identified approximately 150 hits of P. pacificus collagen genes in the available EST, BAC-end, and fosmid-end sequences. However, 1:1 orthologs could only be identified for fewer than 20 collagen genes.     

Chemoattraction in Pristionchus nematodes and implications for insect recognition

Nematodes and insects are the two dominant animal taxa in species numbers, and nematode-insect interactions constitute a significant portion of interspecies associations in a diversity of ecosystems. It has been speculated that most insects represent mobile microhabitats in which nematodes can obtain food, mobility, and shelter. Nematode-insect associations can be classified as phoretic (insects used for transportation, not as food), necromenic (insect used for transportation, then carcass as food), and entomopathogenic (insect is killed and used as food). To determine how nematodes target their hosts, we analyzed the chemosensory response and behavioral parameters of closely related Pristionchus nematodes that form species-specific necromenic associations with scarab beetles and the Colorado potato beetle. We found that all four studied Pristionchus species displayed unique chemoattractive profiles toward insect pheromones and plant volatiles with links to Pristionchus habitats. Moreover, chemoattraction in P. pacificus differs from that of C. elegans not only in the types of attractants, but also in its tempo, mode, and concentration response range. We conclude that Pristionchus olfaction is highly diverse among closely related species and is likely to be involved in shaping nematode-host interactions.     

Distinct patterns of genetic variation in Pristionchus pacificus and Caenorhabditis elegans, two partially selfing nematodes with cosmopolitan distribution

Hermaphroditism has evolved several times independently in nematodes. The model organism Caenorhabditis elegans and Pristionchus pacificus are self-fertile hermaphrodites with rare facultative males. Both species are members of different families: C. elegans belongs to the Rhabditidae and P. pacificus to the Diplogastridae. Also, both species differ in their ecology: C. elegans is a soil-dwelling nematode that is often found in compost heaps. In contrast, field studies in Europe and North America indicate that Pristionchus nematodes are closely associated with scarab beetles. In C. elegans, several recent studies have found low genetic diversity and rare out-crossing events. Little is known about diversity levels and population structure in free-living hermaphroditic nematodes outside the genus Caenorhabditis. Taking a comparative approach, we analyse patterns of molecular diversity and linkage disequilibrium in 18 strains of P. pacificus from eight countries and four continents. Mitochondrial sequence data of P. pacificus isolates reveal a substantially higher genetic diversity on a global scale when compared to C. elegans. A mitochondrial-derived hermaphrodite phylogeny shows little geographic structuring, indicating several worldwide dispersal events. Amplified fragment length polymorphism and single strand conformation polymorphism analyses demonstrate a high degree of genome-wide linkage disequilibrium, which also extends to the mitochondrial genome. Together, these findings indicate distinct patterns of genetic variation of the two species. The low level of genetic diversity observed in C. elegans might reflect a recent human-associated dispersal, whereas the P. pacificus diversity might reflect a long-lasting and ongoing insect association. Thus, despite similar lifestyle characteristics in the laboratory, the reproductive mode of hermaphroditism with rare facultative males can result in distinct genetic variability patterns in different ecological settings.     

Pristionchus pacificus, a nematode with only three juvenile stages, displays major heterochronic changes relative to Caenorhabditis elegans.

The nematode Pristionchus pacificus (Diplogastridae) has been described as a satellite organism for a functional comparative approach to the model organism Caenorhabditis elegans because genetic, molecular, and cell-biological tools can be used in a similar way in both species. Here we show that P. pacificus has three juvenile stages, instead of the usual four found in other nematodes. Embryogenesis is lengthened and many developmental events that take place during the first juvenile stage in C. elegans occur during late embryogenesis in P. pacificus. Video imaging and transmission electron microscopy revealed no embryonic moult. The timing of later developmental events relative to the moults differs between P. pacificus and C. elegans. In addition, the post-embryonic blast-cell divisions display a specific change in timing between the two species, resulting in heterochrony between different cell lineages, such as vulval and gonadal lineages. Developmental events appear to come into register during the last larval stage. Thus, differences in developmental timing between P. pacificus and C. elegans represent a deep heterochronic change. We designate the three juvenile stages of P. pacificus as J1 to J3. Comparison with other species of the family Diplogastridae indicates that this pattern represents an apomorphic character for the monophylum Diplogastridae.     

Comparative genetics: a third model nematode species

Recent studies have introduced Oscheius sp. CEW1 as a third nematode species accessible to genetic analysis, joining the better known Caenorhabditis elegans and Pristionchus pacificus. A group of vulva-defective mutants in Oscheius has been identified, with defects not seen in C. elegans.     

Novel cell-cell interactions during vulva development in Pristionchus pacificus

Vulva development differs between Caenorhabditis elegans and Pristionchus pacificus in several ways. Seven of 12 ventral epidermal cells in P. pacificus die of apoptosis, whereas homologous cells in C. elegans fuse with the hypodermal syncytium. Vulva induction is a one-step process in C. elegans, but requires a continuous interaction between the gonad and the epidermis in P. pacificus. Here we describe several novel cell-cell interactions in P. pacificus, focusing on the vulva precursor cell P8.p and the mesoblast M. P8.p in P. pacificus, unlike its homologous cell in C. elegans, is incompetent to respond to gonadal signaling in the absence of other vulva precursor cells, but can respond to lateral signaling from a neighboring vulval precursor. P8.p provides an inhibitory signal that determines the developmental competence of P(5,7).p. This lateral inhibition acts via the mesoblast M and is regulated by the homeotic gene Ppa-mab-5. In Ppa-mab-5 mutants, M is misspecified and provides inductive signaling to the vulval precursor cells, including P8.p. Taken together, vulva development in P. pacificus displays novel cell-cell interactions involving the mesoblast M and P8.p. In particular, P8.p represents a new ventral epidermal cell type, which is characterized by novel interactions and a specific response to gonadal signaling.     

Pristionchus pacificus: a well-rounded nematode

Nematodes pervade Earth's biosphere and occupy innumerable ecological niches. The role of Caenorhabditis elegans as a model for developmental processes has encouraged us to cultivate a second nematode, Pristionchus pacificus, as a comparative counterpoint to address questions in development, behavior and ecology in nematode evolution. We hope that this endeavor, now more than a decade underway, will allow us to project findings onto other comparative models for biological processes. To this end, our laboratory has made an extensive genetic map and mutant screens to understand changes in developmental programs. Recently, we have been capitalizing on the whole genome sequence of P. pacificus to describe more thoroughly the molecular basis for these changes, as well as to better integrate our molecular knowledge with the biodiversity of Pristionchus species.     

Pristionchus uniformis,should I stay or should I go? Recent host range expansion in a European nematode

Pristionchus pacificus has been developed as a model system in evolutionary developmental biology, evolutionary ecology, and population genetics. This species has a well-known ecological association with scarab beetles. Generally, Pristionchus nematodes have a necromenic association with their beetle hosts. Arrested dauer larvae invade the insect and wait for the host's death to resume development. Only one Pristionchus species is known to frequently associate with a non-scarab beetle. Pristionchus uniformis has been isolated from the chrysomelid Leptinotarsa decemlineata, also known as the Colorado potato beetle, in Europe and North America, but is also found on scarab beetles. This unusual pattern of association with two unrelated groups of beetles on two continents requires the involvement of geographical and host range expansion events. Here, we characterized a collection of 81 P. uniformis isolates from North America and Europe and from both scarab beetles and L. decemlineata. We used population genetic and phylogenetic analyses of the mitochondrial gene nd2 to reconstruct the genetic history of P. uniformis and its beetle association. Olfactory tests on beetles chemical extracts showed that P. uniformis has a unique chemoattractive profile toward its beetle hosts. Our results provide evidence for host range expansion through host-switching events in Europe where P. uniformis was originally associated with scarab beetles and the nematode's subsequent invasion of North America.     

A bacterial artificial chromosome-based genetic linkage map of the nematode Pristionchus pacificus

To understand the evolution of developmental processes, nonmodel organisms in the nematodes, insects, and vertebrates are compared with established model systems. Often, these comparisons suffer from the inability to apply sophisticated technologies to these nonmodel species. In the nematode Pristionchus pacificus, cellular and genetic analyses are used to compare vulva development to that of Caenorhabditis elegans. However, substantial changes in gene function between P. pacificus and C. elegans limit the use of candidate gene approaches in studying P. pacificus mutations. To facilitate map-based cloning of mutations in P. pacificus, we constructed a BAC-based genetic linkage map. A BAC library of 13,440 clones was generated and completely end sequenced. By comparing BAC end and EST sequences between the "wild-type" strain P. pacificus var. California and the polymorphic strain P. pacificus var. Washington, 133 single-stranded conformational polymorphisms were identified. These markers were tested on a meiotic mapping panel of 46 randomly picked F(2) animals after a cross of the two strains, providing the first genetic linkage map of P. pacificus. A mapping strategy using two selected markers per chromosome was devised and the efficiency of this approach was illustrated by the mapping of the Ppa-unc-1/Twitchin gene.     

Natural variation in Pristionchus pacificus dauer formation reveals cross-preference rather than self-preference of nematode dauer pheromones

Many free-living nematodes, including the laboratory model organisms Caenorhabditis elegans and Pristionchus pacificus, have a choice between direct and indirect development, representing an important case of phenotypic plasticity. Under harsh environmental conditions, these nematodes form dauer larvae, which arrest development, show high resistance to environmental stress and constitute a dispersal stage. Pristionchus pacificus occurs in a strong association with scarab beetles in the wild and remains in the dauer stage on the living beetle. Here, we explored the circumstances under which P. pacificus enters and exits the dauer stage by using a natural variation approach. The analysis of survival, recovery and fitness after dauer exit of eight P. pacificus strains revealed that dauer larvae can survive for up to 1 year under experimental conditions. In a second experiment, we isolated dauer pheromones from 16 P. pacificus strains, and tested for natural variation in pheromone production and sensitivity in cross-reactivity assays. Surprisingly, 13 of the 16 strains produce a pheromone that induces the highest dauer formation in individuals of other genotypes. These results argue against a simple adaptation model for natural variation in dauer formation and suggest that strains may have evolved to induce dauer formation precociously in other strains in order to reduce the fitness of these strains. We therefore discuss intraspecific competition among genotypes as a previously unconsidered aspect of dauer formation.     

sem-4/spalt and egl-17/FGF have a conserved role in sex myoblast specification and migration in P. pacificus and C. elegans

Evolutionary comparisons between Caenorhabditis elegans and the satellite organism Pristionchus pacificus revealed major differences in the regulation of nematode vulva development. For example, Wnt signaling is part of a negative signaling system that prevents vulva formation in P. pacificus, whereas it plays a positive role in C. elegans. We wondered if the genetic control of the second major part of the nematode egg-laying system, the sex muscles, has diverged similarly between P. pacificus and C. elegans. The sex muscles derive from the mesoblast M, which has an identical lineage in both species. Here, we describe a large-scale mutagenesis screen for mutations that disrupt the M lineage and the sex myoblast (SM) sublineage. We isolated and characterized mutations that result in a failure of proper SM fate specification and SM migration and showed that the corresponding genes encode Ppa-sem-4 and Ppa-egl-17, respectively. Ppa-sem-4 mutants have additional defects in the specification of the vulva precursor cells P(5, 7).p and experimental studies in the Ppa-egl-17 mutant background indicate a complex set of gonad-dependent and gonad-independent mechanisms required for SM migration. Mutations in Cel-sem-4 and Cel-egl-17 cause similar defects. Thus, the molecular mechanisms of SM cell specification and migration are conserved between P. pacificus and C. elegans.     

Pristionchus pacificus vulva formation: polarized division, cell migration, cell fusion, and evolution of invagination

Tube formation is a widespread process during organogenesis. Specific cellular behaviors participate in the invagination of epithelial monolayers that form tubes. However, little is known about the evolutionary mechanisms of cell assembly into tubes during development. In Caenorhabditis elegans, the detailed step-to-step process of vulva formation has been studied in wild type and in several mutants. Here we show that cellular processes during vulva development, which involve toroidal cell formation and stacking of rings, are conserved between C. elegans and Pristionchus pacificus, two species of nematodes that diverged approximately 100 million years ago. These cellular behaviors are divided into phases of cell proliferation, short-range migration, and cell fusion that are temporally distinct in C. elegans but not in P. pacificus. Thus, we identify heterochronic changes in the cellular events of vulva development between these two species. We find that alterations in the division axes of two equivalent vulval cells from Left-Right cleavage in C. elegans to Anterior-Posterior division in P. pacificus can cause the formation of an additional eighth ring. Thus, orthogonal changes in cell division axes with alterations in the number and sequence of cell fusion events result in dramatic differences in vulval shape and in the number of rings in the species studied. Our characterization of vulva formation in P. pacificus compared to C. elegans provides an evolutionary-developmental foundation for molecular genetic analyses of organogenesis in different species within the phylum Nematoda.