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.     

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.     

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.     

Operon structure and trans-splicing in the nematode Pristionchus pacificus

In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.     

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.     

Phylogeny of the nematode genus <Emphasis Type="Italic">Pristionchus</Emphasis> and implications for biodiversity,biogeography and the evolution of hermaphroditism

Background  

The nematode Pristionchus pacificus has originally been developed as a satellite organism for comparison to Caenorhabditis elegans. A 10X coverage of the whole genome of P. pacificus is available, making P. pacificus the first non- Caenorhabditis nematode with a fully sequenced genome. The macroevolutionary comparison between P. pacificus and C. elegans has been complemented by microevolutionary studies of closely related strains and species within the genus Pristionchus. In addition, new understanding of the biology of Pristionchus from field studies, demonstrating a close association with various scarab beetles and the Colorado potato beetle, supports consideration of this nematode in studies of ecosystems. In the course of field studies on four continents more than 1,200 isolates were established from 15,000 beetle specimens representing 18 Pristionchus species. Two remarkable features of the Pristionchus – beetle association are the high species specificity of the interaction and the interception of the beetle's sex communication system for host recognition by the nematodes, as suggested by chemotaxis studies. Evolutionary interpretations of differences in developmental, behavioral and ecological patterns require a phylogenetic framework of the genus Pristionchus.     

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.     

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.     

Haplotype diversity of the nematode Pristionchus pacificus on Réunion in the Indian Ocean suggests multiple independent invasions

Pristionchus pacificus has been established as a nematode model system in evolutionary developmental biology and evolutionary ecology. Field studies in North and South America, Asia, Africa and Europe indicated that nematodes of the genus Pristionchus live in association with scarab beetles. Here, we describe the first account of soil‐ and beetle‐associated nematodes on an island setting by investigating the island of Réunion in the Indian Ocean. Réunion has high numbers of endemic insects and is one among several attractive islands for biodiversity studies. Being of volcanic origin, Réunion is 2–3 million years old, making it the youngest of the Mascareigne islands. We show that beetle‐ and soil‐derived nematodes on Réunion are nearly exclusively hermaphroditic, suggesting that selfing is favoured over gonochorism (outcrossing) during island colonization. Among members of four nematode genera observed on Réunion, Pristionchus pacificus was the most prevalent species. A total of 76 isolates, in association with five different scarab beetles, has been obtained for this cosmopolitan nematode. A detailed mitochondrial haplotype analysis indicates that the Réunion isolates of P. pacificus cover all four worldwide clades of the species. This extraordinary haplotype diversity suggests multiple independent invasions, most likely in association with different scarab beetles. Together, we establish Réunion as a case study for nematode island biogeography, in which the analysis of nematode population genetics and population dynamics can provide insight into evolutionary and ecological processes. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 170–179.     

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.     

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.     

Mustard biofumigation disrupts biological control by Steinernema spp. nematodes in the soil

Mustard green manures or seed meal high in glucosinolates, which produce a natural biofumigant upon incorporation into the soil, form an alternative to synthetic fumigants. However, the non-target impacts of these biofumigants in the field are unclear. We examined the effectiveness of soil incorporation of Brassica carinata seed meal both in controlling the plant-parasitic Columbia root-knot nematode (Meloidogyne chitwoodi), and on the biological control exerted by the entomopathogenic nematodes Steinernema feltiae and Steinernema riobrave on root-knot nematodes and the Colorado potato beetle (Leptinotarsa decemlineata). Singly, both the seed meal and Steinernema spp. reduced root-knot nematode damage to potato tubers and increased marketable tuber yields. However, there was a negative interaction between the two bioagents such that their combination did not further improve suppression of plant-parasitic nematodes. Thus, mustard seed meal applications harmful to the target root-knot nematode also disrupted the ability of Steinernema spp. to act as biocontrol agents. Further, we observed modest disruption of the biological control of potato beetles following biofumigation. But, the potato beetles were less likely to lay eggs on potato plants grown in mustard-amended soil, suggesting a counteracting benefit of mustard application. Multiple, complementary controls must be integrated to replace the very effective pest suppression typical of synthetic soil fumigants. Our study suggests significant interference between biofumigation and biocontrol agents in the soil, presenting challenges in combining these two environmentally friendly approaches to managing plant-parasitic nematodes and other pests.     

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.     

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.     

Cultivation of the rhabditid Poikilolaimus oxycercus as a laboratory nematode for genetic analyses

Vulva formation is a paradigm for evolutionary developmental biology in nematodes. Not only do the number of vulval precursor cells (VPCs) differ between members in the Rhabditidae and Diplogastridae, they are also sculpted via different developmental mechanisms, either by cell fusion in most Rhabditidae or by programmed cell death in the Diplogastridae. In this context, the species Poikilolaimus oxycercus is the only known species in the family Rhabditidae to have a subset of the Pn.p cells commit programmed cell death during the patterning of the VPCs. Our current study introduces P. oxycercus as a new laboratory organism. There are discrete laboratory strains that are genetically polymorphic from each other as well as heterogeneous within each strain. In order to cultivate this gonochoristic nematode into an experimental model with a tractable genetic system, we produced two inbreeding tolerant, near-isogenic strains capable of producing viable progeny with each other. We also described P. oxycera's morphology by scanning electron microscopy (SEM), basic life history traits, hybrid viability, and mating behavior. P. oxycercus females have no preference for inter- or intra-strain matings, and can mate with multiple males in a relatively short time period, suggesting a propensity for maintaining heterozygosity through promiscuity. Interestingly, all sexes from three species in the genus Poikilolaimus show five 4',6-diamidino-2-phenylindole (DAPI) staining bodies in their germ line cells. This could indicate that Poikilolaimus species possess five bivalent chromosomes in their germ lines, in contrast to the hermaphroditic Caenorhabditis elegans or Pristionchus pacificus, which have six chromosomes.     

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.     

Toxicity of 2,4-diacetylphloroglucinol (DAPG) to Plant-parasitic and Bacterial-feeding Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG) is produced by some isolates of the beneficial bacterium Pseudomonas fluorescens. DAPG is toxic to many organisms, and crop yield increases have been reported after application of DAPG-producing P. fluorescens. This study was conducted to determine whether DAPG is toxic to selected nematodes. The plant-parasitic nematodes Heterodera glycines, Meloidogyne incognita, Pratylenchus scribneri and Xiphinema americanum, and the bacterial-feeding nematodes Caenorhabditis elegans, Pristionchus pacificus, and Rhabditis rainai, were immersed in concentrations ranging from 0 to 100 μg/ml DAPG. Egg hatch and viability of juveniles and adults were determined. DAPG was toxic to X. americanum adults, with an LD(50) of 8.3 μg/ml DAPG. DAPG decreased M. incognita egg hatch, but stimulated C. elegans hatch during the first hours of incubation. Viability of M. incognita J2 and of C. elegans J1 and adults was not affected. There were no observed effects on the other nematodes. The study indicated that DAPG is not toxic to all nematodes, and did not affect the tested species of beneficial bacterial-feeding nematodes. Augmentation of DAPG-producing P. fluorescens populations for nematode biocontrol could be targeted to specific nematode species known to be affected by this compound and by other antibiotics produced by the bacteria, or these bacteria could be used for other possible effects, such as induced plant resistance.