Conservation of large foci formation in arrested oocytes of <Emphasis Type="Italic">Caenorhabditis</Emphasis> nematodes

Within the rhabditid phylogeny of nematodes, the great majority of species are gonochoristic, having evolved as obligate male/female species. In contrast, the well-studied nematode model system, Caenorhabditis elegans, is androdioecious, utilizing a hermaphroditic/male reproductive system. We have previously determined that in the arrested oocytes of old-aged C. elegans hermaphrodites with depleted sperm, large cytoplasmic ribonucleoprotein foci form. The formation of these foci is reversible, as they dissociate within 3 h after a male mates with the hermaphrodite, resupplying it with sperm. The functional significance of these oocyte foci is not known and previously has not been clear for a hermaphroditic species in which oocytes of young adults wait only approximately 23 min to be fertilized. One hypothesis is that the foci function to maintain maternal mRNAs in oocytes while fertilization is delayed. In this paper, we examine four gonochoristic rhabditid species: Caenorhabditis remanei, Caenorhabditis sp. CB5161, Caenorhabditis sp. PS1010, and Rhabditella axei DF5006. We demonstrate that in three of these four species, ovulation arrests in unmated females until mating occurs and large cytoplasmic foci develop in arrested oocytes. The oocyte foci contain nuclear pore proteins and, in C. remanei at least, the RNA-binding protein MEX-3 as well as RNA. We speculate that these foci maintain the integrity of ooctyes, possibly maintaining the stability or translational repression of maternal mRNAs in unmated females. We further speculate that their presence in oocytes of old-aged C. elegans hermaphrodites is due to conservation from an ancestral gonochoristic state.     

Conservation of long-range synteny and microsynteny between the genomes of two distantly related nematodes

Background  

Comparisons between the genomes of the closely related nematodes Caenorhabditis elegans and Caenorhabditis briggsae reveal high rates of rearrangement, with a bias towards within-chromosome events. To assess whether this pattern is true of nematodes in general, we have used genome sequence to compare two nematode species that last shared a common ancestor approximately 300 million years ago: the model C. elegans and the filarial parasite Brugia malayi.     

Hsp-90 and the biology of nematodes

Background  

Hsp-90 from the free-living nematode Caenorhabditis elegans is unique in that it fails to bind to the specific Hsp-90 inhibitor, geldanamycin (GA). Here we surveyed 24 different free-living or parasitic nematodes with the aim of determining whether C. elegans Hsp-90 was the exception or the norm amongst the nematodes. We combined these data with codon evolution models in an attempt to identify whether hsp-90 from GA-binding and non-binding species has evolved under different evolutionary constraints.     

Molecular evolution in Panagrolaimus nematodes: origins of parthenogenesis, hermaphroditism and the Antarctic species P. davidi

Background  

As exemplified by the famously successful model organism Caenorhabditis elegans, nematodes offer outstanding animal systems for investigating diverse biological phenomena due to their small genome sizes, short generation times and ease of laboratory maintenance. Nematodes in the genus Panagrolaimus have served in comparative development and anhydrobiosis studies, and the Antarctic species P. davidi offers a powerful paradigm for understanding the biological mechanisms of extreme cold tolerance. Panagrolaimus nematodes are also unique in that examples of gonochoristic, hermaphroditic and parthenogenetic reproductive modes have been reported for members of this genus. The evolutionary origins of these varying reproductive modes and the Antarctic species P. davidi, however, remain enigmatic.     

nGASP – the nematode genome annotation assessment project

Background  

While the C. elegans genome is extensively annotated, relatively little information is available for other Caenorhabditis species. The nematode genome annotation assessment project (nGASP) was launched to objectively assess the accuracy of protein-coding gene prediction software in C. elegans, and to apply this knowledge to the annotation of the genomes of four additional Caenorhabditis species and other nematodes. Seventeen groups worldwide participated in nGASP, and submitted 47 prediction sets across 10 Mb of the C. elegans genome. Predictions were compared to reference gene sets consisting of confirmed or manually curated gene models from WormBase.     

The evolutionary position of nematodes

Background  

The complete genomes of three animals have been sequenced by global research efforts: a nematode worm (Caenorhabditis elegans), an insect (Drosophila melanogaster), and a vertebrate (Homo sapiens). Remarkably, their relationships have yet to be clarified. The confusion concerns the enigmatic position of nematodes. Traditionally, nematodes have occupied a basal position, in part because they lack a true body cavity. However, the leading hypothesis now joins nematodes with arthropods in a molting clade, Ecdysozoa, based on data from several genes.     

Effects of Caenorhabditis elegans (Nematoda: Rhabditidae) on yield and quality of the cultivated mushroom Agaricus bisporus

Thirteen species of saprobic rhabditid nematodes (11 genera) were identified from samples of compost and casing material collected from mushroom farms in the British Isles. Caenorhabditis elegans, the most frequently found saprobe, was mass-produced monoxenically and its effects on the cultivated mushroom, Agaricus bisporus (strain U3) were studied. C. elegans did not multiply in well-prepared, pasteurised, spawned compost, whereas casing material proved to be a highly suitable environment for its reproduction. An initial casing inoculum of 10⁶ nematodes/crate of compost (7.5 kg), caused a significant reduction in mushroom yield. Losses in total mushroom yields of 11%, 20% and 26% were caused by initial inoculum rates of 10⁶, 10⁷and 2 × 10⁷ nematodes/crate, respectively. Yields were negatively correlated with the initial nematode inoculation level and regression equations were derived. The nematode treatments caused fewer mushrooms to be produced and an absence of the usual distinctive flushing patterns. C. elegans caused considerable deterioration in mushroom quality and characteristic distortion of mushrooms. Individual sporophores were mis-shapen, notched and had brown or violet coloured grills. Up to 3.8%, 6.7% and 10.8% of total weight and 3.5%, 5.4% and 8% of total numbers of mushrooms were distorted at the three highest nematode inoculum rates tested. Weights and numbers of distorted mushrooms were positively correlated with the initial nematode population. C. elegans commonly colonised sporophores.     

Application of a Mathematical Model to Describe the Effects of Chlorpyrifos on Caenorhabditis elegans Development

Background

The nematode Caenorhabditis elegans is being assessed as an alternative model organism as part of an interagency effort to develop better means to test potentially toxic substances. As part of this effort, assays that use the COPAS Biosort flow sorting technology to record optical measurements (time of flight (TOF) and extinction (EXT)) of individual nematodes under various chemical exposure conditions are being developed. A mathematical model has been created that uses Biosort data to quantitatively and qualitatively describe C. elegans growth, and link changes in growth rates to biological events. Chlorpyrifos, an organophosphate pesticide known to cause developmental delays and malformations in mammals, was used as a model toxicant to test the applicability of the growth model for in vivo toxicological testing.

Methodology/Principal Findings

L1 larval nematodes were exposed to a range of sub-lethal chlorpyrifos concentrations (0–75 µM) and measured every 12 h. In the absence of toxicant, C. elegans matured from L1s to gravid adults by 60 h. A mathematical model was used to estimate nematode size distributions at various times. Mathematical modeling of the distributions allowed the number of measured nematodes and log(EXT) and log(TOF) growth rates to be estimated. The model revealed three distinct growth phases. The points at which estimated growth rates changed (change points) were constant across the ten chlorpyrifos concentrations. Concentration response curves with respect to several model-estimated quantities (numbers of measured nematodes, mean log(TOF) and log(EXT), growth rates, and time to reach change points) showed a significant decrease in C. elegans growth with increasing chlorpyrifos concentration.

Conclusions

Effects of chlorpyrifos on C. elegans growth and development were mathematically modeled. Statistical tests confirmed a significant concentration effect on several model endpoints. This confirmed that chlorpyrifos affects C. elegans development in a concentration dependent manner. The most noticeable effect on growth occurred during early larval stages: L2 and L3. This study supports the utility of the C. elegans growth assay and mathematical modeling in determining the effects of potentially toxic substances in an alternative model organism using high-throughput technologies.     

A Discrete Time Model for the Analysis of Medium-Throughput C. elegans Growth Data

Background

As part of a program to predict the toxicity of environmental agents on human health using alternative methods, several in vivo high- and medium-throughput assays are being developed that use C. elegans as a model organism. C. elegans-based toxicological assays utilize the COPAS Biosort flow sorting system that can rapidly measure size, extinction (EXT) and time-of-flight (TOF), of individual nematodes. The use of this technology requires the development of mathematical and statistical tools to properly analyze the large volumes of biological data.

Methodology/Principal Findings

Findings A Markov model was developed that predicts the growth of populations of C. elegans. The model was developed using observations from a 60 h growth study in which five cohorts of 300 nematodes each were aspirated and measured every 12 h. Frequency distributions of log(EXT) measurements that were made when loading C. elegans L1 larvae into 96 well plates (t = 0 h) were used by the model to predict the frequency distributions of the same set of nematodes when measured at 12 h intervals. The model prediction coincided well with the biological observations confirming the validity of the model. The model was also applied to log(TOF) measurements following an adaptation. The adaptation accounted for variability in TOF measurements associated with potential curling or shortening of the nematodes as they passed through the flow cell of the Biosort. By providing accurate estimates of frequencies of EXT or TOF measurements following varying growth periods, the model was able to estimate growth rates. Best model fits showed that C. elegans did not grow at a constant exponential rate. Growth was best described with three different rates. Microscopic observations indicated that the points where the growth rates changed corresponded to specific developmental events: the L1/L2 molt and the start of oogenesis in young adult C. elegans.

Conclusions

Quantitative analysis of COPAS Biosort measurements of C. elegans growth has been hampered by the lack of a mathematical model. In addition, extraneous matter and the inability to assign specific measurements to specific nematodes made it difficult to estimate growth rates. The present model addresses these problems through a population-based Markov model.     

Postembryonic RNAi in <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis>: a nematode insect parasite and host for insect pathogenic symbionts

Background  

Heterorhabditis bacteriophora is applied throughout the world for the biological control of insects and is an animal model to study interspecies interactions, e.g. mutualism, parasitism and vector-borne disease. H. bacteriophora nematodes are mutually associated with the insect pathogen, Photorhabdus luminescens. The developmentally arrested infective juvenile (IJ) stage nematode (vector) specifically transmits Photorhabdus luminescens bacteria (pathogen) in its gut mucosa to the haemocoel of insects (host). The nematode vector and pathogen alone are not known to cause insect disease. RNA interference is an excellent reverse genetic tool to study gene function in C. elegans, and it would be useful in H. bacteriophora to exploit the H. bacteriophora genome project, currently in progress.     

The twisted pharynx phenotype in <Emphasis Type="Italic">C. elegans</Emphasis>

Background  

The pharynx of C. elegans is an epithelial tube whose development has been compared to that of the embryonic heart and the kidney and hence serves as an interesting model for organ development. Several C. elegans mutants have been reported to exhibit a twisted pharynx phenotype but no careful studies have been made to directly address this phenomenon. In this study, the twisting mutants dig-1, mig-4, mnm-4 and unc-61 are examined in detail and the nature of the twist is investigated.     

Evolution of a polymodal sensory response network

Background  

Avoidance of noxious stimuli is essential for the survival of an animal in its natural habitat. Some avoidance responses require polymodal sensory neurons, which sense a range of diverse stimuli, whereas other stimuli require a unimodal sensory neuron, which senses a single stimulus. Polymodality might have evolved to help animals quickly detect and respond to diverse noxious stimuli. Nematodes inhabit diverse habitats and most nematode nervous systems are composed of a small number of neurons, despite a wide assortment in nematode sizes. Given this observation, we speculated that cellular contribution to stereotyped avoidance behaviors would also be conserved between nematode species. The ASH neuron mediates avoidance of three classes of noxious stimuli in Caenorhabditis elegans. Two species of parasitic nematodes also utilize the ASH neuron to avoid certain stimuli. We wanted to extend our knowledge of avoidance behaviors by comparing multiple stimuli in a set of free-living nematode species.     

Diversification and adaptive sequence evolution of <Emphasis Type="Italic">Caenorhabditis</Emphasis>lysozymes (Nematoda: Rhabditidae)

Background  

Lysozymes are important model enzymes in biomedical research with a ubiquitous taxonomic distribution ranging from phages up to plants and animals. Their main function appears to be defence against pathogens, although some of them have also been implicated in digestion. Whereas most organisms have only few lysozyme genes, nematodes of the genus Caenorhabditis possess a surprisingly large repertoire of up to 15 genes.     

Identification of a gonadotropin-releasing hormone receptor orthologue in Caenorhabditis elegans

Background  

The Caenorhabditis elegans genome is known to code for at least 1149 G protein-coupled receptors (GPCRs), but the GPCR(s) critical to the regulation of reproduction in this nematode are not yet known. This study examined whether GPCRs orthologous to human gonadotropin-releasing hormone receptor (GnRHR) exist in C. elegans.     

Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay

Background

Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative.

Methodology/Principal Findings

Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 µM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC₅₀ of 2 µM.

Conclusions/Significance

The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems.     

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.