Nanoparticle imaging and diagnostic of Caenorhabditis elegans intracellular pH

A novel diagnostic tool has been developed for the characterization of intracellular pH (pHi) in the model organism Caenorhabditis elegans. This tool exploits the chemical stability of colloidal silica and the pH sensitivity of certain fluorescent dyes. Once ingested, the fluorescent colloidal dispersion yields a reliable visual indication of pH without the use of chemical fixatives or damaging the nematode. The pH-sensitive silica nanoparticles were visualized by confocal microscopy, and the fluorescence spectra from the internally referenced colloidal particulates were measured. By comparing the fluorescence profile of colloids in wild-type (N2) and mutant (eat-3) C. elegans against a calibration series, the intestinal pHi could be established in each population. The rapid characterization of pHi using this inexpensive nonintrusive technique has significant implications for disease research where C. elegans is used as a model organism.     

Atypical (RIO) protein kinases from Haemonchus contortus--promise as new targets for nematocidal drugs

Almost nothing is known about atypical kinases in multicellular organisms, including parasites. Supported by information and data available for the free-living nematode, Caenorhabditis elegans, and other eukaryotes, the present article describes three RIO kinase genes, riok-1, riok-2 and riok-3, from Haemonchus contortus, one of the most important parasitic nematodes of small ruminants. Analyses of these genes and their products predict that they each play critical roles in the developmental pathways of parasitic nematodes. The findings of this review indicate prospects for functional studies of these genes in C. elegans (as a surrogate) and opportunities for the design of a novel class of nematode-specific inhibitors of RIO kinases. The latter aspect is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock.     

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 natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans

Animal predators can track prey using their keen sense of smell. The bacteriovorous nematode Caenorhabditis elegans employs sensitive olfactory sensory neurons that express vertebrate-like odor receptors to locate bacteria. C. elegans displays odor-related behaviors such as attraction, aversion and adaptation, but the ecological significance of these behaviors is not known. Using a combination of food microbiology and genetics, we elucidate a possible predator–prey relationship between C. elegans and lactic acid bacteria (LAB) in rotting citrus fruit. LAB produces the volatile odor diacetyl as an oxidized by-product of fermentation in the presence of citrate. We show that C. elegans is attracted to LAB when grown on citrate media or Citrus medica L, commonly known as yuzu, a citrus fruit native to East Asia, and this attraction is mediated by the diacetyl odor receptor, ODR-10. We isolated a wild LAB strain and a wild C. elegans-related nematode from rotten yuzu, and demonstrate that the wild nematode was attracted to the diacetyl produced by LAB. These results not only identify an ecological function for a C. elegans olfactory behavior, but contribute to the growing understanding of ecological relationships between the microbial and metazoan worlds.     

The evolution of plasticity of dauer larva developmental arrest in the nematode Caenorhabditis elegans

Organisms can end up in unfavourable conditions and to survive this they have evolved various strategies. Some organisms, including nematodes, survive unfavourable conditions by undergoing developmental arrest. The model nematode Caenorhabditis elegans has a developmental choice between two larval forms, and it chooses to develop into the arrested dauer larva form in unfavourable conditions (specifically, a lack of food and high population density, indicated by the concentration of a pheromone). Wild C. elegans isolates vary extensively in their dauer larva arrest phenotypes, and this prompts the question of what selective pressures maintain such phenotypic diversity? To investigate this we grew C. elegans in four different environments, consisting of different combinations of cues that can induce dauer larva development: two combinations of food concentration (high and low) in the presence or absence of a dauer larva-inducing pheromone. Five generations of artificial selection of dauer larvae resulted in an overall increase in dauer larva formation in most selection regimes. The presence of pheromone in the environment selected for twice the number of dauer larvae, compared with environments not containing pheromone. Further, only a high food concentration environment containing pheromone increased the plasticity of dauer larva formation. These evolutionary responses also affected the timing of the worms’ reproduction. Overall, these results give an insight into the environments that can select for different plasticities of C. elegans dauer larva arrest phenotypes, suggesting that different combinations of environmental cues can select for the diversity of phenotypically plastic responses seen in C. elegans.     

Automated High-Throughput Quantification of Mitotic Spindle Positioning from DIC Movies of Caenorhabditis Embryos

The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes.To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics.Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye.     

Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans

DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.     

The Impact of Bacterial Diet on the Migration and Navigation of Caenorhabditis elegans

Can diet have a significant impact on the ability of organisms to sense and locate food? Focusing on the bacterial feeder Caenorhabditis elegans, we investigated what effect preconditioning on a range of bacterial substrates had on the subsequent chemotaxis process involved in the nematode locating other bacterial populations. Remarkably, we found that C. elegans, initially fed on a diet of Escherichia coli OP50, was significantly impaired in finding E. coli OP50 populations, compared to other available bacterial populations (P < 0.001). We found similar results for another bacterial feeding nematode species, suggesting that a general “substrate legacy” may operate across a wide range of organisms. We discuss this important finding with respect to the variation in response exhibited within a given nematode population, and the impact nematode migration has on bacterial dispersal in the environment.     

Interactions of anthelmintic drugs in Caenorhabditis elegans neuro-muscular ion channel mutants

Due to the increasing development of anthelmintic resistance in nematodes worldwide, it is important to search for anthelmintic compounds with new modes of action and also to investigate the possibility to combine compounds with possible synergistic effects. There might also be the chance to take advantage of the fact that nematode populations which have developed resistance against one anthelmintic class might respond hypersusceptibly to another drug class. The aim of this study was to investigate responses of Caenorhabditis elegans populations with mutations in neuro-muscular ion channels to different anthelmintic classes. Furthermore, potential synergistic effects between two anthelmintic compounds from different classes, i.e. emodepside and tribendimidine, were studied. Although there was neither a synergistic nor an antagonistic effect between emodepside and tribendimidine, other types of interactions could be identified. The C. elegans GABA_A-receptor (GABA_A-R) unc-49 mutants, showing decreased emodepside susceptibility, were more susceptible to tribendimidine than wild-type C. elegans. In contrast, the reverse phenomenon – hypersusceptibility to emodepside in tribendimidine resistant acetylcholine-receptor (AChR) loss of function mutants – was not observed. Moreover, the slo-1 mutant strain (completely emodepside resistant) also showed hypersusceptibility to piperazine. Interestingly, neither the GABA_A-R unc-49 mutants nor the AChR mutants showed decreased susceptibility against piperazine, although there were some studies that indicated an involvement of GABA_A-R or AChR in the piperazine mode of action. In conclusion, the present study provides evidence suggesting that interactions between commercially available anthelmintic drugs with different modes of action might be a relatively common phenomenon but this has to be carefully worked out for each anthelmintic and each anthelmintic drug combination. Moreover, results obtained in C. elegans will have to be confirmed using parasitic nematodes in the future.     

Complete killing of Caenorhabditis elegans by Burkholderia pseudomallei is dependent on prolonged direct association with the viable pathogen

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. Much remains to be known about the contributions of genotypic variations within the bacteria and the host, and environmental factors that lead to the manifestation of the clinical symptoms of melioidosis.

Methodology/Principal Findings

In this study, we showed that different isolates of B. pseudomallei have divergent ability to kill the soil nematode Caenorhabditis elegans. The rate of nematode killing was also dependent on growth media: B. pseudomallei grown on peptone-glucose media killed C. elegans more rapidly than bacteria grown on the nematode growth media. Filter and bacteria cell-free culture filtrate assays demonstrated that the extent of killing observed is significantly less than that observed in the direct killing assay. Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection.

Conclusions/Significance

A combination of genetic and environmental factors affects virulence. In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.     

Caenorhabditis elegans N-glycan Core β-galactoside Confers Sensitivity towards Nematotoxic Fungal Galectin CGL2

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galβ1,4Fucα1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galβ1,4Fucα1,6GlcNAc trisaccharide at 1.5 Å resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.     

Isolation ofCaenorhabditis elegans mutants lacking alcohol dehydrogenase activity

Alcohol dehydrogenase (ADH) and the genes encoding this enzyme have been studied intensively in a broad range of organisms. Little, however, has been reported on ADH in the free-living nematodeCaenorhabiditis elegans. Extracts of wild-typeC. elegans contain ADH activity and display a single band of activity on a native polyacrylamide gel. Reaction rate for alcohol oxidation is more rapid with higher molecular weight alcohols as substrate than with ethanol. Primary alcohols are preferred to secondary alcohols.C. elegans is sensitive to allyl alcohol, a compound that has been used to select for ADH-null mutants of several organisms. Allyl alcohol-resistant mutant strains were selected from ethylmethanesulfonate (EMS)-mutagenized nematode populations. ADH activity was measured in extracts from eight of these strains and was found to be low or nondetectable. These results form a basis for molecular and genetic characterization of ADH expression inC. elegans.     

Shifts in the Distribution of Mass Densities Is a Signature of Caloric Restriction in Caenorhabditis elegans

Although the starvation response of the model multicellular organism Caenorhabditis elegans is a subject of much research, there is no convenient phenotypic readout of caloric restriction that can be applicable to large numbers of worms. This paper describes the distribution of mass densities of populations of C. elegans, from larval stages up to day one of adulthood, using isopycnic centrifugation, and finds that density is a convenient, if complex, phenotypic readout in C. elegans. The density of worms in synchronized populations of wildtype N2 C. elegans grown under standard solid-phase culture conditions was normally distributed, with distributions peaked sharply at a mean of 1.091 g/cm³ for L1, L2 and L3 larvae, 1.087 g/cm³ for L4 larvae, 1.081 g/cm³ for newly molted adults, and 1.074 g/cm³ at 24 hours of adulthood. The density of adult worms under starvation stress fell well outside this range, falling to a mean value of 1.054 g/cm³ after eight hours of starvation. This decrease in density correlated with the consumption of stored glycogen in the food-deprived worms. The density of the worms increased when deprived of food for longer durations, corresponding to a shift in the response of the worms: worms sacrifice their bodies by retaining larvae, which consume the adults from within. Density-based screens with the drug Ivermectin on worms cultured on single plates resulted in a clear bimodal (double-peaked) distribution of densities corresponding to drug exposed and non-exposed worms. Thus, measurements of changes in density could be used to conduct screens on the effects of drugs on several populations of worms cultured on single plates.