Optimizing culture conditions for free-living stages of the nematode parasite Strongyloides ratti.

The rat parasitic nematode Strongyloides ratti (S. ratti) has recently emerged as a model system for various aspects of parasite biology and evolution. In addition to parasitic parthenogenetic females, this species can also form facultative free-living generations of sexually reproducing adults. These free-living worms are bacteriovorous and grow very well when cultured in the feces of their host. However, in fecal cultures the worms are rather difficult to find for observation and experimental manipulation. Therefore, it has also been attempted to raise S. ratti on Nematode Growth Media (NGM) plates with Escherichia coli OP50 as food, exactly as described for the model nematode Caenorhabditis elegans. Whilst worms did grow on these plates, their longevity and reproductive output compared to fecal cultures were dramatically reduced. In order to improve the culture success we tested other plates occasionally used for C. elegans and, starting from the best performing one, systematically varied the plate composition, the temperature and the food in order to further optimize the conditions. Here we present a plate culturing protocol for free-living stages of S. ratti with strongly improved reproductive success and longevity.

Life cycle stage-resolved proteomic analysis of the excretome/secretome from Strongyloides ratti--identification of stage-specific proteases.

A wide range of biomolecules, including proteins, are excreted and secreted from helminths and contribute to the parasite's successful establishment, survival, and reproduction in an adverse habitat. Excretory and secretory proteins (ESP) are active at the interface between parasite and host and comprise potential targets for intervention. The intestinal nematode Strongyloides spp. exhibits an exceptional developmental plasticity in its life cycle characterized by parasitic and free-living generations. We investigated ESP from infective larvae, parasitic females, and free-living stages of the rat parasite Strongyloides ratti, which is genetically very similar to the human pathogen, Strongyloides stercoralis. Proteomic analysis of ESP revealed 586 proteins, with the largest number of stage-specific ESP found in infective larvae (196), followed by parasitic females (79) and free-living stages (35). One hundred and forty proteins were identified in all studied stages, including anti-oxidative enzymes, heat shock proteins, and carbohydrate-binding proteins. The stage-selective ESP of (1) infective larvae included an astacin metalloproteinase, the L3 Nie antigen, and a fatty acid retinoid-binding protein; (2) parasitic females included a prolyl oligopeptidase (prolyl serine carboxypeptidase), small heat shock proteins, and a secreted acidic protein; (3) free-living stages included a lysozyme family member, a carbohydrate-hydrolyzing enzyme, and saponin-like protein. We verified the differential expression of selected genes encoding ESP by qRT-PCR. ELISA analysis revealed the recognition of ESP by antibodies of S. ratti-infected rats. A prolyl oligopeptidase was identified as abundant parasitic female-specific ESP, and the effect of pyrrolidine-based prolyl oligopeptidase inhibitors showed concentration- and time-dependent inhibitory effects on female motility. The characterization of stage-related ESP from Strongyloides will help to further understand the interaction of this unique intestinal nematode with its host.

Development of free-living stages of Strongyloides ratti under different temperature conditions.

It is well known that the Strongyloides species have two different developmental courses-direct and indirect development-and selection of these courses is affected by various environmental factors. This study examined the effect of temperature on the development of first-stage larvae (L1s) of Strongyloides ratti, to clarify how larvae adapt and survive at unsuitable temperatures. It was revealed that L1s cultured at 4 or 10 °C for 120 h could not develop because of growth arrest or delay. However, L1s could develop after transfer to culture at 25 °C for 48 h. Although larvae cultured at 25 °C take indirect development, larvae subjected to low-temperature stimulation (at 4 or 10 °C) take direct development into infective third-stage larvae (L3s), and only 1 min of low-temperature stimulation was sufficient to induce direct development. Morphological study of low-temperature-stimulated L3s revealed that those stimulated at 4 °C (L3-4) showed less development, but those stimulated at 10 °C (L3-10) developed as well as the control (no low-temperature stimulation). Furthermore, we revealed that L3-10 showed similar infectivity to the control when they were injected subcutaneously into rats as the final host, which indicated that L3-10 grew normally. We conclude that S. ratti has a survival strategy of growth arrest or delay if excreted in cold conditions. Moreover, even if they start development after transfer to suitable conditions, they memorize low-temperature stimulation, which leads them to direct development thereafter so that they can immediately infect the final host.

In vitro anthelmintic activity of the essential oils of Zanthoxylum zanthoxyloides and Newbouldia laevis against Strongyloides ratti.

The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: γ-terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: ß-caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC(50) and IC(90)) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC(50) values (19.5 and 18.2 µg/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC(50) = 2.5 µg/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC(50) = 46 µg/ml), N. laevis (IC(50) = 51 µg/ml), and the control [levamisole (IC(50) = 36 µg/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC(50) values higher than 50 µg/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.

Anthelmintic Activity and Cytotoxic Effects of Compounds Isolated from the Fruits of Ozoroa insignis Del. (Anacardiaceae).

Ozoroa insignis Del. is an ethnobotanical plant widely used in traditional medicine for various ailments, including schistosomiasis, tapeworm, and hookworm infections. From the so far not investigated fruits of Ozoroa insignis, the anthelmintic principles could be isolated through bioassay-guided isolation using Caenorhabditis elegans and identified by NMR spectroscopic analysis and mass spectrometric studies. Isolated 6-[8(Z)-pentadecenyl] anacardic (1), 6-[10(Z)-heptadecenyl] anacardic acid (2), and 3-[7(Z)-pentadecenyl] phenol (3) were evaluated against the 5 parasitic organisms Schistosoma mansoni (adult and newly transformed schistosomula), Strongyloides ratti, Heligmosomoides polygyrus, Necator americanus, and Ancylostoma ceylanicum, which mainly infect humans and other mammals. Compounds 1-3 showed good activity against Schistosoma mansoni, with compound 1 showing the best activity against newly transformed schistosomula with 50% activity at 1µM. The isolated compounds were also evaluated for their cytotoxic properties against PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) cell lines, whereby compounds 2 and 3 showed antiproliferative activity in both cancer cell lines, while compound 1 exhibited antiproliferative activity only on PC-3 cells. With an IC50 value of 43.2 µM, compound 3 was found to be the most active of the 3 investigated compounds.

The effect of infection history on the fitness of the gastrointestinal nematode Strongyloides ratti.

SUMMARY: Hosts in nature will often acquire infections by different helminth species over their lifetime. This presents the potential for new infections to be affected (particularly via the host immune response) by a host's history of previous con- or hetero-specific infection. Here we have used an experimental rat model to investigate the consequences of a history of primary infection with either Nippostrongylus brasiliensis, Strongyloides venezuelensis or S. ratti on the fitness of, and immunological response to, secondary infections of S. ratti. We found that a history of con-specific, but not hetero-specific, infection reduced the survivorship of S. ratti; the fecundity of S. ratti was not affected by a history of either con- or hetero-specific infections. We also found that a history of con-specific infection promoted Th2-type responses, as shown by increased concentrations of total IgE, S. ratti-specific IgG1, rat mast cell protease II (RMCPII), IL4 (but decreased concentrations of IFNgamma) produced by mesenteric lymph node cells in response to S. ratti antigen. Additionally, S. ratti-specific IgG1 was positively related to the intensity of both primary and secondary infections of S. ratti. Hetero-specific primary infections were only observed to affect the concentration of total IgE and RMCPII. The overall conclusion of these experiments is that the major immunological effect acting against an infection is induced by the infection itself and that there is little effect of prior infections of the host.

Genes important in the parasitic life of the nematode Strongyloides ratti.

Parasitic nematodes are important pathogens of humans and other animals. The genus Strongyloides has both a parasitic and a free-living adult generation. S. ratti infections of its rat host are negatively affected by the host immune response, such that a month after infection, worms are lost from the hosts. Here we have investigated the changes in parasite gene expression that occur as the anti-S. ratti immune pressure increases. Existing S. ratti expressed sequence tags were used to construct a microarray consisting of 2227 putative genes. This was probed with cDNA prepared from parasites subject to low or high immune pressures. There are significant changes in the gene expression of S. ratti when subject to different immune pressures. Most of the genes whose expression changes have no significant alignment to known genes. These data together with previous S. ratti EST data were then used to identify genes that we hypothesise are central to the parasitic life of S. ratti and, perhaps, other parasitic nematodes. These analyses have identified genes likely to play a key role in the parasitic life of S. ratti; these genes should be the priority for further investigation.

Microarray analysis of gender- and parasite-specific gene transcription in Strongyloides ratti.

The molecular mechanisms by which parasitic nematodes reproduce and have adapted to life within a host are unclear. In the present study, microarray analysis was used to explore differential transcription among the different stages and sexes of Strongyloides ratti, a parasitic nematode of brown rats. Specifically, gender-biased transcription between free-living females and free-living males, and parasitic-biased transcription between parasitic females and free-living females was determined. Of the estimated 3,688 distinct transcripts represented on the microarray, 743 (20%) exhibited male-biased transcription of >1.4-fold (2(0.5)), 689 (19%) female-biased transcription, 418 (11%) parasitic-biased transcription and 305 (8%) free-living-biased transcription. Among those transcripts that exhibited the highest levels of differential transcription, an orthologue of major sperm protein was identified in males, distinct aspartic protease orthologues in either parasitic or in free-living females, and orthologues of hsp-17 chaperone in parasitic females. These 3,688 transcripts were separated into 12 clusters, such that the pattern of transcription between life-stages and biological replicates was similar among transcripts within a cluster and dissimilar between clusters. Using annotation inferred from Caenorhabditis elegans, gene ontology terms over-represented in one or more clusters were identified and showed that female-biased transcription was associated with genes involved in reproductive processes and larval development, male-biased transcription was linked to genes involved in metabolism, and free-living-biased transcription related to genes involved in the regulation of body fluids and response to external stimulus. The association of gene ontology with parasite-biased transcription was less clear. The present findings for S. ratti provide a basis for a detailed exploration of differentially regulated molecules and might assist in the search for novel drug or vaccine targets in parasitic nematodes.

Experimental evolution of parasite life-history traits in Strongyloides ratti (Nematoda).

Evolutionary ecology predicts that parasite life-history traits, including a parasite's survivorship and fecundity within a host, will evolve in response to selection and that their evolution will be constrained by trade-offs between traits. Here, we test these predictions using a nematode parasite of rats, Strongyloides ratti, as a model. We performed a selection experiment by passage of parasite progeny from either early in an infection ('fast' lines) or late in an infection ('slow' lines). We found that parasite fecundity responded to selection but that parasite survivorship did not. We found a trade-off mediated via conspecific density-dependent constraints; namely, that fast lines exhibit higher density-independent fecundity than slow lines, but fast lines suffered greater reduction in fecundity in the presence of density-dependent constraints than slow lines. We also found that slow lines both stimulate a higher level of IgG1, which is a marker for a Th2-type immune response, and show less of a reduction in fecundity in response to IgG1 levels than for fast lines. Our results confirm the general prediction that parasite life-history traits can evolve in response to selection and indicate that such evolutionary responses may have significant implications for the epidemiology of infectious disease.

An expressed sequence tag analysis of the life-cycle of the parasitic nematode Strongyloides ratti.

14,761 expressed sequence tags (ESTs) were generated, representing five stages during the parasitic and free-living phases of the life-cycle of the parasitic nematode Strongyloides ratti. These ESTs formed 4152 clusters, of which 97% contained 10 or fewer ESTs and 66% were singletons. These 4152 clusters are likely to represent approximately 20% of S. ratti's genes. The clusters' consensus sequences were used to assign each cluster to one of three databases: (i) Caenorhabditis elegans and C. briggsae sequences; (ii) other nematode sequences; (iii) non-nematode sequences. This approach has identified putative nematode-specific genes, that may be targets for developing approaches for parasitic nematode control. Approximately 25% of the clusters have no significant alignments and may therefore represent novel genes. The EST representation between the libraries was used to analyse stage-specific or -biased expression in silico. This showed that 81% of clusters are present in only one library and 12% are present in any two libraries, indicating substantial stage-specificity of gene expression. The 30-most abundantly expressed clusters were analysed in further detail. Many of these have significantly different parasitic- or free-living-specific or -biased expression. Many of the parasitic-specific genes are, as yet, uncharacterised: one of these represents 25% of all ESTs obtained from the parasitic stage.

daf-7 and the development of Strongyloides ratti and Parastrongyloides trichosuri.

daf-7 is a key ligand in one of the three pathways that control dauer larva development in Caenorhabditis elegans. Given the similarities between dauer larvae of free-living nematodes and third stage infective larvae of animal parasitic nematodes, we hypothesised that daf-7 may be involved in the development of these infective larvae. To investigate this, we cloned daf-7 orthologues from Strongyloides ratti and Parastrongyloides trichosuri and analysed their RNA level by semi-quantitative RT-PCR during the S. ratti and P. trichosuri life cycles and in a range of in vitro and in vivo conditions. We found that, in both species, the RNA level of daf-7 was low in free-living stages but peaked in the infective L3 (iL3) stage with little or no expression in the parasitic stages. This contrasts with the daf-7 RNA level in C. elegans, which peaks in L1, decreases thereafter, and is absent in dauer larvae. The RNA level of daf-7 in infective larvae was reduced by larval penetration of host skin or development in the host, but not by a shift to the body temperature of the host.

Strongyloides ratti: chemokinesis of glycolytic enzyme- and lectin-treated third-stage infective larvae in vitro.

The infective third-stage larvae (L3s) of Strongyloides ratti, a parasitic nematode in rodents, showed two types of chemokinesis on a gradient of sodium chloride (NaCl) in an in vitro agarose tracking assay. The types were a consistent directional avoidance behavior under unfavorable environmental conditions and a reduced avoidance behavior under favorable conditions. We examined the effects of treatments with glycolytic enzymes and lectins by analyzing the avoidance behavior. L-Fucose dehydrogenase, hyaluronidase, beta-glucosidase, alpha-mannosidase, beta-galactosidase, concanavalin A, wheat germ agglutinin and soybean agglutinin exhibited inhibitory or enhancive effects on chemokinesis. We also confirmed the sites of the amphids of L3s aside from the mouth at the anterior end by scanning electron microscopy, and that concanavalin A-binding sites existed in the vicinity of the amphids using lectin-histochemistry. The carbohydrate moieties in the amphids of S. ratti L3s may play an important role as chemosensors in perceiving environmental cues.

Mating in the nematode parasite Strongyloides ratti: proof of genetic exchange.

Strongyloides, a parasitic nematode, has a complex life cycle that represents a transition between free-living and purely parasitic ways of life. By using minisatellite DNA fingerprinting, we have detected a large number of polymorphic loci in cloned lines of S. ratti. In controlled crosses between free-living adults of S. ratti, we show that genetic exchange, consistent with normal sexual reproduction, occurs. This is in contrast to earlier cytological studies which claimed that reproduction in this organism was by pseudogamy, and thus that inheritance was maternal only.

Developmental switching in the parasitic nematode Strongyloides ratti.

Strongyloides ratti is a nematode parasite of rats. It is able to undergo two types of development outside the host: heterogonic (free-living adults and sexual reproduction) and homogonic (direct larval development). Homogonic development has a number of similarities with the development of the dauer stage of free-living nematodes, including Caenorhabditis elegans. Using isofemale lines of the parasite, factors that control this developmental choice have been investigated. Isofemale lines can be selected for both heterogonic and homogonic development, but are still able to respond to environmental conditions. By using temperature shift experiments it has been possible to determine when larvae become developmentally committed. All larvae are developmentally committed after 24 h at 19 degrees C.

The control of morph development in the parasitic nematode Strongyloides ratti.

The parasitic nematode Strongyloides ratti has a complex life cycle. The progeny of the parasitic females can develop into three distinct morphs, namely directly developing infective third-stage larvae (iL3s), free-living adult males and free-living adult females. We have analysed of the effect of host immune status (an intra-host factor), environmental temperature (an extra-host factor) and their interaction on the proportion of larvae that develop into these three morphs. The results are consistent with the developmental decision of larvae being controlled by at least two discrete developmental switches. One is a sex-determination event that is affected by host immune status and the other is a switch between alternative female morphs that is affected by both host immune status and environmental temperature. These findings clarify the basis of the life cycle of S. ratti and demonstrate how such complex life cycles can result from a combination of simple developmental switches.

An alternative protocol for the serial passage and maintenance of a homogonic strain of Strongyloides ratti in female Sprague-Dawley rats.

A method which does not involve the tedious use of watch glass coprocultures for obtaining filariform infective (L3) larvae of Strongyloides ratti from faecal pellets of infected Sprague-Dawley rats is described. The alternative method utilises Baermannization (18 h) of faecal pellets to yield rhabditiform (L1) larvae of S. ratti and their subsequent culture for 72 h at 19 degrees C in tissue-culture-flasks containing only dechlorinated tap water to yield infective filariform (L3) larvae. The yields and infectivity of the L3 larvae obtained from the two methods were essentially similar.

Steady-state content of glycolytic/tricarboxylic acid-cycle intermediates, adenine nucleotide pools and the cellular redox-status in the infective (L3) larvae of (homogonic) Strongyloides ratti.

Infective (L3) larvae of Strongyloides ratti (homogonic strain) were freeze-clamped (-196 degrees C) and the steady-state content of the glycolytic, Krebs tricarboxylic acid (KTA)-cycle intermediates and adenine nucleotides analysed. Comparison of the mass-action ratios (MARs) of the glycolytic enzymes with their apparent equilibrium constants (K9eq) indicate that phosphoglucomutase, glucosephosphate isomerase, triosephosphate isomerase, phosphoglyceromutase and phosphopyruvate hydratase reactions were all at or near equilibrium, whilst hexokinase, phosphofructokinase and pyruvate kinase were displaced from equilibrium. The S. ratti aldolase and myokinase appear to be somewhat displaced from equilibrium and thus may have pseudoregulatory roles. The adenylate energy charge (AEC), ATP/ADP ratio and the available adenylate energy (AAE) indices were 0.9 +/- 0.04, 8.76 +/- 1.5 and 397 +/- 43, respectively. The free [NAD+]/[NADH+H+] ratio of the cytoplasmic compartment of S. ratti L3 larvae calculated employing the steady-state content of the oxidised and reduced substrates of lactate dehydrogenase (E.C. 1.1.1.27) and the combined glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.12)/3-phosphoglycerate kinase (E.C. 2.7.2.3) system were ca. 523 and 1200, respectively. The free[NAD+]/[NADH+H+] ratio in the mitochondrial compartment of S. ratti L3 larvae calculated using the malate dehydrogenase (E.C. 1.1.1.37) equilibrium was found to be 1962:1. The data is discussed with respect to the predominantly aerobic nature of the energy metabolism of the L3 larvae.

Chemical mutagenesis of the parasitic nematode Strongyloides ratti to isolate ivermectin resistant mutants.

We describe a strategy for the mutagenesis of the free-living adult generation of Strongyloides ratti and selection of worms carrying new mutations in the subsequent F2 generation of infective larvae. We demonstrate that this strategy is successful via the selection of infective larvae that are resistant to the anthelmintic ivermectin at a concentration of 10 ng/ml. The majority of these larvae were unable to give rise to patent infections when used to infect parasite naive rats, implying that the majority of the ivermectin resistance mutations confer pleiotropic defects on parasitic, but not on free-living, development.

Nematocidal activities of thiabendazole and ivermectin against the larvae of Strongyloides ratti and S. venezuelensis.

With the aim of developing therapeutic agents for strongyloidosis, the disease caused by infection with Strongyloides stercoralis, we established a novel assay technique using S. ratti and S. venezuelensis as models for S. stercoralis. The newly developed assay technique was found to more accurately represent treatment-induced larval paralysis than existing assays. Our method uses paper disks impregnated with the test solution, which even allows materials that are sparingly soluble in water to be tested. An inverted microscope was used to observe the larval states, and these states were recorded using a digital camera. We observed the activities of ivermectin and thiabendazole against larvae and calculated larval motility and velocity. These two factors were then combined to determine the overall viability of larvae at selected concentrations. The activities of the anthelmintics were compared by calculating the concentrations at which 50% viability was demonstrated, or in other words, the concentration at which paralysis was caused in 50% of the individuals (50% paralysis concentration; PC(50)). Evaluations after 24h of exposure yielded the following reproducible PC(50) values for ivermectin and thiabendazole, respectively: S. ratti, 2.4 and 140 microM; and S. venezuelensis, 2.3 and 190 microM. After treatment with ivermectin, there was a tendency for larval motility to be greater than that of the controls at low concentrations, a result that might be associated with its mechanism of action.

Conserved miRNAs are candidate post-transcriptional regulators of developmental arrest in free-living and parasitic nematodes.

Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions, Caenorhabditis elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, Pristionchus pacificus, and Strongyloides ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs, and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer versus nondauer stages and infective larvae versus free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved "dauer-infective" expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest and long-term survival in free-living and parasitic nematodes.