Chemogenomic approach to identifying nematode chemoreceptor drug targets in the entomopathogenic nematode Heterorhabditis bacteriophora.

Parasitic nematodes constitute one of the major threats to human health, causing diseases of major socioeconomic importance worldwide. Recent estimates indicate that more than 1 billion people are infected with parasitic nematodes around the world. Current measures to combat parasitic nematode infections include anthelmintic drugs. However, heavy exposure to anthelmintics has selected populations of livestock parasitic nematodes that are no longer susceptible to the drugs, rendering several anthelmintics useless for parasitic nematode control in many areas of the world. The rapidity with which anthelmintic resistance developed in response to these drugs suggests that increasing the selective pressure on human parasitic nematodes will also rapidly generate resistant worm populations. Therefore, development of new anthelmintics is of major importance before resistance becomes widespread in human parasitic nematode populations. G-Protein Coupled Receptors (GPCRs) represent an important target for many pharmacological interventions due to their ubiquitous expression in various cell types. GPCRs contribute to numerous physiological processes, and their ligand binding sites located on cell surfaces make them accessible targets and attractive substrates in terms of druggability. In fact, ∼35 % of Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved drugs target GPCRs and their associated proteins, with over 300 additional drugs targeting GPCRs at the clinical trial stage. Nematode Chemosensory GPCRs (NemChRs) are unique to nematodes, and therefore represent ideal substrates for target-based drug discovery. Here we set out to identify NemChRs that are transcriptionally active inside the host, and to use these NemChRs in a reverse pharmacological screen to impede parasitic development. Our data identified several NemChRs, and we focused on one that was expressed in neuronal cells and exhibited the highest fold change in transcription after host activation. Next, we performed homology modelling and molecular dynamics simulations of this NemChR in order to conduct a virtual screening campaign to identify candidate drug targets which were ranked and selected for experimental testing in bioassays. Taken together, our results identify and characterize a candidate NemChR drug target, and provide a chemogenomic pipeline for identifying nematicide substrates.

Combined use of ivermectin, dimethyl sulfoxide, mineral oil and nematophagous fungi to control Rhabditis spp.

Rhabditis spp., is a nematode known to cause otitis externa, an infection difficult to control, in cattle reared within tropical regions. The objective of this study was to assess the combined use of ivermectin 1%, dimethyl sulfoxide 1% and mineral oil 100% containing nematophagous fungi of both Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34) species to control in vitro Rhabditis spp. Thus, 12 experimental groups were designed with eight replicates each: G1 (nematodes + AC001); G2 (nematodes + NF34); G3 (nematodes + ivermectin 1%/positive control); G4 (nematodes + dimethyl sulfoxide 1%/positive control); G5 (nematodes + mineral oil 100%/positive control); G6 (nematodes + AC001 + ivermectin 1%); G7 (nematodes + NF34 + ivermectin 1%); G8 (nematodes + AC001 + mineral oil 100%); G9 (nematodes + NF34 + mineral oil 100%); G10 (nematodes + AC001 + dimethyl sulfoxide 1%); G11 (nematode + NF34 + dimethyl sulfoxide 1%); G12 (nematode + distilled water/negative control). The results demonstrated that all experimentally treated groups differed statistically (p < 0.01) from the control group. In the present study, the use of dimethyl sulfoxide 1% and mineral oil 100% in conjunction with conidia fungi portrayed noteworthy outcomes, which represents a future premise for the combined use of nematophagous fungi within these vehicles in both controlling Rhabditis spp.

Ethanolic extracts of Inula viscosa, Salix alba and Quercus calliprinos, negatively affect the development of the entomopathogenic nematode, Heterorhabditis bacteriophora - A model to compare gastro-intestinal nematodes developmental effect.

Heterorhabditis bacteriophora can represent a model system for herbal medication against gastro-intestinal strongylid parasites in determining the recovery and development due to their unique parasitic infectious cycle. The fact that plant extracts impair nematode development is known but their differential impact on stages of the life cycle of H. bacteriophora has never been investigated. We examined the developmental stages resumed from eggs, young juveniles (J1-3), infective juveniles (IJs), young and adult hermaphrodites of H. bacteriophora upon exposure to crude ethanolic extracts of Inula viscosa, Salix alba, and Quercus calliprinos at concentrations of 600, 1200, and 2400ppm. Our results showed that plant extracts were highly toxic to the survival of the eggs and young juveniles J1 to J3 at all concentrations. The plant extracts inhibited their development and were associated with low reproduction parameters (i.e. fecundity and viability of eggs). The IJs, J4, young and developed hermaphrodites displayed concentration-dependent negative effect on development with less egg count, poor vulval muscle development, loss of egg laying capacity and progeny development by matricidal hatching. Plant extract of I. viscosa at low (600ppm) concentration did not impair vulval development. These results suggest that these plant extracts show potential for the control of parasitic rhabditids.

Highly Potent Extracts from Pea (Pisum sativum) and Maize (Zea mays) Roots Can Be Used to Induce Quiescence in Entomopathogenic Nematodes.

Root exudates can play an important role in plant-nematode interactions. Recent studies have shown that the root cap exudates obtained from several plant species trigger a state of dormancy or quiescence in various genera of nematodes. This phenomenon is not only of fundamental ecological interest, but also has application potential if the plant-produced compound(s) could be used to control harmful nematodes or help to prolong the shelf-life of beneficial entomopathogenic nematodes (EPNs). The identification of the compound(s) involved in quiescence induction has proven to be a major challenge and requires large amounts of active material. Here, we present a high-throughput method to obtain bioactive root extracts from flash-frozen root caps of green pea and maize. The root cap extract obtained via this method was considerably more potent in inducing quiescence than exudate obtained by a previously used method, and consistently induced quiescence in the EPN Heterorhabditis megidis, even after a 30-fold dilution. Extracts obtained from the rest of the root were equally effective in inducing quiescence. Infective juveniles (IJs) of H. megidis exposed to these extracts readily recovered from their quiescent state as soon as they were placed in moist soil, and they were at least as infectious as the IJs that had been stored in water. Excessive exposure of IJs to air interfered with the triggering of quiescence. The implications of these results and the next steps towards identification of the quiescence-inducing compound(s) are discussed from the perspective of applying EPN against soil-dwelling insect pests.

An investigation on the chemotactic responses of different entomopathogenic nematode strains to mechanically damaged maize root volatile compounds.

Entomopathogenic nematodes (EPNs) respond to a variety of stimuli when foraging. In a laboratory investigation, we tested the chemotactic responses of 8 EPN strains (Steinernema and Heterorhabditis) to three mechanically damaged maize root compounds (linalool, α-caryophyllene and ß-caryophyllene). We hypothesized that the EPN directional response to the tested volatile compounds would vary among the species and volatile compound and may be related to foraging strategies. The nematodes with an intermediate foraging strategy (Steinernema feltiae) proved to be less active in their movement toward volatile compounds in a comparison with the ambushers (Steinernema carpocapsae) and cruisers (Steinernema kraussei and Heterorhabditis bacteriophora); ß-caryophyllene was found to be the most attractive substance in our experiment. The results of our investigation showed that the cruisers were more attracted to ß-caryophyllene than the ambushers and intermediates. The foraging strategy did not affect the movement of the IJs toward the other tested volatile compounds or the control. Our results suggest that the response to different volatile cues is more a strain-specific characteristic than a different host-searching strategy. Only S. carpocapsae strain B49 displayed an attraction to linalool, whereas S. kraussei showed a retarded reaction to ß-caryophyllene and α-caryophyllene in our experiment. The EPN strains showed only a weak attraction to α-caryophyllene, suggesting that this volatile compound could not have an important role in the orientation of IJs to the damaged roots of maize plants. These results expand our knowledge of volatile compounds as the cues that may be used by EPNs for finding hosts or other aspects of navigation in the soil.

Effects of barium and cadmium on the population development of the marine nematode Rhabditis (Pellioditis) marina.

Offshore oil and gas drilling often involves the use of fluids containing barium and traces of other heavy metals. These may affect the environment, but information on their toxicity to benthic biota remains scant. Here, we present results of a 10-day bioassay with the marine nematode Rhabditis (Pellioditis) marina at different loads of barium (0-10 ,000 ppm nominal concentrations) and cadmium (0-12 ppm) in the range of concentrations reported from drilling-impacted sediments. Barium did not affect the fitness and population development of R. (P.) marina at concentrations up to 300 ppm, but did cause a decrease in population abundance and an increase in development time from concentrations of 400-2000 ppm onwards. Increased mortality occurred at 4800 ppm Ba. For cadmium, LOEC and EC50 values for total population abundance were 2.95 and 8.82 ppm, respectively. Cd concentrations as low as 2.40 to 2.68 caused a decrease in the abundance of adult nematodes, indicating that assays covering more generations would likely demonstrate yet more pronounced population-level effects. Our results indicate that oil and gas drilling activities may potentially have important implications for the meiobenthos through the toxicity of barium and associated metals like cadmium.

Identification and characterization of a novel gene involved in the trans-specific nematicidal activity of Photorhabdus luminescens LN2.

Photorhabdus luminescens subsp. akhurstii LN2 from Heterorhabditis indica LN2 showed nematicidal activity against axenic Heterorhabditis bacteriophora H06 infective juveniles (IJs). Transposon mutagenesis identified an LN2 mutant that supports the growth of H06 nematodes. Tn5 disrupted the namA gene, encoding a novel 364-residue protein and involving the nematicidal activity. The green fluorescent protein-labeled namA mutant was unable to colonize the intestines of H06 IJs.

Antifungal and anthelmintic activities of Cleistopholis patens (Annonaceae).

Basic CH2Cl2 extract of the trunk bark of Cleistopholis patens (Annonaceae) exhibited antifungal activities against Candida albicans, C. parapsilosis, and C. glabrata using an agar well-diffusion assay method. Bioassay-guided fractionation of the extract led to the isolation of 8-hydroxysampangine. The methanolic extract displayed anthelmintic activity against Rhabditis pseudoelongata. Purification of the neutral CH2Cl2 extract yielded bornyl-p-transcoumarate and bornyl-p-cis-coumarate.

[Enhancing the anthelminthic activity roots of Annona sengalensis]. / Mise en evidence de l'activite antihelminthique des racines d'Annona senegalensis.

INTRODUCTION: Annona senegalensis is a plant largely used in traditional medicine in Africa. The roots showed an antiparsitic activity. The purpose of this work is to determine through bioassay on Rhabditis pseudoelongata the activity of the roots of Annona senegalensis and characterise if any substances responsible for this activity. MATERIAL AND METHODS: The vegetable material consists of roots of Annona senegalensis collected in Casamance at 1999. The tests of activity are carried out on Rhabditis pseudoelongata, stock L. Lamy, Institut Pasteur, Paris. Powder of roots (1070 g) was extracted with methanol. From this rough extract, we carried out two extractions with the dichloromethane of which one after passages in alkaline and acid medium. Various extracts were subjected to a chromatography on column of silica gel or from alumina gel; some compounds were isolated by high performance liquid chromatography . The identification of compounds was deed using spectrometric techniques (mass, Ultraviolet, Infrared) and nuclear magnetic resonance. The tests of activity related to three extracts and seven molecules isolated from the roots. The percentage of dead worm is determined after two hours of contact with demineralised water containing the sample to be tested. RESULTS: The neutral extract chloromethylenic showed an important activity on Rhabditis pseudoelongata. Five acetogenines were insulated starting from this extract chloromethylenic (gigantetronenine, squamocine, glaucanisine, glaucanetine, goniothalamicine) and two alkaloids starting from the alkaline extract chloromethylenic (liriodenine and norolivéroline). The tests on Rhabditis pseudoelongata of these compounds showed an activity of the squamocine with a IC50 of 0.1 microg/ml and IC90 0.3 microg/ ml more powerful than that of the levamisole, reference substance (IC50 of 0.8 microg/ml and IC90 13 microg/ml). CONCLUSION: The study of the biological activity of the roots of Annona senegalensis made it possible to determine the anthelminthic activity of the extract chloromethylenic from where the squamocine was isolated. The presence of this molecule could explain the anthelminthic activity of this plant.

The effects of chromium VI on the fitness and on the beta-tubulin genes during in vivo development of the nematode Steinernema feltiae.

The entomopathogenic nematode (EPN), Steinernema feltiae, is a commonly occurring nematode in the soil in Ireland. Consequently, we have conducted investigations as to the utility of this species as a candidate organism for the detection of chromium in Irish soils. These experiments have demonstrated that S. feltiae can survive and reproduce in the presence of high concentrations of chromium VI. It was observed that concentrations as high as 1000 ppm have little effect on the ability of this organism to produce large numbers of progeny. Nematodes were not observed to reproduce above 1800 ppm. However, an increase in development times for the nematode in vivo was noted at concentrations of 400 ppm upwards. This paper also illustrates the effects upon the beta-tubulin genes within nematode populations exposed to chromium VI in vivo. DNA sequencing has shown that elevated levels of variations occur among the population treatments, although these variations do not appear to be dependent upon chromium concentration. These findings constitute this organism appropriate for further investigation for the development of sub-lethal end points and biomarkers for the detection and biomonitoring of chromium VI contamination in soil.

Effects of the nematode Phasmarhabditis hermaphrodita and of venom from the endoparasitic wasp Pimpla hypochondriaca on survival and food consumption of the pest slug Deroceras reticulatum; implications for novel biocontrol strategies.

BACKGROUND: Controlling pests through disruption of biochemical pathways by physiologically active compounds/factors from animals and plants represents an expanding field of research. The authors investigated whether such factors in venom from the wasp Pimpla hypochondriaca (Retzius) can affect the viability and food consumption of the slug Deroceras reticulatum (Müller), and whether they can improve the efficacy of nematode-induced slug mortality. RESULTS: Exposure of slugs to 4 mL of water containing 500, 1000 and 5000 Phasmarhabditis hermaphrodita (Schneider) resulted in significant increases in mortality (with hazard ratios of 3.5, 3.9 and 5.8 respectively) and significant reductions in total food consumption and mean food consumption each day for 21 days. Injection of slugs with 4, 8 or 12 microL of P. hypochondriaca venom resulted in significant increases in mortality (with hazard ratios of 3.3, 4.5 and 9.0 respectively) and significant reductions in total food consumption compared with the controls. However, there was no significant effect of venom on the mean food consumption on individual days of the 21 day assay period, although significant reductions occurred for the 8 and 12 microL doses up to day 10. Injecting slugs with 4 microL of venom prior to exposure to 500 nematodes had no synergistic effect on either mortality or food consumption compared with either of the individual treatments. CONCLUSIONS: Pimpla hypochondriaca venom contains factors capable of killing and reducing food consumption by D. reticulatum. The utilization of these factors as components of integrated pest management strategies is discussed.

Effects of sublethal abiotic stressors on population growth and genetic diversity of Pellioditis marina (Nematoda) from the Westerschelde estuary.

Understanding the effects of anthropogenic pollutants at the ecosystem level requires a proper understanding of the toxicological effects at the population level. Species living in estuaries resist highly fluctuating conditions, and are often exposed to sublethal concentrations of pollutants coming from industrial and domestic wastes. In the Westerschelde estuary, the most upstream sampled population of the nematode Pellioditis marina is genetically less diverse than elsewhere. It experiences lower salinities and higher Cd concentrations than more downstream populations in the estuary. In the present study, we investigate whether these environmental conditions may explain the lower genetic diversity in the most upstream location. To this end we followed the development of genetically diverse P. marina populations under experimental conditions during 14 days. Genetic diversity was assessed in the F1, F2 and F5 generation by screening mitochondrial cytochrome oxidase c subunit 1 variation with the single-strand conformation polymorphism method (SSCP) and nucleotide sequencing. Our results show that sublethal Cd concentrations reduce population development of P. marina at suboptimal salinities, and that low salinity conditions induce responses at the genetic level. Nevertheless, the genetic effects were not persistent over generations, which emphasize the need for longer multigenerational experiments.

Bioactive properties of plant species ingested by chimpanzees (Pan troglodytes schweinfurthii) in the Kibale National Park, Uganda.

We measured the biological activities of a selected sample (84 crude extracts) of 24 species eaten by wild chimpanzees (Pan troglodytes schweinfurthii) in the Kibale National Park, western Uganda, to assess their potential chemotherapeutic values. Antibacterial, antimalarial, and/or antileishmania activities were observed in some crude extracts, and five of these extracts showed a significant cytotoxicity against human tumor cells. Active compounds isolated from three plant parts occasionally ingested by chimpanzees (Diospyros abyssinica (Ebenaceae) bark, Uvariopsis congensis (Annonaceae) leaves, and Trichilia rubescens (Meliaceae) leaves) showed highly significant medicinal properties. Two novel antiparasitic limonoids were isolated from Trichilia rubescens and their molecular structures were determined. In addition to elucidating the natural equilibrium maintained between hosts and pathogens, our investigation of the diet of wild chimpanzees may serve as a guideline to discovering plants with bioactive properties that should be preserved from destruction because of their health maintenance value for great ape populations.

Glycogen: its importance in the infectivity of aged juveniles of Steinernema carpocapsae.

Infective juveniles (IJs) of Steinernema carpocapsae (All) are able to remain relatively highly infective even when they have almost exhausted their neutral lipid reserves. This is not seen in other steinernematid species so we proposed that carbohydrate may be important for infectivity in aging IJs of S. carpocapsae. The present study investigated glycogen utilization in IJs of 4 entomopathogenic nematodes, S. carpocapsae, S. riobravis (Biosys 355), S. feltiae (UK76) and S. glaseri (NC), stored in distilled water at 25 degrees C. The 4 species had appreciable amounts of glycogen; from ca. 8% dry weight in S. riobravis to ca. 18% in S. glaseri. Infective juveniles of S. carpocapsae and S. riobravis survived for 120-135 days and utilized ca. 90% of their glycogen reserve at an almost constant rate during a 112-day storage period. Steinernema feltiae and S. glaseri lived for much longer (> 450 days) and during a 250-day storage period their glycogen content decreased by 27 and 40%, respectively. In contrast to the other 3 species, the rate of lipid decline preceded that of glycogen in S. carpocapsae. The rate of glycogen decline in S. carpocapsae IJs incubated with the glycolytic inhibitor, iodoacetamide (10(-4) M) was significantly reduced (P < 0.05) compared with untreated nematodes, and the infectivity of inhibitor-treated aged (> 80 days) IJs was reduced compared with controls. Incubating aged (80-day) IJs of S. carpocapsae (mean neutral lipid content ca. 10% of initial level) with 10(-4) M iodoacetamide for 24 h significantly reduced (P < 0.05) their infectivity compared with freshly harvested inhibitor-treated IJs and untreated controls. Following an 11-day recovery period, the infectivity of inhibitor-treated aged IJs recovered significantly (P < 0.05). The evidence suggests that glycogen is an important source of energy for maintaining infectivity in aged IJs of S. carpocapsae.

Synthesis and antiparasitic activities of amidinic azolated derivatives.

A set of heterocyclic N-acetamidinium hydrochlorides were prepared from the corresponding N-acetonitriles. The antiparasitic screening showed that, while all amidines are practically inactive, some nitriles present leishmanicide properties.