Nematode Growth Research Articles

(Homo)glutathione deficiency impairs root-knot nematode development in Medicago truncatula.

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes.

Plant regeneration from Crotalaria spectabilis hairy roots which showed inhibited growth of root-knot nematodes (Meloidogyne hapla and M. incognita) in vitro

Hairy roots were induced from leaf segments of Crotalaria spectabilis, which is used as a green manure crop antagonistic to nematodes, by infection with a mikimopine type wild strain of Agrobacterium rhizogenes A13 (MAFF02-10266). For initial proliferation of hairy roots after induction, addition of gibberellin A3 to the medium was indispensable. These roots exhibited vigorous growth and abundant lateral branching on half-strength Murashige and Skoog (1/2MS) medium without plant growth regulators. The adventitious shoots were induced from 24% of root segments 2 months after transfer onto B5 medium containing 5 mg l－1 6-benzyladenine. The axillary buds excised from these shoots produced roots 1 month after transfer onto 0.4% (wv－1) gellan gum-solidified 1/2MS medium containing 30 mM fructose without plant growth regulators. Regenerated plants were successfully grown under greenhouse conditions. Infection of hairy roots of C. spectabilis with root-knot nematodes Meloidogyne hapla and M. incognita led to the growth inhibition of these nematodes.

Effect of a Neem preparation on reproduction of the nematode Globodera rostochiensis and growth of potato

An experiment was conducted under glasshouse conditions to test the effects of neem-based product in the form of drenches and potato tuber treatments on the growth of potato and population density of the potato cyst nematode Globodera rostochiensis. The soil applications of the biopreparation improved the plant growth and yield of potato, being greatest with 1% Neem Azal. The biggest increase in the yield occurred in the Oxamyl application (15.8%). The biopreparation in concentrations from 0.2% to 1% increased the yield of potatoes from 0.3% to 9.7% compared to the untreated inoculated control. The soil application was relatively more effective in increasing of the yield (0.3%-9.7%) than the potato tuber treatments (0.3%-2.3%). The nematode reproduction was reduced 83.3% with Oxamyl and from 59.8% to 71.0% with Neem Azal treatments.

Effect of local tree seeds in the control of root knot nematode Meloidogyne javanica (Treub) chitwood and growth promotion of chickpea (Cicer arietinum L.) and mung bean (Vigna radiata L.)

Seeds of local trees, such <i>Azadirachta indica</i> A. Juss, <i>Adenanthera pavonina</i> L., <i>Leucaena leucocephala</i> (Lam.) de Wit and <i>Eucalyptus</i> spp., were used as aqueous extract at 25, 50 and 100 % concentration to control the activity of <i>Meloidogyne javanica</i> (Treub) Citwood. All seed extracts showed lethal effect on <i>M. javanica</i> eggs, and a gradual decrease in egg hatching and an increase in mortality of second-stage juveniles were observed with the increase in extract concentration. <i>L. leucocephala</i> was found to be most effective in reducing egg hatching, whereas 100 % mortality of juveniles was observed in the case of <i>A. indica</i> seed extract. Number of knots was significantly reduced at 100 % concentration when seeds of chick pea and mung bean were treated and soil was drenched with <i>A. pavonina</i> and <i>Eucalyptus</i> spp. seed extract.

HdfR is a regulator in Photorhabdus luminescens that modulates metabolism and symbiosis with the nematode Heterorhabditis

The bacterium, Photorhabdus luminescens, is an insect pathogen that also maintains a mutualistic interaction with nematodes from the family Heterorhabditis. Photorhabdus luminescens is carried in the gut of the infective juvenile (IJ), a nematode stage that infects soft-cuticled insect larvae in the soil. Photorhabdus luminescens is released into the insect blood to convert the insect into a biomass that is able to support nematode growth and development. Nematode reproduction continues for 2-3 generations before the nematodes enter an alternative developmental pathway leading to a new generation of IJs that are efficiently colonized by P. luminescens in a process called transmission. Transmission begins with the adherence of P. luminescens to the rectal gland cells (RGC) located in the gut of the hermaphrodite. The bacteria enter, and replicate within, the RGC resulting in vacuolization, and ultimately lysis, of the RGC. In this way P. luminescens are released throughout the body cavity of the hermaphrodite to encounter, and colonize, the developing IJs. In this study we show that the LysR-type regulator, HdfR, is required for normal transmission in P. luminescens TTO1. Transcriptome analysis revealed that HdfR regulates the expression of 124 genes, including genes involved in arginine metabolism, hydroxyphenylacetate catabolism and pigment production. Using fluorescence microscopy we show that the ΔhdfR mutant is able to attach to, and grow within, the hermaphrodite as well as wild-type bacteria but subsequent steps in transmission are delayed. Therefore, HdfR plays an important role in coordinating the interaction between P. luminescens and its nematode partner during transmission.

Multiplicação de Bacillus subtilis em vinhaça e viabilidade no controle da meloidoginose, em cana-de-açúcar

Objetivou-se, com este trabalho, avaliar o controle da meloidoginose e o crescimento da cana-de-açúcar, em função da aplicação de Bacillus subtilis ao solo após multiplicação em vinhaça. Bioensaios foram conduzidos em laboratório com avaliação do crescimento de B. subtilis em meio líquido com interesse na melhor composição de meio de cultura, a partir da vinhaça. No experimento em casa de vegetação foi utilizado solo coletado em área de cultivo de cana com histórico de alta infestação com Meloidogyne spp. No solo acondicionado em vasos efetuou-se o plantio da variedade de cana-de-açúcar RB 72454, cujos tratamentos foram: controle; vinhaça pura (50 m³ ha-1); Bacillus subtilis em suspensão aquosa (50 m³ ha-1); B. subtilis multiplicado na vinhaça para 50 m³ ha-1 e 100 m³ ha-1. A multiplicação de Bacillus subtilis em meio de cultura à base de vinhaça (25%) foi significativamente superior em comparação com o meio de cultura caldo nutriente. A aplicação de B. subtilis em suspensão aquosa promoveu o crescimento e a redução da reprodução dos nematóides em cana-de-açúcar durante o experimento. A aplicação de B. subtilis multiplicado na vinhaça não proporcionou os benefícios de estímulo ao crescimento e controle da meloidoginose na cana-de-açúcar, encontrados com a aplicação apenas da bactéria no solo.

Change of some defense compounds of cucumber treated with Bacillus cereus and salicylic acid against Meloidogyne javanica

In this research, the effects of antagonist bacterium (Bacillus cereus) and salicylic acid (SA) as chemical inducer alone and in combination against root knot nematode (Meloidogyne javanica) as the most important nematode in Iran, on cucumber roots (cv. superdominus) was investigated. The used concentrations were 109 cfu/ml for bacteria and 5 mM for SA. In greenhouse experiments some parameters were evaluated including nematode and plant growth criteria in treated plants with the two above materials. Results of this experiment showed that combination of biocontrol agent and chemical inducer decreased galling, egg masses and egg numbers on roots of cucumber. In biochemical analysis, also, some of defense compounds such as hydrogen peroxide and peroxidase activities were measured. Results showed that hydrogen peroxide level and peroxidase activity increased after inoculation until 4th and 5th days respectively and then decreased gradually. Soil drenching of B. cereus and SA caused maximum induction in roots. Key words: Bacillus cereus, salicylic acid, Meloidogyne javanica, peroxidase, hydrogen peroxide.

Interaction of Silver Nanoparticles with Biological Surfaces of Caenorhabditis elegans

Silver nanoparticles (AgNPs) are being used in an increasing number of industrial and commercial applications; this has resulted in an increased release of AgNPs into the environment. Understanding the interaction of AgNPs with biological surfaces is important, as such understanding will facilitate predictions of the further effects of nanoparticles on biological systems. This study highlights the interaction of citrate-coated silver nanoparticles (cAgNPs) with the biological surfaces of the nematode C. elegans. General toxicity, as proxied by factors such as mortality and reproduction, was evaluated in nematode growth medium (NGM), which provides a more homogeneous distribution of cAgNPs than in K-medium. The survival and reproduction of C. elegans evidenced a clear reduction in up to 100 mg/L and 10 mg/L of cAgNPs, respectively. We also noted significant interactions of cAgNPs with the biological surfaces of C. elegans. Severe epidemic edema and burst were detected in the exposure group, which may be associated with secondary infections in soil ecosystems. We observed no evidence of cAgNPs intake by C. elegans. This is, to the best of our knowledge, the first report to investigate the nanotoxicity of cAgNPs as related to biological surfaces of C. elegans; further research is needed to study the fate of cAgNPs inside of C. elegans.

STR-33, a Novel G Protein-coupled Receptor That Regulates Locomotion and Egg Laying in Caenorhabditis elegans

Despite their predicted functional importance, most G protein-coupled receptors (GPCRs) in Caenorhabditis elegans have remained largely uncharacterized. Here, we focused on one GPCR, STR-33, encoded by the str-33 gene, which was discovered through a ligand-based screening procedure. To characterize STR-33 function, we performed UV-trimethylpsolaren mutagenesis and isolated an str-33-null mutant. The resulting mutant showed hypersinusoidal movement and a hyperactive egg-laying phenotype. Two types of egg laying-related mutations have been characterized: egg laying-deficient (Egl-d) and hyperactive egg laying (Egl-c). The defect responsible for the egg laying-deficient Egl-d phenotype is related to Gα(q) signaling, whereas that responsible for the opposite, hyperactive egg-laying Egl-c phenotype is related to Gα(o) signaling. We found that the hyperactive egg-laying defect of the str-33(ykp001) mutant is dependent on the G protein GOA-1/Gα(o). Endogenous acetylcholine suppressed egg laying in C. elegans via a Gα(o)-signaling pathway by inhibiting serotonin biosynthesis or release from the hermaphrodite-specific neuron. Consistent with this, in vivo expression of the serotonin biosynthetic enzyme, TPH-1, was up-regulated in the str-33(ykp001) mutant. Taken together, these results suggest that the GPCR, STR-33, may be one of the neurotransmitter receptors that regulates locomotion and egg laying in C. elegans.

Effects of associated bacteria on the pathogenicity and reproduction of the insect-parasitic nematodeRhabditis blumi(Nematoda: Rhabditida)

Three bacteria, Alcaligenes faecalis , Flavobacterium sp., and Providencia vermicola , were isolated from dauer juveniles of Rhabditis blumi . The pathogenic effects of the bacteria against 4th instar larvae of Galleria mellonella were investigated. Providencia vermicola and Flavobacterium sp. showed 100% mortality at 48h after haemocoelic injection, whereas A. faecalis showed less than 30% mortality. Dauer juveniles showed 100% mortality against G. mellonella larvae, whereas axenic juveniles, which do not harbor associated bacteria, exhibited little mortality. All of the associated bacteria were used as a food source for nematode growth, and nematode yield differed with bacterial species. Among the bacterial species, P.vermicola was most valued for nematode yield, showing the highest yield of 5.2× 10(4) nematodes/mL in the plate. In bacterial cocultures using two of the three associated bacteria, one kind stimulated the other. The highest total bacterial yield of 12.6g/L was obtained when the inoculum ratio of P. vermicola to A. faecalis was 10:1. In air-lift bioreactors, the nematode growth rate increased with an increasing level of dissolved oxygen. The maximum nematode yield of 1.75× 10(5)nematodes/mL was obtained at 192h with an aeration rate of 6 vvm.

Growth and propagation of entomogenous nematodes, Steinernema spp., on several kinds of media prepared by chicken liver and intestine.

Growth and propagation of entomogenous nematodes, Steinernema spp., on several kinds of media prepared by chicken liver and intestine.

The Effect of Unsaturated Fatty Acids added to Culture Media on the Growth of the Pine Wood Nematode, Bursaphelenchus xylophilus

The Effect of Unsaturated Fatty Acids added to Culture Media on the Growth of the Pine Wood Nematode, Bursaphelenchus xylophilus

Submerged Monoxenic Culture Medium Development for Heterorhabditis bacteriophora and its Symbiotic Bacterium Photorhabdus luminescens: Protein Sources

Most medium formulations for improving culture of entomopathogenic nematodes (EPN) based on protein sources have used enriched media like animal feed such as dried egg yolk, lactalbumin, and liver extract, among other ingredients. Most results, however, showed unstable yields and longer production time. Many of the results do not show the detailed parameters of fermentation. Soy flour, cotton seed flour, corn gluten meal, casein powder, soytone, peptone, casein hydrolysates, and lactalbumin hydrolysate as protein sources were tested to determine the source to support optimal symbiotic bacteria and nematode growth. The protein hydrolysates selected did not improve bacterial cell mass compared with the yeast extract control, but soy flour was the best, showing 75.1% recovery and producing more bacterial cell number (1.4×10⁹/ml) than all other sources. The highest yield (1.85×10⁵ IJs/ml), yield coefficient (1.67×10⁶ IJs/g medium), and productivity (1.32×10⁷ IJs/l/day) were also achieved at enriched medium with soybean protein.

A semi-fluid gellan gum medium improves nematode toxicity testing

This study examined an alternative test medium for nematodes that use gellan gum as the gelling agent instead of agar. The semi-fluid consistency of the gel-like component nematode growth gellan gum (CNGG) supports three-dimensional distribution of the nematodes and food bacteria, but still allows free movement of the former. Moreover, flexible preparation of the medium and easy recovery of the test organisms are possible. Here, the effects of the nematicides ivermectin (pharmaceutical) and aldicarb (pesticide) and of the metal cadmium on the growth and reproduction of the free-living nematodes Caenorhabditis elegans and Panagrolaimus cf. thienemanni were studied in CNGG media. Results were compared to those obtained with the standard liquid test media in order to evaluate the applicability of CNGG for nematode toxicity testing. The sensitivity of P. cf. thienemanni to all three substances was found to be higher than that of C. elegans, but both nematodes showed the highest sensitivity to ivermectin exposure. This raises concerns about the risk posed by the pharmaceutical to non-target nematodes. In contrast to ivermectin bioassays carried out in CNGG medium, those conducted in liquid medium resulted in wide-ranging variability between and within replicates. Thus, CNGG seems to be particularly valuable for testing hydrophobic substances with a high sorption affinity as it favors their sorption to food bacteria and minimizes contact with the surfaces of the test vessels. However, the medium was less suitable for deriving toxicity thresholds for cadmium and may likewise not be an appropriate choice for testing other metals. The medium introduced herein was shown to be appropriate for sublethal nematode toxicity testing and likely provides a convenient environment for testing other nematode species. Besides improved testing of hydrophobic substances, CNGG also offers advantages for long-term studies, such as full life-cycle experiments, in which fresh medium is regularly needed. Moreover it may be beneficial for testing other poorly soluble or insoluble substances, such as nanoparticles.

EFFECT OF PAECILOMYCES LILACINUS AND PLANT GROWTH PROMOTING RHIZOBACTERIA ON MELOIDOGYNE INCOGNITA INOCULATED BLACK GRAM, VIGNA MUNGO PLANTS

ABSTRACT An experiment was carried out to study the interaction of root knot nematode, Meloidogyne incognita, Paecilomyces lilacinus and plant growth promoting rhizobacteria, Bradyrhizobium on the growth of black gram, Vigna mungo. In the present investigation it was revealed that Meloidogyne incognita resulted in significant decrease in the growth of black gram, root-nodule development, nitrogen contents of root and shoot, and nitrogenase activity at all inoculum levels. Treatment of Bradyrhizobium and Paecilomyces lilacinus resulted in significantly lesser damage to plant growth of blackgram than the plants treated with bacteria at the time of inoculation with rootknot nematode, M. incognita fallowed by plants where bacteria and fungus was applied 10 days after nematode inoculation. .Treatment of Bradyrhizobium significantly increased the nitrogen content of root and shoot in all the treatments. Nitrogenase activities in nematode infected plants was higher in plants treated with P. lilacinus and Bradyrhizobium before and at the time of nematode inoculation in comparison to plants which were treated by P. lilacinus and Bradyrhizobium 10 days after nematode inoculation.

Concentration- and time-dependent behavioral changes in Caenorhabditis elegans after exposure to nicotine

Nicotine induces profound behavioral responses in the model organism Caenorhabditis elegans. We tested the effect of a broad range of concentrations of nicotine (from 0.001mM to 30mM in nematode growth medium) on C. elegans locomotor behavior. We also followed the time-course influence on the sensitivity of C. elegans to nicotine (from 0min to 300min). A low concentration (0.001mM) of this alkaloid causes a reduction of the speed of movement. By contrast, moderate concentrations (0.01 and 0.1mM) induced acceleration of the mean speed of locomotion of C. elegans. High doses of nicotine (above 1mM) induced slowing down of the movements and, finally, paralysis. Time-dependent analysis revealed that the stimulating effect of nicotine abolished the slowing down of C. elegans in control experiments after 30min in the presence of 0.001, 0.1 and 10mM nicotine. In the presence of 0.1mM nicotine, the stimulation phase lasted up to 70min. The evidence indicates that nicotine can have dual effects on the speed of locomotion, which is dependent on differences in its dosage and treatment time.

The Novel Hydroxylamine Derivative NG-094 Suppresses Polyglutamine Protein Toxicity in Caenorhabditis elegans

Aggregation-prone polyglutamine (polyQ) expansion proteins cause several neurodegenerative disorders, including Huntington disease. The pharmacological activation of cellular stress responses could be a new strategy to combat protein conformational diseases. Hydroxylamine derivatives act as co-inducers of heat-shock proteins (HSPs) and can enhance HSP expression in diseased cells, without significant adverse effects. Here, we used Caenorhabditis elegans expressing polyQ expansions with 35 glutamines fused to the yellow fluorescent protein (Q35-YFP) in body wall muscle cells as a model system to investigate the effects of treatment with a novel hydroxylamine derivative, NG-094, on the progression of polyQ diseases. NG-094 significantly ameliorated polyQ-mediated animal paralysis, reduced the number of Q35-YFP aggregates and delayed polyQ-dependent acceleration of aging. Micromolar concentrations of NG-094 in animal tissues with only marginal effects on the nematode fitness sufficed to confer protection against polyQ proteotoxicity, even when the drug was administered after disease onset. NG-094 did not reduce insulin/insulin-like growth factor 1-like signaling, but conferred cytoprotection by a mechanism involving the heat-shock transcription factor HSF-1 that potentiated the expression of stress-inducible HSPs. NG-094 is thus a promising candidate for tests on mammalian models of polyQ and other protein conformational diseases.

Outcrossing and crossbreeding recovers deteriorated traits in laboratory cultured Steinernema carpocapsae nematodes

The nematode Steinernema carpocapsae infects and kills many pest insects in agro-ecosystems and is commonly used in biocontrol of these pests. Growth of the nematodes prior to distribution for biocontrol commonly results in deterioration of traits that are essential for nematode persistence in field applications. To better understand the mechanisms underlying trait deterioration of the efficacy of natural parasitism in entomopathogenic nematodes, we explored the maintenance of fitness related traits including reproductive capacity, heat tolerance, virulence to insects and ‘tail standing’ (formerly called nictation) among laboratory-cultured lines derived from natural, randomly mating populations of S. carpocapsae. Laboratory cultured nematode lines with fitness-related trait values below wild-type levels regained wild-type levels of reproductive and heat tolerance traits when outcrossed with a non-deteriorated line, while virulence and ‘tail standing’ did not deteriorate in our experiments. Crossbreeding two trait-deteriorated lines with each other also resulted in restoration of trait means to wild-type levels in most crossbred lines. Our results implicate inbreeding depression as the primary cause of trait deterioration in the laboratory cultured S. carpocapsae. We further suggest the possibility of creating inbred lines purged of deleterious alleles as founders in commercial nematode growth.

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 FindingsIn 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/SignificanceA 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.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage <em>Caenorhabditis elegans</em>

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.