Free-living Nematode Panagrellus Redivivus Research Articles

Development of a low-cost technology for mass production of the free-living nematode Panagrellus redivivus as an alternative live food for first feeding fish larvae.

The free-living nematode Panagrellus redivivus is a suitable food source for first feeding fish. In the present report, a new method for the mass production of P. redivivus is presented. The technique involves multiplication of the nematode in monoxenic (single microorganism: Saccharomyces cerevisiae) solid culture (fluid media supported by 1- to 4-cm(3) sponge cubes) in autoclavable plastic bags (size range: 50 x 30 cm to 75 x 67 cm). Two growing media were tested: oat-meal medium (OM), which is an oat-based medium (16.7% oat-meal flour in 0.8% saline solution), and purified ingredient medium (PIM), a semi-synthetic medium (1.64% meat peptone, 0.94% yeast extract, 12.6% corn starch, 0.24% glucose, 1.48% sunflower oil, in 0.8% saline solution). The bags were inoculated with 350 nematodes/g medium. After an average period of 12 days (11-13 days) at 25 degrees C, the average yield (number of nematodes/g medium) was 241 x 10(3) for OM and 333 x 10(3) for PIM in 12-l bags (50 x 30 cm). The production scale has currently reached a bag volume of 50 l (75 x 67 cm); using PIM and the conditions described above, it was possible to harvest more than 1.3 x 10(9) nematodes/bag (291 x 10(3) nematodes/g medium). In PIM, when sun flower oil was replaced with the same amount of fish oil or cod liver oil, yields of 259 x 10(3) and 290 x 10(3) nematodes/g medium, respectively, were attained. The technology for mass production and formulation of P. redivivus should enable fish-hatchery operators to rely on a cheap, standardised, and permanently available live food product for first feeding fish larvae.

Occurrence of dopamine in Panagrellus redivivus and Meloidogyne incognita

AbstractDopamine-like immunoreactivity was detected in various parts of the nervous system of adult males and females of the free-living nematode Panagrellus redivivus , including the nerve ring and some cephalic processes, the ventral nerve cord, and the tail region. Positive immunoreactivity for dopamine was not found in infective second stage juveniles (J2) of the root-knot nematode Meloidogyne incognita. This putative neurotransmitter was detected by HPLC in homogenates of both species, the dopamine content in P.redivivus being eight times greater than in M. incognita. No other catecholamine compounds were detected in these species by HPLC. The development of a more sensitive immunocytochemical assay for dopamine will be required to determine the location of dopamine in the nervous system of M. incognita J2.

Larval pigmentation, survival and growth of Penaeus indicus fed the nematode Panagrellus redivivus enriched with astaxanthin and various lipids

The highly unsaturated fatty acid (HUFA) content of the free-living nematode Panagrellus redivivus was successfully increased when three different lipid sources (cod-liver oil, marilla oil and capelin oil) from marine animals were used in the nematode culture medium. The lipid enrichment significantly increased the ω3 HUFA content, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), of the nematodes compared with lipid non-enriched nematodes. Penaeus indicus larvae fed the lipid-enriched nematodes had significantly (P < 0.05) greater larval survival (69–77%) until metamorphosis than those fed non-enriched nematodes (54%). This nematode species was also enriched with astaxanthin to determine the effects of this carotenoid on pigmentation, survival and growth during the larval development of P. indicus. The pigment, delivered via nematodes, significantly (P < 0.05) improved larval coloration and survival (88%) compared with that of placebo-pigmented nematodes (78%). However, there was no strong evidence to show the benefit of either pigment and/or lipid enrichment on larval growth and development of P. indicus. This study has shown that the nutritional value of the nematodes can be enhanced by the addition of fish oil into the culture medium. Supplementation of EPA and DHA, together with synthetic astaxanthin, allow the nematodes to be used as a sole diet for the larval culture of P. indicus.

A novel cystathionine β-synthase from Panagrellus redivivus (Nematoda)

The free-living nematode Panagrellus redivivus can be used as a biochemical model for parasitic nematodes in the search for new chemotherapeutic agents. A novel cystathionine β-synthase has been purified 3600-fold from the cytosol of P. redivivus. The enzyme catalyses the synthesis of cystathionine from homocysteine plus serine or cysteine. The enzyme, native M r 71.7 kDa, pI 4.7, is a dimer and also catalyses the replacement of the β-SH group of cysteine with 2-mercaptoethanol to yield a thioether, S-(2-hydroxyethyl) cysteine and H 2S. This reaction proceeds much faster than cystathionine synthesis and L-cysteine cannot be replaced by D-cysteine, L-cystine, N-acetyl L-cysteine, cysteamine or D,L-homocysteine. 2-Mercaptoethanol in the assay can be replaced by monothiolglycerol and to a lesser extent by cysteamine. The absolute K m values for L-cysteine and 2-mercaptoethanol were 0.13 ± 0.05 mM and 1.72 ± 0.24 mM, respectively, the absolute V max was 55 ± 4.9 μmol·min −1·mg protein −1. The enzyme had a pH optimum of approx. 8.5 and did not require metal ions for activity. The enzyme was inhibited by a series of substrate analogues, anthelmintics and plant phenols. The P. redivivus enzyme differs markedly from its mammalian equivalent and suggests distinctive differences in sulphur amino acid metabolism in nematodes.

Nitric oxide mediates the inhibitory effects of SDPNFLRFamide, a nematode FMRFamide-related neuropeptide, in Ascaris suum.

1. The physiological effects of two Phe-Met-Arg-Phe-NH2 (FMRFamide)-related neuropeptides isolated from the free-living nematode Panagrellus redivivus, SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2), were examined using neuromuscular preparations from the parasitic nematode Ascaris suum. 2. PF1 and PF2 hyperpolarized muscle membrane and induced sustained flaccid paralysis, independent of external Cl-, in both innervated and denervated preparations. 3. PF1 reversed spastic contractions induced by the cholinomimetic levamisole, elevated K+, or the excitatory nematode FMRFamide-related neuropeptides KNEFIRFamide or KHEYLRFamide. 4. PF1 reversal of levamisole contraction was blocked by pretreatment with agents that interfere with nitric oxide (NO) synthesis (e.g., N-nitro-L-arginine), whereas sodium nitroprusside, which releases NO in solution, mimicked PF1 and PF2. 5. NO synthase activity, monitored by the conversion of [3H]arginine to [3H]citrulline, was twice as abundant in A. suum hypodermis as in muscle, but was not present in reproductive tissue. The relative abundance of NO synthase activity in these tissues was similar to the observed specific binding of [3H]PF1. 6. These results suggest that the inhibitory effects of PF1 and PF2 on nematode somatic muscle are mediated by NO, and that the hypodermis may serve a role in this process analogous to that of the endothelium in vertebrate vasculature.

Competitive interactions between two nematophagous fungi during infection and digestion of the nematode Panagrellus redivivus

Individuals of the free-living nematode Panagrellus redivivus were infected with conidia of the endoparasitic nematophagous fungus Drechmeria coniospora. At different stages of infection the nematodes were added to an agar-plate culture of the predatory fungus Arthrobotrys oligospora which contained traps. Initially, nematodes infected with D. coniospora were invariably captured, penetrated and colonized by A. oligospora. Then both fungi used the nematode contents for development of hyphal biomass outside the nematode. But inside nematodes in which infection by D. coniospora was advanced development of A. oligospora was markedly hampered. In dual-infected nematodes (colonized from opposite ends), the hyphae of A. oligospora were often observed to be dead or degenerate when in close proximity to living mycelium of D. coniospora, indicating that the latter acts as an antagonist against A. oligospora under these conditions. The interaction between trophic hyphae of D. coniospora and A. oligospora involved thickening of the cell wall of A. oligospora at the sites of contact and degradation of the hyphal compartments and shrivelling of the cell wall of A. oligospora. The observed interactions are discussed in relation to mycoparasitism and possible ecological function.

Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora

When grown in liquid cultures allowing the formation of nematode traps, the fungus Arthrobotrys oligospora produced two extracellular proteases hydrolysing the chromogenic substrate Azocoll. The protease activity was separated into two fractions (FI and FII) using anion-exchange chromatography. In bioassays, protease(s) present in FII immobilized the free-living nematode Panagrellus redivivus indicating that the enzyme(s) might be involved in the infection of nematodes. A protease designated PII was purified from FII to apparent homogeneity by hydrophobic interaction and size-exclusion chromatography, resulting in an approximately 15-fold increase in specific activity. The purified enzyme was glycosylated, had a molecular mass of approximately 35 kDa (gel filtration) and an isoelectric point of pH 4.6. PII immobilized P. redivivus in bioassays and hydrolysed proteins of the purified cuticle. The enzyme hydrolysed several protein substrates including casein, bovine serum albumin and gelatin, but not native collagen. Examination of substrate specificity with synthetic peptides showed that PII readily hydrolysed tripeptides with aromatic or basic amino acids including N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-4-nitroanilide (Bz-Phe-Val-Arg-NA) and succinyl-glycyl-glycyl-L-phenylalanine-4-nitroanilide (Suc-Gly-Gly-Phe-NA). Mono-peptides were hydrolysed at considerably slower rates. PII had an optimum activity between pH 7 and 9 and was susceptible to autodegradation. PII was inhibited by several serine protease inhibitors including phenylmethylsulfonyl fluoride (PMSF), chymostatin and antipain. The protease was N-terminally blocked, but the sequence of one internal peptide showed a high homology with a region containing the active site histidine residue of the subtilisin family of serine proteases.

A nematode FMRFamide-like peptide, SDPNFLRFamide (PF1), relaxes the dorsal muscle strip preparation ofAscaris suum

PF1 (SDPNFLRFamide) is a FMRFamide-like peptide extracted from the free-living nematode Panagrellus redivivus. Here we show that this peptide causes a hyperpolarization of somatic muscle cells of the parasitic nematode Ascaris suum and a relaxation of the somatic muscle strip preparation. We have assessed whether or not the relaxation of Ascaris dorsal muscle strip by PF1 is due to (i) inhibition of the release of the excitatory neuromuscular junction transmitter acetylcholine (ACh), (ii) potentiation of the release of the inhibitory neuromuscular junction transmitter gamma-aminobutyric acid (GABA) or (iii) a direct inhibitory action of the peptide on the muscle cells. Under the experimental conditions described here, tonic ACh release does not seem to be involved in determining the resting membrane potential or resting tone of the Ascaris dorsal muscle strip and thus inhibition of tonic ACh release is unlikely to explain the relaxation elicited by the peptide. Furthermore, PF1 (100 nM-1 microM) inhibited the contraction of the muscle strip elicited by bath application of ACh, suggesting either a direct inhibitory action of the peptide on the muscle cells or a potentiation of GABA release. In electrophysiological experiments, the reversal potential for the PF1 hyperpolarization was not the same as that for GABA. Thus, PF1 hyperpolarizes Ascaris muscle by a mechanism that does not involve stimulation of GABA release from inhibitory pre-synaptic terminals.

Effect of the nematode Panagrellus redivivus on growth and enzyme production by Phanerochaete velutina and Stereum hirsutum

Different strains of two basidiomycetes, Stereum hirsutum and Phanerochaete velutina, were treated with dead and living populations of the free-living nematode Panagrellus redivivus. Responses to treatment differed within and between the two fungal species: growth of S. hirsutum was reduced, whereas that of P. velutina was stimulated. P. velutina displayed increased protease and reduced esterase and acid phosphatase activities, whereas S. hirsutum displayed increased protease and acid phosphatase activity. In S. hirsutum, laccase, tyrosinase and cellulase production increased in response to living nematodes, and amylase increased in response to dead nematodes. Spot tests indicated spatial localization of laccase and acid phosphatase in different hyphal regions of both species.

Enhancement of the Fatty Acid Composition of the Nematode Panagrellus redivivus Using Three Different Medial

AbstractThe free living nematode Panagrellus redivivus has shown promise as a live food for marine shrimp and fish. This study evaluated the effect of three media on the fatty acid (FA) profile of nematodes. The media tested were: wheat flour (WF), wheat flour plus yeast (WFY), and wheat flour plus yeast plus fish oil (WFFO).The total lipid content of nematodes grown on WFFO (14.9%) was significantly higher than the lipid content of nematodes grown on WF (2.7%) and WFY (4.4%). The lipid of nematodes grown on WFFO also contained a higher percentage of n‐3 highly unsaturated fatty acids (HUFA) (11.2%), especially 20:5(n‐3) (7.4%) and 22:6(n‐3) (3.3%), than nematodes grown on WF (4.8%) and WFY (5.7%). Nematodes grown on WFFO medium had similar lipids as Artemia, especially desirable n‐3 HUFA's.Total daily yield of nematodes was highest on WFY. There was no difference in yields of nematodes on WF and WFFO. This study has shown that the nutritional value of nematodes can be influenced by the media on which they are cultured. With a proper medium, cultured nematodes can provide the inexpensive, consistently nutritious live food that can be considered for larval culture of marine crustaceans and fish.

Panagrellus redivivus: Failure to find evidence for the occurrence and biosynthesis of sialic acids

The occurrence of sialic acids in the free-living nematode Panagrellus redivivus was studied by periodate oxidation/[ 3H]sodium borohydride reduction of about 10 7 nematodes. In parallel, the capability of sialic acid biosynthesis was examined by metabolic labeling of the same number of nematodes with N-[ 3H]acetylmannosamine. In both experiments, radioactivity was incorporated into the nematodes. Mild acid hydrolysis, however, did not release radioactively labeled sialic acids or derivatives as tested by radio thin-layer chromatography, suggesting that P. redivivus does not contain or synthesize sialic acids.

Strategies for detoxification of aldehydic products of lipid peroxidation in helminths

Crude extracts from a number of helminths including Schistosoma intercalatum and Fasciola hepatica were able to detoxify known aldehydic products of lipid peroxidation. A major route for alk-2-enal and alka-2,4-dienal detoxification in parasitic helminths was via glutathione conjugation and glutathione transferase appeared to be responsible for the activity. As yet uncharacterised NADPH-linked systems may provide an important secondary pathway for detoxification of alk-2-enals and alka-2,4-dienals in parasitic helminths. The free-living nematode Panagrellus redivivus had higher active NADH/NADPH-linked aldehyde reduction systems compared to parasitic helminths. The NADH linked and NADPH linked reductions in P. redivivus were mitochondrial and cytosolic activities respectively. NADH/NADPH-linked systems may be responsible for alkanal reduction in helminths as there is no evidence of conjugation of alkanals with glutathione. P. redivivus and Haemonchus contortus were also able to oxidise aldehydes via NAD/NADP-linked systems.

The use of a novel motility assay for the isolation of bioactive peptides from the free-living nematode Panagrellus redivivus

1. 1. An assay for the quantitative measurement of the motility of the nematode Panagrellus redivivus has been developed. 2. 2. A number of fractions showing bioactivity with P. redivivus have been isolated from the worms using size and partition chromatography. 3. 3. These fractions have been shown to contain peptides and a number of them are currently being sequenced. 4. 4. These results demonstrate the feasibility of isolating bioactive peptides from nematodes purely on the basis of their bioactivity rather than on the basis of their immunoreactivity with antisera raised against known bioactive peptides.

Glutathione transferase in the free-living nematode Panagrellus redivivus

The free-living nematode Panagrellus redivivus was found to have relatively high cytosolic glutathione transferase activity. Chromatofocusing indicated that at least four GSH transferase forms were present in the nematode cytosol. An endogenous molecular factor interfered with the binding of the cytosolic GSH transferases to a glutathione affinity matrix and binding only occurred after a partial purification step. The major resolved GSH transferase form B was a basic enzyme that showed no biochemical homology to the three mammalian multigene GSH transferase families and appeared to interact with plant phenols, possible natural substrates. A minor GSH transferase form AT showed a biochemical relationship to the mammalian α family including catalytic activity with a model lipid hydroperoxide substrate.

The possible absence of cytochrome P-450 linked xenobiotic metabolism in helminths

The distribution of cytochrome P-450, b 5 and associated oxidations, aniline hydroxylase, 7-ethoxycoumarin O-deethylase and 4-nitroanisole O-demethylase were studied in the parasitic nematode Heligmosomoides polygyrus, its mammalian host, Mus musculus and the free-living nematode Panagrellus redivivus. Cytochrome P-450, its associated oxidations and cytochrome b 5 could not be detected in whole homogenates or subcellular fractions (mitochondrial, microsomal and soluble fractions) of either nematode under a variety of assay conditions which included attempted induction with sodium phenobarbital. P. redivivus was able to reduce 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene and azobenzene, but activity was localised in the soluble fraction unlike the mammalian cytochrome P-450 linked reduction of azobenzene, which is predominantly microsomal. The implications of these results in terms of chemotherapy are discussed.

Interaction of nematodes with nematophagus fungi: induction of trap formation, attraction and detection of attractants

Induction of trap formation in Arthrobotrys oligospora, A. conoides and Monacrosporium cystosporum was studied during a 24 h period in the presence of the free-living nematodes Panagrellus redivivus on various nutrient media. A definite pattern of trap formation was observed in these fungi. Low nutrient mineral salt medium had the most pronounced effect on trap formation. Attraction and repulsion of P. redivivus towards these three nematophagous and three non-nematophagous fungi was studied. The nematodes were attracted towards three nematophagous and one non-nematophagous fungi, the other two repelled them. Attractants of the nematophagous fungi were determined by thin-layer chromatography. In the case of A. oligospora and M. cystosporum, four spots were traced, whereas in A. conoides, five spots were detected.

Isolation and characterization of a nematode transposable element from Panagrellus redivivus.

We have isolated a transposable element, designated PAT-1, from the free-living nematode Panagrellus redivivus. P. redivivus strain C15 was found to have a high spontaneous mutation frequency compared to the standard Caenorhabditis elegans laboratory strain N2. To characterize the genetic lesions occurring in spontaneous C15 mutants, we molecularly cloned the homolog of the C. elegans unc-22 gene from wild-type P. redivivus and two strains carrying spontaneous mutations in this gene. One of these mutations resulted from the insertion of a 4.8-kilobase segment of repetitive DNA. This repetitive element (PAT-1) varies in copy number (10-50 copies) and location in different P. redivivus strains and is absent from C. elegans. The element could be useful as a transformation vector for C. elegans. Our approach is a general one that could be used to isolate additional nematode transposons from other species.

Anaerobic Metabolism in the Free-Living Nematode Panagrellus redivivus

Under anaerobic conditions, the free-living nematode Panagrellus redivivus produces a range of acidic and neutral end products. The main acidic end product was lactate with lesser amounts of alanine, acetate, and propionate; the major neutral end product was ethanol with smaller amounts of glycerol and traces of acetoin. Apart from carbon dioxide, no other end products could be detected. During recovery from anaerobiosis, P. redivivus was found to be able to remetabolize the accumulated end products. The coproduction of ethanol and glycerol may provide a mechanism for reoxidizing mitochondrial NADH during anaerobiosis.

Acetaldehyde formation by mitochondria from the free-living nematode Panagrellus redivivus.

The mitochondrial fraction from the free-living nematode Panagrellus redivivus decarboxylates pyruvate to produce significant amounts of acetaldehyde. Acetaldehyde formation is stimulated by thiamin pyrophosphate, shows a sharp optimum at pH 6.8 and is greater under anaerobic than aerobic conditions. The pyruvate decarboxylase activity cofractionates with, and is probably a partial reaction of, the pyruvate dehydrogenase complex. Acetaldehyde production is modulated by NAD+, ATP and acetyl-CoA and is greatly stimulated by lipoic acid. The pyruvate decarboxylase system is extremely sensitive to thiol-group inhibitors and is inhibited by oxygen in the presence of pyruvate.

Estimation of mutation rates induced by large doses of gamma, proton and neutron irradiation of the X-chromosome of the nematode Panagrellus redivivus

The radiation-resistant free-living nematode Panagrellus redivivus was used to study mutation rates in oocytes, following gamma, proton and neutron irradiation in the dose range 45–225 grays. γ-Radiation produced approximately 0.001 lethal X-chromosomes per gray over the range tested. Proton or neutron irradiation produced approximately 0.003 lethal X-chromosomes per gray at lower doses, with the mutation rate dropping to 0.001 lethal X-chromosome per gray at the higher doses. These results suggest a dose-dependent mutation-repair system. Cell lethality was also examined. γ-Radiation produced the greatest amount of cell lethality at all doses, while neutron irradiation had no cell lethal effect at any of the doses examined.