Free-living Nematode Turbatrix Aceti Research Articles

Improving nematode culture techniques and their effects on amino acid profile with considerations on production costs

The purpose of this study was to investigate the effectiveness of 11 different culture media for production of the free-living nematode Turbatrix aceti. Several other harvesting methods were tested in addition to mass production. A further focus was the investigation of amino acid alterations caused by the application of various media during the culture of T. aceti and two additional nematode species, Panagrellus redivivus and Caenorhabditis elegans. Finally, a cost analysis for the production of T. aceti was generated and its outcome compared to the production of conventional live feed organisms. Altogether 11 liquid culture media were tested for mass production of the nematode Turbatrix aceti using a minimum of effort in terms of labour and costs. Six harvesting methods, including filtration as well as active swimming of T. aceti were evaluated. Additional to the culture of T. aceti in four of the above-mentioned media, the nematodes P. redivivus and C. elegans were cultured on two different solid media. Cost analysis for the production of T. aceti includes those of the media, the equipment, as well as the labour costs for culture and harvest. An average density of approx. 30 × 106 ± 8.13 × 106 nematodes L−1 was achieved for T. aceti. The most efficient method (20 μm filtration) allowed harvesting 85.3 ± 2.7% of the nematodes from the medium without disturbing the particles. Lowest efficiency was achieved by combining sedimentation and filtration, accomplishing a harvest of 42.1 ± 5.8%. The amino acid profile of all three nematode species turned out to be both stable and very similar. Amino acid enrichment had little effect. The costs for producing one million T. aceti individuals ranged between 5.39 and 6.19 €, where labour costs accounted for 73 to 84% of the total production costs. In conclusion, T. aceti appears to be very robust, easy to handle, as well as cheaper to cultivate compared to other live-feed organisms. Therefore, its use in commercial aquaculture should be given future consideration.

Bacillus thuringiensisstrains affect population growth of the free-living nematodeTurbatrix aceti

Summary Microbial toxins from Bacillus thuringiensis israelensis, B.t. kurstaki, and B.t. morrisoni reduced population growth of the free-living nematode Turbatrix aceti. Worm populations declined more after treatment with B.t. israelensis than the other tested strains. Population growth of the nematode was inhibited by repeated exposure to B.t. israelensis toxin at three-day intervals during a two-week period. Exposure of the nematodes to the toxin for eight hours caused more mortality than lesser periods of time. Microbial toxins from B. thuringiensis may be a source of agents for nematode control.

Mineral content of the free-living nematode turbatrix aceti and ruminant parasite trichostrongylus colubriformis

1. The parasitic nematode Trichostrongylus colubriformis and free-living nematode Turbatrix aceti were examined for element content. 2. T. colubriformis contained significantly less phosphorus, potassium, manganese and boron than T. aceti. 3. T. colubriformis contained significantly more nitrogen and copper than T. aceti. 4. Comparative study of the chemical composition of parasitic vs free-living nematodes may facilitate in vitro cultivation of the parasitic forms.

Prostanoids of nematodes: Prostaglandins and thromboxane b 2 from Trichostrongylus Colubriformis and Turbatrix Aceti

1. Prostaglandins A 2, E 1, E 2, 6-keto-F 1α, F 2α and thromboxane B 2 were found in extracts prepared from the parasitic nematode Trichostrongylus colubriformis. 2. These same prostanoids plus prostaglandins B 2 and D 2 were found in extracts from the free-living nematode Turbatrix aceti. 3. Though prostaglandins B 2, D 2, 6-keto-F 1α and F 2α occurred in all extracts from T. aceti, and 6-keto-F 1α and f 1α were in all extracts from T. colubriformis, the other prostanoids were absent from some extracts and there were great variations between extracts in the amounts of given prostanoids recovered. 4. This constitutes the first fully-documented report of prostanoids from nematodes.

The effect of aging on cell-free protein synthesis in the free-living nematode Turbatrix aceti

The age-related reduction in cell-free synthesis in the free-living nematode Turbatrix aceti is due to a defect in the ribosomes. Addition of young ribosomal wash or use of young medium does not improve the activity of old, run-off ribosomes in the presence of phenylalanine and poly(U). It appears that some of the old ribosomes are incapable of binding the EF-1-GTP-aminoacyl-tRNA complex. These ineffective ribosomes are present in the 80 S (monosomal) fraction. Old ribosomes obtained from polysomes appear to bind normally.

Age-related changes in the excision repair capacity of Turbatrix aceti

Excision repair capacity was measured in the free-living nematode Turbatrix aceti, a model aging system. Excision repair was assessed by both repair synthesis activity and the actual removal of pyrimidine dimers from the genome. The young organisms removed lesions more rapidly and completely than the old for all fluences tested. Repair synthesis began and peaked earlier for young nematodes than old. The data consistently indicated a decline in DNA excision repair capacity with age in the nematode.

Production and excision repair of cyclobutane-type pyrimidine dimers in the DNA of Turbatrix aceti.

The production and removal of 254 nm ultraviolet-induced pyrimidine dimers was measured in the DNA of the free-living nematode Turbatrix aceti. Approximately 0.0035 per cent pyrimidine dimers are produced per J/m2. Following a fluence of 100 Jm2, approximately 50 per cent of the dimeric photoproducts were excised within 60 min. The number of pyrimidine dimers excised did not change with increasing U.V. fluence, indicating saturation of the U.V. repair system in T. aceti. The results indicate a highly efficient and selective repair system in Turbatrix aceti for dimeric photoproducts.

Relation between growth rate and lifespan in α-tocopherol cultured Turbatrix aceti

When α-tocopherol was added to axenic cultures of the free-living nematode Turbatrix aceti, larvae grew at a faster rate than untreated controls. Additional studies showed that α-tocopherol must be present during the first 72 hours of development to produce this effect. α-Tocopherol supplementation increases Iifespan of these nematodes as well as increasing their rate of development.

39] Fructose-bisphosphate aldolase from Ascaris suum

Publisher Summary This chapter describes an assay method and the purification procedure for fructose-bisphosphate aldolase from Ascaris suum . The assay is based on the oxidation of NADH by dihydroxyacetone phosphate (DHAP) in a coupled system containing triosephosphate isomerase and α-glycerophosphate dehydrogenase. The purification process involves extraction, ammonium sulfate precipitation, substrate elution from phosphocellulose column, diethylaminoethyl (DEAE)-Sephadex chromatography, and crystallization. Crystalline ascarid muscle aldolase is homogeneous by polyacrylamide gel electrophoresis and sodium dodecyl sulfate-gel electrophoresis reveals one protein band. The enzyme has a molecular weight of approximately 155,000–160,000 and is composed of four subunits of identical molecular weight. Ascarid aldolase exhibits a high degree of homology in amino acid composition to vertebrate muscle aldolase. Immunological studies indicate lack of common immunological determinants between rabbit aldolases A and B and ascarid aldolase. Free-living nematode Turbatrix aceti aldolase differs in amino acid composition from Ascaris suum to the same extent as it differs from that of aldolases from mammals.

Effect of α-tocopherol on the lifespan of Turbatrix aceti

The influence of α-tocopherol on the lifespan of the free-living nematode Turbatrix aceti was examined. For control nematodes the mean lifespan was 45.40 days: for α-tocopherol treated cultures it was 60.68 days. The maximum lifespan of the control nematodes was 112 days, as compared to 120 days for the α-tocopherol treated group. Examination of the survival curve revealed that the α-tocopherol treatment significantly influenced survival both at the beginning of development (0–18 days) and at the end of the lifespan, or senescent period. When nematodes were transferred out of α-tocopherol medium into control medium at 0 to 24 hours, there was a significant decrease in their lifespan. When this transfer was made at 96 hours, there was no effect on lifespan. In supplementation studies, α-tocopherol was added at one, four and 30 days. Addition of α-tocopherol at one day significantly increased lifespan, but there was no effect when supplementation was started at four or 30 days. The results suggest the α-tocopherol must be present at the time when cells are developing and enlarging to influence lifespan.

Altered enolase in aged Turbatrix aceti results from conformational changes in the enzyme.

Young- and old-type enolases (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) from the free-living nematode Turbatrix aceti can be unfolded in 1.25 M guanidine hydrochloride and subsequently refolded with essentially a quantitative recovery. After refolding, both enolases form an identical or near-identical third type of the enzyme as determined by spectral criteria, sensitivity to heat, immunotitration, and rate of inactivation by bacterial protease. By the same criteria, the refolded enolase is closer in conformation to the native old form of the enzyme than to the young form. The results prove that young and old enolases are conformational isomers and that an in vivo transformation from young to old enzyme takes place by conformational changes without covalent modification. The process may be related to the previously demonstrated slowing of enolase turnover in T. aceti. Errors in sequence cannot be involved in the age-related alteration of the enzyme.

Multiple forms of isocitrate lyase in the matrix of Turbatrix aceti mitochondria

The localization of five separate forms of isocitrate lyase (EC 4.1.3.1) in the free-living nematode Turbatrix aceti was determined by analyzing their distribution among subcellular fractions with continuous (Tris-acetate, pH 7.5) polyacrylamide gel electrophoresis. Enzyme activity on gels was detected either by substrate-dependent Schiff's-aldehyde staining or absorbance of phenylhydrazone at 324 nm. Following rate sedimentation of worm homogenates, the highest specific activity for isocitrate lyase was recovered in the pellet containing intact mitochondria. Glyoxysomes (microbodies) were not observed by electron microscopy in this or any other fraction. Selective removal of mitochondrial outer membranes (and hence components in the intermembrane space) was accomplished by two different procedures: (1) passage of mitochondria in a hypersomotic medium through a French pressure cell at 1500 psi, or (2) treatment with 0.6 mg digitonin mg protein. Electron microscopy revealed essentially complete removal for the former procedure, but only partial removal following digitonin treatment. Equilibrium centrifugation on sucrose gradients was necessary to strip the residual outer membranes from the digitonin-treated mitochondria. Mitoplasts resulting from the two procedures were subfractionated into matrix and inner membrane components by high-pressure disruption (14,000 psi) and subsequent rate sedimentation (144,000 g, 60 min). Identical electrophoretic patterns were found using both slab and disc gels, whether stained with Schiff's reagent or scanned at 324 nm, in samples taken from clarified homogenates, intact mitochondria, mitoplasts, or matrix fractions. The results indicate that all five forms of the enzyme occur together in the mitochondrial matrices. Their individual functions are not yet known, but they may be involved in the regulation of isocitrate metabolism common to the tricarboxylic acid and glyoxylate cycles occurring within the same mitochondria.

Neutral and acid proteinases from free living nematode Turbatrix aceti. Partial purification and characterization

1. 1. Neutral and acid proteinases from Turbatrix aceti were isolated and partially purified, 400–500-fold and characterized. 2. 2. Neutral proteinase is a thiol proteinase with a molecular weight of 22000 and pH optimum of 6.5. It exhibits chymotrypsin-like specificity and is inhibited by p-mercuribenzoate and β-mercaptoethanol. 3. 3. Neutral proteinase is also inhibited by a specific irreversible inhibitor of serine proteinases—diisopropylphosphofluoridate with second-order rate constant of inactivation = 21 min −1 M −1. However, it is not effected by high molecular weight inhibitors of serine proteinases. 4. 4. Acid proteinase is a typical carboxyl proteinase with a molecular weight of 60,000 and pH optimum of 3.5. It is inhibited by a specific inhibitor of carboxyl proteinases 1,2-epoxy-3-( p-nitrophenoxy) propane.

Isolation of an acyl-CoA carboxylase from the tapeworm spirometra mansonoides

An acyl-CoA carboxylase, which catalyzes the carboxylation of acetylpropionyl-, and butyryl-CoA, has been isolated from the tapeworm Spirometramansonoides. The enzyme has an absolute requirement for ATP, Mg2+, and HCO3− and, in addition, requires K+ for full catalytic activity. The enzyme has been purified 50-fold by a combination of calcium phosphate gel adsorption, ion-exchange column chromatography, and gel filtration. In its substrate specificity, K+ requirement, molecular size, and antigenic behavior, the tapeworm enzyme is similar to the acyl-CoA carboxylase of another helminth— the free-living nematode Turbatrixaceti.

Acyl-coenzyme A carboxylase of the free-living nematode Turbatrix aceti. 2. Its catalytic properties and activation by monovalent cations.

A highly purified acyl-CoA carboxylase from the nametode Turbatrix aceti catalyzes the ATP, Mg2+, and HCO3- dependent alpha-carboxylation of acetyl-CoA, propionyl-CoA, and butyryl-CoA at the respective rates of 6.8, 39.7, and 9.1 mumol per min per mg. The enzyme is inhibited by avidin and sulfhydryl reagents. It is activated up to 30-fold by the monovalent cations K+, Rb+, Cs+, or NH4+, with the apparent activation constants of 11.0, 4.1, 10.0, and 6.7 mM, respectively. In the presence of K+, the apparent Km for ATP increases 5-fold, and the Km for HCO3- decreases 4-fold, whereas the Km for propionyl-CoA remains constant. Of the partial reactions, the ATP-32P exchange reaction and the carboxylation of free biotin have a nearly absolute requirement for K+. By contrast, the [14C]acetyl-CoA-malonyl-CoA exchange reaction proceeds without K+ at 80% of its maximum rate. The data indicate that K+ primarily stimulates the first half of the carboxylation reaction, i.e., the ATP-dependent carboxylation of the biotinyl residue.

Acyl-coenzyme A carboxylase of the free-living nematode Turbatrix aceti. 1. Its isolation and molecular characteristics.

A biotin containing enzyme which carboxylates acetyl-CoA has been isolated from the nematode Turbatrix aceti and purified to homogeneity as judged by the criteria of polyacrylamide gel electrophoresis and ultracentrifugation. The enzyme has a sedimentation coefficient of 18.0 S and a molecular weight of 667 000. It is composed of four protomers having a molecular weight of 140 000 each. Each protomer, in turn, consists of two distinct polypeptide chains (molecular weights 82 000 and 58 000) and one biotinyl prosthetic group which is linked to the 82 000 peptide. The amino acid composition of the nematode carboxylase has also been determined.

Purification of fructose-1.6-diphosphate aldolase from the free-living nematode Turbatrix aceti. Comparison of properties with those of other class I aldolases

1. 1. A fructose-1.6-diphosphate aldolase from the nematode Turbatrix aceti was purified to homogeneity. 2. 2. Purity was verified by electrophoretic and immunological criteria. 3. 3. The enzyme has a mol. wt of 158,000 and possesses 4 identical subunits of 40,000 mol. wt each. 4. 4. The enzyme resembles vertebrate aldolase A as it has a proline in its NH 2-terminus and a tyrosine in its COOH-terminus. 5. 5. However, its catalytic properties, electrophoretic mobility and amino acid composition suggest resemblance to vertebrate aldolase C.

Aging in the free-living nematode Turbatrix aceti. Techniques for synchronization and aging of large-scale axenic cultures

Simple procedures are described for aging large cultures of the free-living nematode, Turbatrix aceti, for use in biochemical studies. After growth in 50 ml of axenic defined medium for 14–16 days in 1 l. Roux bottles, young organisms were separated by “screening” through stainless steel filter cloth. Incubation in fresh medium under conditions which were shown to inhibit reproduction (incubation at 36° or addition of fluorodeoxyuridine [FUDR] plus uridine) permitted the cultures to age without contamination by newborn organisms. Development of a clear medium permitted harvesting by simple filtration on a fritted glass Buchner filter. It was observed that the 50 per cent survival of cultures in the presence of FUDR was approximately 27 days and at 36°C was approx. 16 days. Measurement of length indicated that the growth of the organisms in size was near normal during the aging process.

Studies on ageing in Turbatrix aceti

Morphologic and physiologic changes which occur during senescence in the free-living nematode Turbatrix aceti are described. With age areas of the interchordal hypodermis containing nerve elements thickened, electron-dense aggregates formed within the pseudocoelom and age pigment granules accumulated within the intestinal epithelium. Specific gravity did not change with age. Old nematodes which had reproduced showed increased osmotic fragility, but this change was not observed in virgin females. The parameters characterizing senescence in T. aceti are compared with those of Caenorhabditis briggsae, another nematode being used as a model to study biological ageing.

Biochemical studies of the free-living nematode Turbatrix aceti—II. Isolation and partial characterization of polysomes, ribosomes and ribosomal subunits

1. 1. Various methods of extraction of ribosomes and polyribosomes were tried. The method of Earl & Morgan (1968) gave the highest yields. The sedimentation constants of the particles were calculated and it was seen that Mg 2+ was critical for obtaining 80 S and heavier particles. 2. 2. Two methods were tried to obtain ribosomal subunits. Dialysis against low Mg 2+ buffers according to Tashiro & Siekevitz (1965) caused change of the 80 S particles to 60 S. The high KCl treatment according to Martin et al. (1969) yielded two subribosomal particles with sedimentation constants similar to the subunits of other animals. 3. 3. The methods described enable the production of sufficient quantities of polysomes, ribosomes and ribosomal subunits for studies of the protein synthesizing apparatus of Turbatrix aceti.