Ascaris suum: Free amino acids and proteins in the pseudocoelom,seminal vesicle and glandular vas deferens

The free amino acids and proteins of the seminal vesicle and pseudocoelomic fluids in the male Ascaris suum were examined and compared. The seminal fluid contained a high concentration of lysine (lysine: glutamic acid ratio of 5:1) while the pseudocoelomic fluid contained more glutamic acid than lysine with alanine, serine, glycine and proline being the most abundant free amino acids. The proteins present in the seminal fluid differed from those in the pseudocoelomic fluid in both number and molecular weight. The sperm activating substance (SAS) present in homogenates of the glandular vas deferens of male worms is nondialyzable, heat-sensitive and can be precipitated using 45% saturated ammonium sulfate. Active moieties can be recovered following passage of the ammonium sulfate precipitates through ultrafiltration membranes or by applying gland homogenates to an ion exchange column. When subjected to SDS-polyacrylamide gel electrophoresis, the active fractions revealed both low and high molecular weight substances. During attempts to purify a single activating substance, it was noted that the more heterogeneous fractions contained the highest activating capacity. Thus, no precise relationship between the biological activity and the purity of the various fractions was determined.     

Subcellular fractions and the refringent granules of the spermatozoa of Ascaris suum (Nematoda)

Summary Six subcellular fractions were isolated by differential centrifugation of the homogenate of spermatozoa of Ascaris suum. The cellular constituents of pelleted fractions, as identified by electron microscopy, were membranes and membranous organelles (fraction A1), microsomal (A2), cytoplasmic (A3), large refringent granules (B1), small refringent granules (B2) and a detergent-soluble fraction (B3).Polypeptide analysis by SDS-PAGE showed that the 18,400-dalton band, one of the major spermatozoan proteins, is detectable in all of the fractions. However, the cytoplasmic (A1) and refringent-granule (B1) fractions contained the highest level.The isolated refringent granules consisted of 2–6 % lipid while the nonlipid fraction formed an insoluble matrix with a fibrillar network morphology. This fibrillar matrix contained three polypeptides of small molecular weight (7,000–14,000) in addition to the 18,400-dalton polypeptide. These small polypeptides (7,000–14,000 MW) are detectable only in fractions of the refringent granules and are therefore called the refringent-granule proteins (RGP). These RGP are sensitive to tryptic hydrolysis and have solubility properties similar to the protein, ascaridine.Adult Ascaris suum were generously provided by Wilson and Company, Cedar Rapids, Iowa. This study was supported by postdoctoral fellowship 5F32AI05646 from NIH. The assistance of Mr. Douglas Wood is gratefully acknowledged     

In vitro activation and behavior of the ameboid sperm of Ascaris suum (Nematoda)

Summary A system is described for the study of activation and motility of Ascaris spermatozoa in vitro. Activation was accomplished by addition of the sperm-activating substances (SAS), extracted from the male accessory gland, to cells incubated in phosphate-buffered saline (pH 7.4) at 37–39° C under anaerobic conditions (95% N₂, 5% CO₂). Activation is characterized by a change from spherical to ameboid shape with coalescence of the refringent granules. The normal ameboid spermatozoa bear several stubby and needle-like filopodia at the lamellipodial margin. Within the lamellipodium are bundles of microfilament-like structures extending toward the pseudopodial membrane and concentrating within the needle-like filopodia. These filopodia exhibit a pendulous, sweeping motion with subsequent retraction and disappearence within the main lamellipodium. Membranes of the ameboid cells interact at the pseudopodial regions with partial fusion, as suggested by apparent membrane breakdown between interdigitating portions of the pseudopodia. Activation is complete in 5–15 min, is totally inhibited at 4° C and/or by an atmospheric environment, but can be reinitiated by transfer to anaerobic conditions at 22–9° C. Activation also requires favorable pH (6.8–8.7) and continual exposure to sufficiently high sodium concentrations (134–154mM), i.e., lowering of sodium concentration to 10 mM causes irreversible inactivation. Sodium may be replaced by potassium or lithium but not by Tris or sucrose. Proteinases (10 g/ml) can act as activators even though SAS lack detectable proteolytic activity against azoalbumin, azocasein, TAME and BTEE and SAS activation was not inhibited by TLCK or soybean trypsin inhibitor.Adult Ascaris suum were provided through the generosity of Wilson and Company, Cedar Rapids, Iowa, U.SA. This study was supported by grant number 5T01 HD00152 and postdoctoral fellowship 1F 32AI05646 from the National Institute of Health, U.S.A.     

Reversible inhibition of hatching of infective eggs of Ascaris suum (Nematoda)

Hurley L. C. and Sommerville R. I. 1982. Reversible inhibition of hatching of infective eggs of Ascaris suum (Nematoda). International Journal for Parasitology12: 463–465. Dilute solutions of an oxidising agent, iodine, reversibly inhibit hatching of infective eggs of Ascaris suum. The capacity to hatch is restored by exposure to reducing agent, hydrogen sulphide. These observations add to known similarities between hatching of infective eggs and exsheathment of infective larvae. It is proposed that the regulatory mechanisms for both processes are similar.     

Purification and characterization of acid trehalase from muscle of Ascaris suum (Nematoda)

Acid trehalase (EC 3.2.1.28) was isolated from muscle of Ascaris suum by fractionating with ammonium sulfate, acetone and column chromatography on DEAE-cellulose and phenyl sepharose CL-4B. The purified homogeneous preparation of native acid trehalase exhibited a molecular mass of 76 kDa and of 38 kDa on SDS-PAGE. The enzyme has the optimum pH 4.9, pI 4.3, Km of 6.6 mM and Vmax=34.5 nM min(-1) x mg(-1). Besides trehalose, it hydrolyses sucrose, isomaltose and maltose and, to a lesser degree melezitose, and it does not act on cellobiose and lactose. Acid trehalase was activated by MgCl2, KNO3, NaCl, CaCl2, CH2ICOOH and p-chloromercuribenzoate and inhibited by EDTA, ZnSO4 and FeCl3.     

Structural analysis of neutral glycosphingolipids from Ascaris suum adults (Nematoda: Ascaridida)

The neutral glycosphingolipid fraction from adults of the pig parasitic nematode, Ascaris suum, was resolved into four components on thin-layer chromatography. The high-performance liquid chromatography-isolated components were structurally analysed by: methylation analysis; exoglycosidase cleavage; gas-liquid chromatography/mass spectrometry; liquid secondary-ion mass spectrometry; and, in particular, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures were determined as: Glc(β1-1)ceramide, Man(β1-4)Glc(β1-1)ceramide, GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide and Gal(α1-3)GalNAc(β1-4)GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide; and were characterized as belonging to the arthro-series of protostomial glycosphingolipids. No glycosphingolipid component corresponding to ceramide tetrasaccharide was detected during these analyses. The ceramide composition of the parent glycosphingolipids was dominated by the 2-(R)-hydroxy C24:0 fatty acid, cerebronic acid, and C17 sphingoid-bases: 15-methylhexadecasphing-4-enine and 15-methylhexadecaphinganine in approximately equal proportions. The component ceramide monohexoside was characterized by an additional 15-methylhexadecaphytosphingosine. Abbreviations: CDH, ceramide dihexoside; Cer, ceramide; CMH, ceramide monohexoside; CPH, ceramide pentahexoside; CTH, ceramide trihexoside; CTetH, ceramide tetrahexoside; Hex, hexose; HexNAc, N-acetylhexosamine; HPTLC, high-performance thin-layer chromatography; LSIMS, liquid secondary-ion mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; N-, Nz- and A-glyco(sphingo)lipids, neutral, neutralzwitterionic and acidic glyco(sphingo)lipids, respectively This revised version was published online in November 2006 with corrections to the Cover Date.     

Activity of enzymes regulating glycogen metabolism in perfused muscle-cuticle sections of Ascaris suum (Nematoda)

A new perfusion system has been developed in which muscle-cuticle sections of Ascaris suum were perfused, enabling study of enzymes in vitro. Using this technique the activity of the regulatory enzymes glycogen synthase and glycogen phosphorylase was determined, and the level of glycogen in the muscle was assessed. During starvation, 98% of glycogen synthase was in the inactive D-form, and 80% of the glycogen phosphorylase activity was in the active a-form. When the ascarid muscle section was perfused with 27 mM glucose, 13.1% of the glycogen synthase was in the active I-form, whereas phosphorylase a-levels dropped to 46% and glycogen was synthesized at a linear rate of 12 mg/g/hr or 1.23 mumoles/min/g muscle-cuticle. ATP levels (3.71 +/- 0.32 mM) remained unchanged over a 4-hr perfusion period with an adenylate energy charge of 0.82. Fructose supported glycogen synthesis, though not as well as glucose. Galactose, mannose, and trehalose did not support glycogen synthesis. The new perfusion system should be useful in future, similar studies on Ascaris.     

Amino acid and lipid composition of refringent granules from the ameboid sperm of Ascaris suum (Nematoda)

Transformation of the spermatozoon of Ascaris suum from a spheroidal to an ameboid cell is associated with the formation of a motile pseudopodium and coalescence of the intracellular refringent granules. The pseudopodia of the ameboid spermatozoa contain filaments organized into dense patches, bundles, web-like or lace-like networks, as observed by electron microscopy. The morphology and chemistry of the refringent granules were investigated in subcellular fractions enriched for these structures. Isolated refringent granules were heterogeneous in size measuring from 0.5 X 0.6 to 2.3 X 3.5 microns. Each granule is surrounded by a 110 A thick layer. During fusion, the surfaces of the refringent granules form small extensions resembling micropodia. The process of fusion occurs at many sites on a given granule and simultaneous fusion of several granules was commonly observed. Amino acid analyses of the refringent granule proteins (RGP's) indicated: they are rich in aspartic acid or asparagine (48%), leucine (10%), serine (19%) and aromatic amino acids (11%). Gas-liquid chromatographic analyses of alditol acetate derivatives of monosaccharides released by mild acid hydrolysis showed the predominant sugars to be glucose (7.3 micrograms/mg protein), galactose (9.2 micrograms/mg) and N-acetylglucosamine (5.5 micrograms/mg). Lipid analyses indicated a complex mixture of glycerides, ascarosides and waxes, together with a major component that resembled free fatty acid in mobility on TLC.     

Amino acid and lipid composition of refringent granules from the ameboid sperm of Ascaris suum (Nematoda)

Summary Transformation of the spermatozoon of Ascaris suum from a spheroidal to an ameboid cell is associated with the formation of a motile pseudopodium and coalescence of the intracellular refringent granules. The pseudopodia of the ameboid spermatozoa contain filaments organized into dense patches, bundles, web-like or lace-like networks, as observed by electron microscopy.The morphology and chemistry of the refringent granules were investigated in subcellular fractions enriched for these structures. Isolated refringent granules were heterogeneous in size measuring from 0.5×0.6 to 2.3×3.5 m. Each granule is surrounded by a 110 Å thick layer. During fusion, the surfaces of the refringent granules form small extensions resembling micropodia. The process of fusion occurs at many sites on a given granule and simultanenous fusion of several granules was commonly observed.Amino acid analyses of the refringent granule proteins (RGP's) indicated: they are rich in aspartic acid or asparagine (48%), leucine (10%), serine (19%) and aromatic amino acids (11%). Gas-liquid chromatographic analyses of alditol acetate derivatives of monosaccharides released by mild acid hydrolysis showed the predominant sugars to be glucose (7.3 g/mg protein), galactose (9.2 g/mg) and N-acetylglucosamine (5.5 g/mg). Lipid analyses indicated a complex mixture of glycerides, ascarosides and waxes, together with a major component that resembled free fatty acid in mobility on TLC.     

Behavioral and cellular effects of serotonin on locomotion and male mating posture in Ascaris suum (Nematoda)

The site and mode of action of serotonin on locomotion were investigated in the parasitic nematode Ascaris suum. Injection of serotonin into Ascaris immediately caused paralysis in animals that were generating locomotory waveforms. Injected serotonin also increased body length and decreased the number of propagating body waves. Similar injections into the male tail produced a ventral tail curl. Injection of N-acetyl-serotonin had no effect on the generation of locomotory waveforms, but increased the body length and decreased the number of body waves in the waveform. Other biogenic amines were also tested but were much less potent.Serotonin decreased the amplitude of a submaximal acetylcholine-induced muscle contraction and increased the time to attain this contraction. The time course of this effect on the response to ACh was much slower than the action of injected serotonin on locomotory waveforms, suggesting that additional elements are involved in the action of serotonin on locomotory behavior.Serotonin abolished spontaneous slow potentials in VI motor neurons and decreased the frequency of EPSPs in DE2 motor neurons, probably by a pre-synaptic mechanism. In the male tail, serotonin depolarized the male-specific transverse ventral muscle cells, but did not affect either dorsal or ventral longitudinal muscle cells.Abbreviations ACh acetylcholine - cAMP cyclic adenosine monophosphate - DA dopamine - DE dorsal excitatory motor neuron - DI dorsal inhibitory motor neuron - DM dorsal muscle - ELP egg-laying pore - EPSP excitatory post synaptic potential - GABA gamma aminobutyric acid - HRB head restricted behavior - IPSP inhibitory post synaptic potential - 5-HT 5-hydroxytryptamine, serotonin - NA-5-HT N-acetyl-5-hydroxytryptamine - NE norepinephrine - OA octopamine - PBS phosphate buffered saline - PCF pseudocoelomic fluid - tVM malespecific transverse ventral muscle - TRYP tryptamine - VI ventral inhibitory motor neuron     

Centrids, a pair of asymmetrically arranged sense organs in Ascaris suum (Nematoda, Ascaridoidea)

Two prominent, asymmetrically placed cuticular somatic sensilla, called centrids, are reported in Ascaris suum Goeze, 1782, the pig roundworm. The right centrid is situated much more anteriorly on the body than is the left one. The centrids are globular in the fourth-stage larva and obviously void of an apical pore, suggesting at least a tactile function. In adult worms, the centrids are platelike, lacking a globular expansion. The observation on the presence of asymmetrically placed centrids in A. suum gives further impetus to the importance assigned to sense organs in the classification and identification of nematodes. The name centrid was originally chosen to indicate the placement of the papillae in the midbody region of worms. The name centrid, rather than, e.g. postdeirid, is proposed to be used when denoting asymmetrically oriented midbody sensilla among the Ascaridida and papillae, when shown homologous to these, of species within the Rhabditea generally. This proposal is in line with the name "Mittelk?rperpapillen" originally adopted to denote homologous sensillae in Cucullanidae (Seuratoidea) by T?rnquist in 1931.     

Poly(ADP-ribosylation) in Ascaris suum

Poly(ADP-ribosylation) was demonstrated in the intestinal parasite Ascaris suum, especially in the reproductive tissues. The activity of the ADP-ribosyltransferase was found to depend on divalent cations and to be stimulated by deoxyribonuclease I about 5-fold. The reaction rate was optimal at a temperature of 30 degrees C and at pH about 8.4. The apparent Km value for NAD was estimated to be 0.2mM. The enzyme activity was effectively inhibited by nicotinamide (Ki = 65 microM) benzamide (6 microM), 3-aminobenzamide (10 microM), theophylline (35 microM) and thymidine (50 microM). The type of inhibition by these compounds was found to be competitive with respect to NAD.     

Effect of electric power network frequency magnetic field on embryonic development of Ascaris suum (Nematoda)

Fertilised Ascaris suum eggs were subjected to an alternating electromagnetic field of frequency 50 Hz and density 2 mT for 60 days. The developing embryos in both control and experimental cultures were examined daily under a microscope. The experiment resulted in an accelerated rate of embryogenesis in the eggs incubated in the electromagnetic field, higher rates of malformed embryos as well as much higher mortality rate of L2 larvae.     

Population structure in Ascaris suum (Nematoda) among domestic swine in Denmark as measured by whole genome DNA fingerprinting

We here analyze the population structure in the pig roundworm, Ascaris suum, among domestic pigs in Denmark using a whole-genome DNA fingerprinting technique, "amplified fragment length polymorphism" (AFLP) analysis. With these data, we can extract absolute gene frequency variance components and G-statistics for 135 independent nucleotide polymorphisms. The average proportion of total variance partitioned between Jutland and Zealand is less than 3% of the total variance, implying no restriction in gene flow between worms from different regions in Denmark. The average gene frequency difference between two farms widely separated in Jutland represents 5% of the total genetic variance of these two farms combined. Conversely, worms from different hosts within these two farms are more subdivided, with an average of 12% of the total variance in gene frequencies within farms being distributed between hosts. This result implies substantial single generation inbreeding due to founder effects in the establishment of adult worms in single hosts. Absolute variance components extracted from the gene diversities also showed significant differences, with the among-host variance being greater that the between-farm and between-region values. This little geographical variation is discussed in relation to the hierarchic structure of the Danish swine production system. Comparison of our results with other studies on parasitic roundworms, suggests that patterns of host dispersal effectively control patterns of worm gene flow. Furthermore, the potential spread of anthelminth resistance among A. suum may thus be rapid, due to the flow of infected hosts within the domestic swine stocks in Denmark.