The cercarial tail in Proterometra macrostoma (Digenea: Azygiidae): permeability and fine structure of the tegument.

Permeability of the cercarial tail in Proterometra macrostoma was examined in vitro with 1 mM 3H-glucose, which tails absorb by diffusion alone. Naturally emerged cercariae (bodies withdrawn into tails) were permeable, but they rapidly (3 min) equilibrated with glucose in the bathing medium and maintained steady state for 4 hr. Metabolism of absorbed glucose was not detectable until after 90 min, and radioactivity in bodies dissected from tails after 4 hr was negligible. On the basis of cercarial water content (90% of total weight) and absorbed isotope at steady state, the calculated volume of the equilibrating compartment was 4% of an intact cercaria. This value correlated well with that of the tegument (3-5%), which was 1-2 microm thick as seen by transmission electron microscopy. A continuous, electron-dense basal membrane/lamina separated the tegument from subtegument. We conclude that the glycocalyx and external plasma membrane are freely permeable, whereas the basal membrane is the barrier that effectively isolated the subtegument from exogenous glucose. The basal membrane also may be the primary structure that protects the subtegument and cercarial body from effects of osmotic stress.

Proterometra macrostoma (Digenea: Azygiidae): variations in cercarial morphology and physiology.

Snails, Elimia semicarinata, infected with Proterometra macrostoma were collected monthly in 1990 and 1991 from North Elkhorn Creek near Lexington, Kentucky, and kept on a 12:12 h light-dark cycle for 2 weeks. Cercariae emerging from snails were classified into 8 strains (I-VIII) based on differences in number and distribution of spined and spineless papillae on the tail. Cercariae also had unique patterns of emergence, swimming behaviour and infectivity in 4 species of sunfish. Of 513 infected snails collected in May, 339 had pure infections with the strain frequencies (% of 339): I, 46.6; II, 7.7; III, 12.1; IV, 8.8; V, 0.6; VI, 2.7; VII, 11.8; VIII, 9.7. In the multiple infections, 159 snails shed 2 strains, 14 shed 3, and 1 snail shed 4 strains simultaneously. A comparison of sunfish and parasite populations in Kentucky, Ohio and Michigan indicated that strain frequency in P. macrostoma is regulated by the species composition of the sunfish population.

The rapid development of the glucose transport system in the excysted metacestode of Hymenolepis diminuta.

Temporal changes in glucose transport capacity in excysted scoleces of Hymenolepis diminuta were examined. Assays involved incubation for 1 min in [3H]glucose after pre-incubation for 1 min to 8 h in saline. There were two abrupt increases in uptake velocity, a relatively small one between 15 and 75 min, and a large one between 5 and 6 h, during which the Vmax increased from 0.36 to 2.49 nmol/25 larvae/h. The second increase was unaffected when the pre-incubation saline contained 5 mM glucose, but it was completely blocked when the excysted larvae were pre-incubated in Ca(2+)-free saline. Abrupt glucose transport changes did not occur in intact cysticercoids or in scoleces when the substrate was [3H]leucine or [3H]uracil. Arrhenius plots (log V versus 1/temperature, 10-42 degrees C) were linear for intact cysticercoids, but were biphasic for both scoleces and adults with discontinuities at 20 +/- 1 degrees C. Thus, 'activation' of the excysted scolex seemed to involve a specific, Ca(2+)-dependent increase in number of glucose transporters functioning in the worm surface. The Arrhenius plots indicated that development in the final host does not involve a major change in lipid composition of the parasite's membranes.

Water balance and its relation to fermentation acid production in the intestinal parasites Hymenolepis diminuta (Cestoda) and Moniliformis moniliformis (Acanthocephala).

Water balance and its relation to carbohydrate metabolism was examined in Hymenolepis diminuta in parallel with the putative osmoconformer Moniliformis moniliformis. Worms were removed from rat intestines, weighed, and incubated (37 C) 1 hr in rat serum and various salines, some with mannitol to vary osmotic concentration from 150 to 400 mOsm/L. Worms were removed at 15-min intervals, weighed, and returned to the test solution. Rat serum and a Ringer's saline (pH 7.4 and 300 mOsm/L) with or without 5 mM glucose were isotonic to M. moniliformis, which behaved like an osmometer, shrinking, or swelling in proportion to external osmotic changes. Hymenolepis diminuta rapidly lost 20-25% wet weight in these solutions and regained lost water when 5 mM glucose was added to the saline. Tapeworms maintained constant body weight between 210 and 335 mOsm/L, but they rapidly gained or lost water outside of this range. Glucose metabolism and uptake of [3H]glucose from the medium increased progressively between 210 and 310 mOsm/L, whereas uptake rates of [3H]leucine, 22Na+, and 36Cl- were not affected. Unbuffered saline (initial pH 6.5 and 300 mOsm/L) had a lower pH (5.0) and higher osmolality (307 mOsm/L) after a 1-hr incubation with tapeworms. Such saline was less hypertonic than unconditioned saline to freshly obtained worms. A Ringer's saline (300 mOsm/L) containing 50 mM acetate- was also hypertonic (greater than 20% weight loss) to tapeworms at pH 7.4, but it was hypotonic (greater than 20% weight gain) at pH 5.0. Isotonicity at 300 mOsm/L was achieved with pH 5.0 and 20 mM acetate-, the approximate pH and fermentation acid concentration in an infected rat intestine. Rats infected with tapeworms (12 days old) were fasted for 2 days. Starved worms were smaller but had the same percentage of body water and internal osmolality as controls. These results show that H. diminuta can regulate its body water content and that water balance is closely related to the fermentation acid concentration and pH of the bathing medium.

Fine structure and sugar transport functions of the tegument in Clinostomum marginatum (Digenea: Clinostomatidae): environmental effects on the adult phenotype.

Digenean flukes can be classified into 3 groups according to their location in the host: the lumen of the alimentary canal or associated organ, body cavity or tissue, and external surfaces. We obtained adults of Clinostomum marginatum that had matured in these 3 habitats and compared the fine structure and glucose transporting capacity of their teguments. Adults from the esophagus of herons, Ardea herodias, had thick, smooth teguments and took up glucose by facilitated diffusion, the type of transport that is Na(+)-independent and insensitive to phlorizin. By contrast, the surfaces of adults cultured from metacercariae in body cavities of laboratory mice were amplified 3-5-fold due to numerous irregular projections of the tegument. Glucose transport by these worms was largely Na(+)-dependent and inhibited by phlorizin, indicating active transport. Ectoparasites from herons' mouths had relatively thick, smooth teguments, but these worms always were encrusted with bacteria and yeast that are known to absorb and metabolize glucose. Most of the attached bacteria, and the apparent glucose uptake associated with their presence, were removed by treating the worms with antibiotics prior to transport assays. As facilitated diffusion and active transport are operational simultaneously in metacercariae, the type of transport function, if any, expressed in the adult is determined by environmental conditions associated with the worm's habitat.

Bacteria associated with tegument of Clinostomum marginatum (Digenea).

Adults of Clinostomum marginatum freshly collected from a heron, Ardea herodias, were examined using transmission electron microscopy. Specimens from the mouth of the bird were encrusted with bacteria that were not removed by washing unless the saline contained antibiotics. There was no evidence that the attached bacteria were damaging to the trematode tegument. Three species of Gram-negative bacteria were isolated from the worm surfaces and identified; Achromobacter sp. was present in pure culture on 4 of 6 original cultures and in mixed culture with Edwardsiella tarda and Enterobacter agglomerans in 2 cultures. These species and 3 unidentified species of bacteria were isolated from the oral epithelium of the heron. Microorganisms were not seen attached to the surfaces of worms recovered from the esophagus. Because E. tarda and E. agglomerans were the only species isolated from the heron esophagus, the intimate bacterial-worm association in the heron mouth may be due specifically to Achromobacter sp.

Mechanism of 3-0-methylglucose uptake by Hymenolepis diminuta.

The mechanism by which Hymenolepis diminuta (Cestoda) absorbs 3-0-methylglucose (30MG) in vitro was analyzed. Influxes of 0.1 and 0.01 mM-3H-30MG during incubations ranging from 5 s to 60 min were not affected significantly when 10 mM-unlabeled 30MG was present as an inhibitor. After 60 min in 0.1 mM-3H-30MG, the concentration of labeled substrate within tapeworms (0.04 mumol ml-1 worm water = 0.04 mM) was less than that of the bathing medium. Tapeworms incubated for 1 h with either 5 mM-glucose or 5 mM-beta-methylglucose (beta MG) gained 15-20% more water than did tapeworms in saline alone, but addition of 5 mM-30MG to the saline had no significant effect on weight change. When the 3H-30MG concentration was varied from 0.01 to 10 mM, influxes were a linear function of substrate concentrations. These analyses show that H. diminuta absorbs 30MG by simple diffusion alone. Thus, use of this monosaccharide to estimate the internal concentration of actively transported sugars (e.g. glucose or beta MG) in H. diminuta is invalid.

Hymenolepis diminuta (Cestoda) liberates an inhibitor of proteolytic enzymes during in vitro incubation.

Hymenolepis diminuta liberated measurable amounts of 'Lowry-positive material' (LPM) and protein during incubation for 2 h in vitro. When tapeworms were incubated in the presence of bovine trypsin (BT), or when BT was added to the medium after removing the tapeworms, the enzyme's proteolytic activity was inhibited significantly. Centrifugation of the medium at 30,000 g yielded a pellet composed of tegumental elements, but this fraction did not inhibit BT. The 30,000 g supernatant fraction contained a chemical(s) that inhibited the proteolytic enzymes of the rodent host's intestinal contents (IC). The inhibitor(s) was stable following repeated freeze-thaw cycles, heat labile, and not degraded by BT or IC, and it inhibited the amidase activity of BT in a non-competitive manner.

Cultivation of Clinostomum marginatum (Digenea: Clinostomatidae) metacercariae in vitro, in chick embryo and in mouse coelom.

Metacercariae of Clinostomum marginatum obtained from naturally infected Perca flavescens were cultured by 5 methods. In vitro cultivation at 37 C in twice daily changes of Tyrode's, Na-poor Tyrode's, and Eagle's media failed to produce ovigerous adults after 4-5 days. Metacercariae placed on the chorioallantois of week-old chick embryos at 37 C migrated to the albumen. Only 8 of 123 worms recovered were ovigerous after 4-7 days in ovo. Best success occurred with metacercariae injected in groups of 4 or 6 into the abdominal cavities of male A/J mice. Despite liver lesions and strong inflammatory responses in the host, all 174 worms recovered were ovigerous after 4-8 days. None of 41 mice died prematurely from the procedure or the parasites.

Fine structure and permeability of the metacercarial cyst wall of Clinostomum marginatum (Digenea).

Encysted metacercariae of Clinostomum marginatum (Digenea) were obtained from tissues of yellow perch, Perca flavescens. The outermost wall (host response) as seen under electron microscopy consisted of a single, fibrous tissue layer, 10-25 micron thick. The tissue contained flattened fibrocytes, small fat deposits, and vacuoles embedded between layers of collagen fibers. The cyst cavity was filled with small vesicles, crystals, and debris. No layer corresponding to the primary (parasite-produced) cyst wall secreted by most species of metacercariae was noted. To determine the permeability of the cyst wall, encysted worms were incubated under initial rate conditions with [3H] glucose, with and without the glucose transport inhibitors phlorizin and phloretin. After incubation, the worms were mechanically excysted, washed, and processed to determine glucose uptake rates. Vmax and Kt were greater than those obtained for worms excysted prior to incubation with substrate. Moreover, the presence of phlorizin or phloretin in the incubation medium had no effect on glucose uptake by encysted worms. Thus, the selective permeability of the cyst wall permits free diffusion of glucose to the cutaneous transport systems of the worm, while restricting the movements of phlorizin and phloretin.

Environmental sodium regulates cutaneous sugar transport in a digenean fluke.

Glucose uptake was examined in adult specimens of Proterometra macrostoma (Trematoda: Digenea) recovered from the stomach (endoparasites) and gills (ectoparasites) of longear sunfish, Lepomis megalotis. The endoparasitic forms transported glucose directly through the external body surface by Na+-independent, facilitated diffusion, but the ectoparasites absorbed glucose by free diffusion alone. To determine how this transport function is regulated, cercariae were incubated in solutions having Na+ concentrations normally found in fish gut (50 mM) and in fresh water (0.5 mM). Glucose transport capacity was retained in 50 mM Na+, but disappeared in worms incubated for 1-3 days in 0.5 mM Na+. Returning worms from the latter solution to one containing 50 mM Na+ fully restored glucose transport within a day. By contrast, incubation up to 5 days in 0.5 mM Na+ had no effect on glucose transport in endoparasitic adults of P. macrostoma and cercariae of P. edneyi. Thus, cutaneous sugar transport function in the migrating larva of P. macrostoma is subject to indirect regulation by environmental Na+.

Specificity of the surface aminopeptidase in Moniliformis moniliformis (Acanthocephala).

The hydrolysis of various oligopeptides in solution by intact Moniliformis moniliformis was examined using paper chromatographic analysis of the incubation medium. In the presence of transport inhibitors, the respective peptide sub-units and/or amino acid residues accumulated in the bathing medium. Only peptides with serine, methionine, leucine or alanine at the NH2-terminal end of the peptide were hydrolysed. There was no hydrolysis when these amino acids were located internally or at the COOH-terminus indicating genuine aminopeptidase activity of the class, alpha-aminoacylpeptide hydrolase. Hydrolysis was negligible when the NH2-terminus was arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, phenylalanine, proline, tryptophan, tyrosine, or valine. In separate experiments, mediated uptake of 0.1 mM 3H-leucine by the worms in 2 min was inhibited 100% by 5 mM unlabelled leucine or tri-serine, but only partially inhibited by 5 mM Ser-Gly (66%), 10 mM Ser-Gly (74%), 5 mM Leu-Leu (69%), 10 mM Leu-Leu (70%), 5 mM Leu-Gly (58%) or 5 mM Met-Met (69%). Because the inhibitions produced by 5 mM Leu-Leu plus 5 mM Met-Met (79%) or 5 mM Leu-Leu plus 5 mM Ser-Gly (76%) were not additive, a single enzyme is indicated. The name serine aminopeptidase is proposed because of its preference for serine.

Paracellular water uptake and molecular sieving by the foot epithelium of terrestrial slugs.

A paracellular pathway in the foot epithelium of Lehmannia valentiana can be opened by dehydrating the slug. Movement of water from a wet pad through the opened pathway into the haemolymph of this terrestrial slug is rapid. The sieving properties of this paracellular pathway have been determined using the reference isotope 3HOH and various 14C-labelled solutes. Paracellular uptake of 14C-insulin (Fig. 1) and 3HOH (Fig. 2) is initial rate for at least 3 min. If the wet pad contains 1,000 cpm of 14C per ml of 3HOH, slugs absorb only about 400 cpm of 14C with each ml of 3HOH absorbed representing a sieving ratio of 0.4 for insulin. The sieving ratio of 14C-inulin does not change when the concentration is increased from 0.1 to 2.5 mmol/l. Moreover, the sieving ratio of 14C-inulin was not affected significantly by the nature of the labelling, i.e., 14C-carboxyl vs 14C-methoxy. Sieving ratios for 14C-mannitol (182 Da), 14C-polyethylene glycol (4,000 Da), and 14C-inulin (5,250 Da) were 0.92, 0.63, and 0.39, respectively (Table 1), indicating that sieving is dependent on molecular size. 14C-Dextran (70,000 Da) and blue dextran (200,000 Da) were excluded from the paracellular pathway (Fig. 4). The effective pore size of the paracellular pathway was estimated using the relationships between sieving ratio and molecular weight of 3HOH and the various solutes that can pass through the pathway. The extrapolated pore size is equivalent to that of a sieve having a molecular weight cutoff of about 10,000 Da (Fig 3).

Analysis of contact-rehydration in terrestrial gastropods: absorption of 14C-inulin through the epithelium of the foot.

Contact-rehydration in terrestrial slugs involves a specific drinking behaviour during which water is rapidly absorbed through the integument of the foot. When dehydrated slugs were placed on wet filter paper containing 14C-inulin, they displayed the characteristic drinking posture and absorbed both water and 14C-inulin. Samples of haemolymph from dehydrated slugs after 12 min of contact-rehydration contained about 6 micrograms of 14C-inulin 100 mg-1 of haemolymph (0.24 mmol l-1 14C-inulin in the substrate). The haemolymph of hydrated slugs however contained no detectable radioactivity after 12 min on the filter paper. Electron microscopy revealed that the intercellular spaces between the epithelial cells of the foot were reduced in dehydrated slugs, but were rapidly enlarged during contact-rehydration. It is concluded that contact-rehydration in terrestrial slugs is mediated by bulk flow of water through an epithelial paracellular pathway in the integument of the foot.

Allosteric control of pyrimidine transport in Hymenolepis diminuta: an unusual kinetic isotope effect.

A thymine-uracil transport system is present in the plasma membrane brush-border of the tapeworm, Hymenolepis diminuta. The relation between initial uptake and substrate concentration of either thymine or uracil was sigmoidal when 2-14C-labelled substrates were used. In contrast, absorption kinetics of methyl-14C- and 3H-labelled substrates were hyperbolic. Since there was no metabolism of the labelled substrates during the incubation period, these differences indicate that the sigmoidal kinetics of pyrimidine transport in this organism is an isotopic effect associated with the presence of the 14C label at the 2 position in the pyrimidine ring.

Trypsin inhibition by tapeworms: antienzyme secretion or pH adjustment?

The tapeworm Hymenolepis diminuta releases proteins that inhibit trypsin activity. These proteins may be either antienzymes or nonspecific macromolecules that interfere with trypsin. Saline solutions with initial pH values ranging from 5.5 to 10.0 were all acidified to pH 5.0 by tapeworms. If the initial pH was lower than 5.0, it was raised. Because trypsin activity is inhibited at pH 5.0, this intestinal parasite can protect itself from digestion by regulating its environmental pH or releasing trypsin inhibitors, or both.

Sugar transport by larval and adult Proterometra macrostoma (Digenea) in relation to environmental factors.

Monosaccharide transport systems were identified in rediae and cercariae of Proterometra macrostoma. Glucose transport by cercariae in vitro was accomplished by the bodies, but tails absorbed glucose by diffusion alone. No sugar transport system was detected in adults obtained from laboratory infections of sunfish. Temperature of Elkhorn Creek where infected snails (Goniobasis livescens) were found varies from 28 C in summer to 5 C in winter. Glucose transport by both larval stages was optimal between 30 and 25 C and was negligible below 15 C. Transport by rediae was activated by Na+ and was maximal at 50 mM, the approximate Na+ concentration of snail hemolymph. The redial glucose transport system was inhibited by various sugars and was more sensitive to phlorizin than to phloretin. Rediae accumulated glucose and nonmetabolized 3-O-methyl-glucose against apparent concentration gradients, indicating an active transport system. In contrast, glucose transport by "embryonic" cercariae was completely inhibited by 10 mM Na+. Transport by cercariae aged in creek, water 6 hr became relatively insensitive to Na+, and were less permeable to glucose by diffusion. The cercarial system differed in other characteristics from the redial system, including its high sensitivity to phloretin relative to phlorizin and its lower Vmax. The cercarial system apparently functioned only as a facilitated diffusion system that served to move sugar across the tegument down its chemical gradient. Development of P. macrostoma thus showed functional transformations of the tegument that may be subject to regulation by environmental factors.