Gary L. Uglem

Habitat Specificity and Correlated Aminopeptidase Activity in the Acanthocephalans Neoechinorhynchus cristatus and N. crassus

In natural concurrent infections of the large-scale sucker, Catostomus macrocheilus, the acanthocephalans Neoechinorhynchus cristatus and N. crassus occupied ecologically separate regions. N. crassus attached in the anterior intestine, while N. cristatus attached in the posterior intestine. Subadults of N. cristatus had a mean point of attachment 6 cm anterior to adults and occupied a longer section of intestine than did adults. There was no apparent intraspecies difference in distribution of N. crassus. Only subadults of N. cristatus were able to establish infections in abnormal host fishes. These data suggested that habitat specificity was well defined for both age groups of N. crassus and for adult N. cristatus, but was less so for subadult N. cristatus. Fluorometric aminopeptidase (APase) determinations showed the following: (1) extracts of each species possessed a characteristic and reproducible profile of APase activity; (2) posteriorly situated N. cristatus possessed greater and broader APase activity/mg worm protein than did anteriorly positioned N. crassus; (3) on the other hand, posterior host tissues in the environment of N. cristatus contained less activity than anterior host tissues where N. crassus attached; (4) during maturation of N. cristatus, APase levels decreased. Such a change in N. crassus was not observed. These findings suggest that high levels and a broad range of APase activity in subadult N. cristatus are correlated with its generalized habitat selection. The decrease in APase activity with age paralleled its increased habitat specificity. The demonstration of a biochemical basis for parasite-host specificity is difficult because several chemical as well as physical factors may be involved. Cohabiting parasites seldom have been subjects of such studies. Segregation of cohabitants in the environment may reflect the variation of a physiological adaptation in each species. If it is easily recognized, the adaptation and its relationship to habitat specificity may be studied in the laboratory. In preliminary examinations the acanthocephalan Neoechinorhynchus crassus Van Cleave, 1919, was found in large-scale suckers, Catostomus macrocheilus, of Hangman Creek near Tekoa, Washington, but not in those from the Palouse River east of Potlatch, Idaho. N. cristatus Lynch, 1936, was recovered from the same host from both areas. Concurrent infections by both parasites were common in Hangman Creek suckers, and each species tended to segregate in the intestine. As congeners, they Received for publication 6 January 1972. *From a dissertation submitted by the senior author to the graduate school as partial fulfillment of the requirements for the degree of Doctor of Philosophy. t Present address: Department of Biology, Rice University, Houston, Texas 77001. t Present address: Department of Bacteriology, University of Idaho, Moscow, Idaho 83843. represented an excellent model for study of the niche concept and physiological specificity of helminth parasites. Several studies have shown that helminths undergo a change in chemical composition during larval development (Goodchild and Wells, 1957; Archer and Hopkins, 1958; Hopkins and Hutchison, 1958; Mettrick and Cannon, 1970), suggesting that chemical changes are correlated with habitat specificity of developing worms. Using histochemical methods, Crompton (1963) demonstrated the presence of leucine aminopeptidase in the body wall of the acanthocephalan Polymorphus minutus. We have found that extracts of acanthocephalans and other helminths possess extensive and reproducible aminopeptidase (APase) activity on several a-amino acid-,-naphthylamide substrates. The purpose of this study was to examine changes in APase composition of N. cristatus and N. crassus during development from subadults to adults. A concurrent study of their interand intraspecies intestinal distributions gave insight into the dynamic relationship of ontogeny to habitat specificity. MATERIALS AND METHODS From September 1969 to June 1971, largescale suckers, Catostomus macrocheilus (Girard), were collected at monthly intervals by electric

Evidence for a sodium ion exchange carrier linked with glucose transport across the brush border of a flatworm (Hymenolepis diminuta, Cestoda).

The manner in which the flatworm, Hymenolepis diminuta (Cestoda), regulates the transport of glucose and Na+ across the brush border was examined. While the presence of an unstirred region in the brush border may favor the reabsorption of leaked glucose, some leaked glucose was lost to the ambient medium. This loss was markedly enhanced by preloading the worms with glucose and by removing Na+ from the incubation medium. Since glucose and Na+ influxes are coupled, glucose leakage stimulated the influx of 22Na+. However, this 22Na+ influx was balanced by a simultaneous increased 22Na+ efflux. The presence of phlorizin inhibited both unidirectional fluxes of 22Na+ indicating that efflux of 22Na+ occurred by countertransport; countertransport of [14C] glucose appeared to be negligible. A model has been proposed in which the transport of glucose and compensating transfers of Na+ across the membrane occur via the same carrier.

The life history and larval development of Neoechinorhynchus saginatus Van Cleave and Bangham, 1949 (Acanthocephala : Neoechinorhynchidae).

The life cycle of Neoechinorhynchus saginatus, an intestinal parasite of creek chubs, Semotilus atromaculatus, in eastern North Dakota is reported, with a description of development in the ostracod intermediate host. Eggs ingested by Cypridopsis vidua hatch, liberating the acanthors. These penetrate the intestinal wall and in 3 to 4 days begin metamorphosis in the hemocoel as immobile, unattached acanthellae. Juveniles appear to be fully developed by the 14th day, and feeding experiments indicate that such larvae are infective to chubs on the 16th day. The family Neoechinorhynchidae, order Eoacanthocephala, includes some two dozen species from North American fishes for which the life cycles of five are known. These include Neoechinorhynchus cylindratus, N. emydis, N. rutili, Octospinifer macilentis, and Paulisentis fractus. Only the last species fails to use ostracods as an intermediate host. Neoechinorhynchus saginatus, an intestinal parasite of the creek chub, Semotilus atromaculatus (Mitchill), was described from Wisconsin lakes and streams by Van Cleave and Bangham (1949). Fischthal (1950, 1952) also reported this species in Wisconsin chubs, and Meyer (1954) found it in fallfish, S. corporalis (Mitchill), in Maine. More recently, Voth and Larson (1968) reported N. saginatus from creek chubs of the Goose River, a tributary of the Red River in eastern North Dakota. Although creek chubs range from Montana to eastern Canada and south to the Gulf of Mexico, N. saginatus has been found in only three widely separated localities. Apparently, there have been no previous reports concerning the life cycle of this parasite. MATERIALS AND METHODS Creek chubs collected from the Goose River, near Portland, North Dakota, provided gravid females of N. saginatus. A second tributary (the Forest River) some 60 miles to the north possessed uninfected chubs used in feeding experiments. Received for publication 9 May 1969. * From a thesis submitted by Mr. Uglem to the University of North Dakota in partial fulfillment of the requirements for the degree of Master of Science, 1968. tPresent address: Department of Zoology, University of Idaho, Moscow, Idaho 83843. Ostracods were tested as potential intermediate hosts of N. saginatus since they serve in this capacity in four of the five known life cycles of neoechinorhynchids. Three species of ostracods identified as Cypridopsis vidua (O. F. Muller), Cypria maculata Hoff, and Potamocypris sp., collected from the Forest River, were successfully cultured for 2 years. They were exposed by allowing them to feed from 1 to 2 hr on aqueous suspensions of N. saginatus eggs in small finger bowls. The exposed hosts were then transferred for the duration of the experiment to large finger bowls of water held at 25 C under continuous illumination. These ostracods were examined periodically for developing parasites by making dissections using fine insect pins in a dilute solution of neutral red in 0.6% saline. One species of amphipod (Hyalella azteca), one species of copepod (Cyclops sp.), and four species of gastropods (Physa sp., Gyraulus sp., Lymnaea sp., and Ferrissia sp.) were also exposed to eggs and dissected periodically in an attempt to determine the specificity of the larval parasite. The chubs were inoculated by force-feeding infected ostracods with a polyethylene tube and syringe. Intermittent dissections were made to observe the growth and maturation of the parasites within the fish. Drawings were made from vital stained material with the aid of a camera lucida, by freehand, or from photomicrographs. All measurements are recorded in microns unless otherwise indicated. Permanent slide mounts of the ostracods, 16-day juveniles, and 46-day subadults have been deposited into the University of North Dakota Parasitology Collection with accession numbers 532535. Additional specimens are in the private collection of the senior author.

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

SummaryA paracellular pathway in the foot epithelium ofLehmannia 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 isotope3HOH and various14C-labelled solutes.1.Paracellular uptake of14C-inulin (Fig. 1) and3HOH (Fig. 2) is initial rate for at least 3 min. If the wet pad contains 1,000 cpm of14C per ml of3HOH, slugs absorb only about 400 cpm of14C with cach ml of3HOH absorbed representing a sieving ratio of 0.4 for inulin.2.The sieving ratio of14C-inulin does not change when the concentration is increased from 0.1 to 2.5 mmol/l. Moreover, the sieving ratio of14C-inulin was not affected significantly by the nature of the labelling, i.e.,14C-carboxyl vs14C-methoxy.3.Sieving ratios for14C-mannitol (182 Da),14C-polyethylene glycol (4,000 Da), and14C-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).4.The effective pore size of the paracellular pathway was estimated using the relationships between sieving ratio and molecular weight of3HOH 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).

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 Μm 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 Ktwere 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.

The life cycle of Neoechinorhynchus cristatus Lynch, 1936 (Acanthocephala) with notes on the hatching of eggs.

The life cycle of Neoechinorhynchus cristatus Lynch, 1936, a parasite of Catostomus macrocheilus and other catostomid fishes, is presented. Of five species of ostracods tested as intermediate hosts, development of hatched acanthors to juvenile worms occurred only in Cypridopsis helvetica. Feeding experiments indicated that after 20 days juveniles are infective to suckers. Attempts to infect any of the ostracods with N. crassus or N. venustus were unsuccessful. Based on observations of natural and artificial hatching of eggs, the composition and functional significance of the egg envelopes are discussed. The acanthocephalan Neoechinorhynchus cristatus Lynch, 1936, has been reported from the large-scale sucker, Catostomus macrocheilus, and other catostomid fishes. In northern Idaho, suckers infected with N. cristatus inhabit the same rivers as does the ostracod, Cypridopsis vidua. This widely distributed ostracod serves as intermediate host for N. saginatus from creek chubs, Semotilus atromaculatus, in North Dakota, Maine, and Wisconsin (Uglem and Larson, 1969). The purpose of this study was to examine the life cycle of N. cristatus using C. vidua, and other species of ostracods, as potential intermediae hosts. MATERIALS AND METHODS Large-scale suckers, C. macrocheilus, from the Palouse River east of Potlatch, Idaho, and from Hangman Creek near Tekoa, Washington, provided gravid females of N. cristatus. Bottom samples and submerged vegetation collected from the rivers yielded 5 species of ostracods: Cypridopsis vidua; C. vidua var. obesa; Cypridopsis helvetica; Cypria sp.; and Potamocypris sp. These were cultured in aquaria until sufficient numbers for feeding experiments could be obtained. Observations on the life cycle of N. saginatus and the methods of infecting ostracods and examining them for developing larvae are from Uglem and Larson (1969). Natural hatching of eggs of N. cristatus was observed and compared to artificial hatching induced by drying and wetting egg smears (Manter, 1927, 1928; Moore, 1942). To eliminate possible pressure on the eggs during examination, the cover slip was supported by small chips of glass. All measurements are expressed in microns, as an average of at least 10 observations Received for publication 5 May 1972. * Present address: Department of Biology, Rice University, Houston, Texas 77001. unless indicated otherwise. Drawings were made freehand and from photomicrographs. RESULTS AND DISCUSSION Of the five species of ostracods exposed to eggs of N. cristatus development of the larvae to infective juveniles (cystacanths) occurred only in C. helvetica (Table I). Although acanthors hatched in C. vidua and C. vidua var. obesa and penetrated the gut wall, growth in the hemocoel was incomplete. In the latter species oval-shaped acanthellae were recovered 4 to 7 days postfeeding, but no further development was observed. Apparently, these ostracods provided conditions that initiate hatching and metamorphosis, but lacked those needed for complete larval development. Eggs also hatched in Cypria and Potamocypris liberating active acanthors, but no evidence of penetration of the gut wall was found. No attempt was made to determine the incidence of naturally infected ostracods. Concurrent infections of N. cristatus and N. crassus adults in C. macrocheilus were common. Attempts to complete the larval development of N. venustus (obtained from the fine-scale sucker, C. columbianus) or N. crassus in any o the ostracods were unsuccessful. Development of these parasites was limited to hatching only (Table I). The life cycle of N. cristatus is basically similar to other neoechinorhynchids with only minor variations. Eggs measuring 56 by 27 are released by females and pass out of the intestine of the sucker with the feces. An infective egg consists of an unhatched or shelled acanthor, about 42 by 14, and four

Localization of facilitated diffusion and active glucose transport in cysticercoids of Hymenolepis diminuta (Cestoda)

Abstract Active glucose transport by adults of Hymenolepis diminuta is known to be Na+-dependent and more sensitive to phlorizin than to phloretin. We found that infective cysticercoids apparently take up glucose by facilitated diffusion. This type of transport was distinguished by insensitivity to Na+ and phlorizin, but inhibition by phloretin. The preferred substrate was glucose followed by galactose > β-methylglucoside > α-methylglucoside; mannose, glucosamine, 3-O-methylglucose, melibiose and 2-deoxyglucose did not interact with the system. Vmax and Kt for glucose transport were 9.9 ±2.46 nmoles 25 larvae/h, and 0.8 ± 0.27 m M , respectively. In an attempt to localize the transporters, the cysticercoid wall enclosing the scolex was removed by excysting the larvae in vitro. Glucose uptake by the excysted scoleces was similar to adult transport, i.e. Na+-dependent and sensitive to phlorizin rather than phloretin. These results indicate that the withdrawn scolex, like the adult, is capable of active glucose transport. Facilitated diffusion appears to be associated primarily with the cyst wall which is lost when the larva excysts in the vertebrate host

Facilitated diffusion and active transport systems for glucose in metacercariae of Clinostomum marginatum (Digenea)

Abstract Metacercariae of Clinostomum marginatum excysted from yellow perch, Perca flavescens, appear to have two systems for transporting glucose across the tegument, facilitated diffusion and active transport. These systems were distinguished by their differential sensitivities to Na+, phlorizin and phloretin. In Ringers saline for cold-blooded vertebrates, 0.1 m m phlorizin and phloretin were incomplete, but similarly effective inhibitors of glucose uptake in 3 min incubations; worms accumulated in 1 h nonmetabolized 3-O-methylglucose against an apparent concentration difference demonstrating the active transport component. In Na+-free saline, phlorizin sensitivity and active transport capacity disappeared, but a phloretin sensitive, Na+-independent component remained. The Vmax and K1 of the Na+-independent system were 3.0 ± 0.54 μmol/g ethanol-extracted dry wt/h, and 0.8 ± 0.36 m m , respectively. Vmax and K1 of the Na+-dependent system, estimated by subtracting the Na+-independent values from those obtained in Ringers saline, were 1.3 ± 0.27 μ mol/g ethanol-extracted dry wt/h, and0.7 ± 0.36mm, respectively.

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.

Proterometra macrostoma (Trematoda: Azygiidae): Functional morphology of the tegument of the redia

Abstract Scanning and transmission electron microscopy were used to study the tegument of the redia of Proterometra macrostoma . The predominant feature of the tegument is the presence of orderly arranged channels giving the tegument a honeycomb appearance in thin sections. The channels are closely apposed like stacked cylinders and lie perpendicular to the basal lamina. The channels are about 0.75 μm long by 0.2 μm in diameter each opening to the outside by a small pore. Mitochondria are located between the basal lamina and the bases of the channels. The free surface of the tegument is amplified secondarily by annular folds and small unbranched mierovilli. In vitro experiments were then designed to test the biophysical properties of this unusual tegument with respect to the flow of water and solutes through the channels. Addition of 5 m m glucose stimulated the uptake of 14 C-mannitol suggesting that a concentration gradient of 14 C-mannitol was generated in the channels.