Omer R. Larson

Bacteria Associated with the 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.

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.

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.

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.

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 Tyrodes, Na-poor Tyrodes, and Eagles 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.

Cold hardiness and winter survival in the bird flea, Ceratophyllus idius

Abstract Adults of the purple martin flea Ceratophyllus idius Jordan and Rothschild, were sampled from outdoor and −6°C freezer-held populations from early September to mid-April of two consecutive years. Levels of polyols (glycerol, sorbitol) and carbohydrates (glycogen, glucose, trehalose) were monitored and correlated with temperatures in both regimens. Natural autumn cooling and controlled laboratory chilling (3°C every fourth day) produced large increases in glycerol levels with concomitant declines in glycogen. These processes appeared to commence at about +10°C and continued at subzero temperatures until glycerol content averaged 443 ± 14.9 μmol/g wet wt and glycogen fell to an average of 17 ± 1.1 μmol/g. Sorbitol levels remained low at all times (1–3 μmol/g), although glucose and trehalose content showed transitory, 2-fold increases during cold acclimation. Supercooling points declined and stabilized at −29 to −30°C in both regimens. Glycerol levels in freezer-held fleas remained nearly constant throughout the experiment, while outdoor specimens showed monthly fluctuations and a large decline by mid-April. The springtime decrease was not linked to a comparable rise in glycogen content. In outdoor samples, 32.4% of the fleas were still alive in mid-April, approx 10 days prior to the expected return of migrant birds.

The detection of glycerol in overwintering purple martin fleas, Ceratophyllus idius

1. 1. Adult Ceratophyllus idius from eastern North Dakota produced glycerol in the late autumn which may act as a cryoprotective agent enabling some of the fleas to survive the winter. 2. 2. The glycerol was apparently synthesized at the expense of glycogen and glucose, and the reverse reactions occurred with the onset of spring and rising temperatures.

Abnormal Opisthaptor in Neopolystoma orbiculare

During late October 1970, while examining the urinary bladders of several painted turtles (Chrysemys picta) for helminths, an unusual parasite abnormality was observed. One of 12 _ : . E . specimens of Neopolystoma orbiculare (Stunkard 1916) Price 1939 was found possessing only three suckers on its opisthaptor (Fig. 1). Turtles were purchased that autumn from Lemberger Company (since acquired by Mogul-ED) of Oshkosh, Wisconsin. All worms were preserved in alcohol-formalin-acetic acid under coverslip pressure, stained in hematoxylin, and mounted in Canada balsam. The aberrant worm measured 4.0 mm in length with an opisthaptor 0.98 mm in diameter, while the : il i 11 normal specimens ranged from 3.3 to 4.5 : : mm (mean 3.96 mm) with haptoral diameters : from 0.89 to 1.19 mm (mean 1.03 mm). Although spacing between suckers is generous on the anomalous haptor, there are no size or structural differences between suckers of the abnormal and normal worms.