Paul M. Nollen

A scanning electron microscopic investigation of the outer surfaces of Gorgoderina attenuata

Abstract The external surface of Gorgoderina attenuata is characterized by ridges that encircle the worm at regular intervals. On the ridges are protuberances that give a beaded appearance to the tegumental surface. Ridges running perpendicular to the encircling bands are apparent close to both suckers. The raised protuberances of the tegumental areas may serve to increase the absorptive surface. A pit-like cavity was observed on the dorsal surface of the oral sucker. In addition numerous raised structures which appear to be of two types were present on the surface of the oral sucker. One type resembles a depression with a small knob barely visible in the center and the second type consists of button-like patches of tissue. From their structure and location these are tentatively identified as sensory organs.

Megalodiscus temperatus: Scanning electron microscopy of the tegumental surfaces

Abstract The tegumental surface of Megalodiscus temperatus forms cobblestonelike areas with rows of indentations encircling the worm. This pattern merges in several areas into folds and ridges, some of which represent the musculature of the posterior sucker and genital pore. Papillae surrounding the base of the oral sucker appear as two types: one with a bulb-like base and a short apical knob; the second typified by a hair-like structure (cilium?) of variable length projecting from a pit. From their location on the oral sucker and the resemblance to previously described structures, they are presumed to be sensory receptors. A circle of papillae on the closed posterior sucker was also observed. Rod-shaped bacteria were seen adhering to some of the worms observed but they were not found to be in any special association with the tegument or in any specific areas of the worms surface.

Scanning and transmission electron microscopy of oral sucker papillae of Philophthalmus megalurus.

Abstract Edwards H. H. , Nollen P. M. & Nadakavukaren M. J. 1977. Scanning and transmission electron microscopy of the oral sucker papillae of Philophthalmus megalurus. International Journal for Parasitology7: 429–437. Scanning electron microscopy reveals papillae on both the inside and outside of the oral sucker of the eyefluke, Philophthalmus megalurus. In the transmission electron microscope the papillae can be divided into 3 different types with 2 of these types occurring on the outside of the oral sucker. One type of outer papilla contains a bulb cell which is terminated by a cilium having an apparent typical arrangement of microtubules. This type is a presumed sensory receptor having tango- and/or rheoreceptor function. The second type of outer papilla contains a gland cell which secretes electron-dense granules to the outside of the fluke. The third type of papilla is found only on the inside of the oral sucker and contains a bulb cell terminated by a cilium which does not have the typical 9 + 2 arrangement of microtubules. Instead, their numbers increase to over 60 randomly arranged microtubules. This inner papilla is felt to have chemoreceptor function because of its location and the presence of the modified cilium. The bulb cell of the inner papilla contains 2 newly described features. The first is granular, and associated with microtubules; it may be a possible nucleating site for microtubule synthesis. The second structure is a crystalline inclusion of unknown function and origin.

The Taxonomy and Biology of Philophthalmid Eyeflukes

Publisher Summary This chapter discusses the stages of the life cycle of eyeflukes. Eyeflukes in the genus Philophthalmus are found world-wide and occur in both marine and fresh-water habitats. Most live in the orbit of birds in various microhabitats, but a few can be found in the intestine and at least one species in the mouth cavity. Significant information is available on the mating behavior of adults, the host-finding behavior of miracidia, and cyst formation by cercariae. The adult stages are good research subjects because they are readily accessible in the orbit of birds. Light microscopy studies indicate the presence of various surface features on eye fluke rediae, such as sensory papillae, a birth pore, and posterior appendages. Metacercariae are infective immediately upon encystment and rotation within the cyst. Eyefluke metacercarial cysts have their narrow end open to the environment, and they do not survive for long periods of time. Transplant studies are especially facilitated because major surgery is not required to transfer worms from host to host.

Digenetic trematodes: Quinone tanning system in eggshells☆

Abstract Three species of digenetic trematodes with colorless and transparent eggshells, Philophthalmus megalurus , Gorgoderina attenuata , and Megalodiscus temperatus , were compared by histochemical tests with Haematoloechus medioplexus , which has an identifiable quinone tanning system. Precursors for tanning including basic and tyrosine-rich proteins were identified histochemically in all species. Autoradiograms indicated that tyrosine was taken up after in vitro exposure of adult worms by vitelline tissue of G. attenuata and M. temperatus , but at a slower rate in the latter species. Protein synthesis inhibitor studies utilizing cycloheximide showed a reduction of tyrosine incorporation in the vitelline tissue of G. attenuata suggesting that tyrosine is incorporated into protein. Inhibition in M. temperatus was negligible, but evidence from other studies indicated absorption of cycloheximide by the adult may be minimal. Phenolase could not be found in G. attenuata and M. temperatus but was identified in P. megalurus . However, the enzyme system in the latter species seemed to be a partial one capable of oxidizing dihydroxyphenols but not monohydroxyphenols. Histochemical and solubility tests for keratin-type protein in the eggshells were inconclusive. It is suggested that positive identification of keratin as a structural protein of trematode eggshells be withheld until more reliable techniques can be employed. Possible evolutionary implications of the tanned eggshell in the Digenea are discussed.

Studies on the Reproductive System of Hymenolepis diminuta Using Autoradiography and Transplantation

Adult Hymenolepis diminuta exposed in vitro for 3 hr to 3H-thymidine showed incorporation of the isotope on autoradiograms over nuclei of actively dividing cells in the testes, ovary, vitellaria, and developing embryos in the eggs. Timing studies utilizing labeled worms transplanted to uninfected hosts showed that it took 18 hr for spermatogonia to develop to primary spermatocytes, 24 hr to secondary spermatocytes, 36 hr to spermatids, and 48 hr to sperm bundles. Self-insemination was confirmed in single worm transplants of 3 days by the presence of labeled sperm in the seminal receptacles. In multiple worm transplants labeled worms inseminated themselves in each case and cross-inseminated with 92% of the unlabeled worms present.

Mating behaviour of Echinostoma caproni and E. trivolvis in concurrent infections in hamsters

Young adults of Echinostoma caproni and E. trivolvis were recovered from hamsters. Some worms of each species were exposed to [3H]tyrosine for 1 h to label sperm, and these were transplanted singly to uninfected hamsters with various combinations of unexposed worms of either the opposite species or both species. Worms recovered 5 days later were serially sectioned, processed for autoradiography and observed for the location of radioactive sperm. Interspecies mating was detected when E. caproni was the sperm donor and E. trivolvis the recipient, but not the converse. The cross-insemination rate during interspecies mating was very low (13%) when compared to the normal rate of E. caproni intraspecies mating (52%). When single donor adults of either E. caproni or E. trivolvis had a choice of both recipient species, no interspecies mating took place, but both self- and cross-inseminated in a non-restrictive mating pattern typical of echinostome species. After transplantation, both species localized in their normal habitat within the hamster intestine. However, 25% of opposite species recoveries were found within 1 cm of each other, making interspecies mating a possibility.

Autoradiographic Studies on the Reproductive System of Philophthalmus hegeneri Penner and Fried, 1963

Adult Philophthalmus hegeneri exposed in vitro to 3H-thymidine showed incorporation of the isotope on autoradiograms over nuclei of actively dividing cells of the testes, ovary, vitellaria, and developing miracidia within eggs. Autoradiograms of worms exposed for 1 hr to 3H-tyrosine showed heavy labeling over the ovary, vitelline cells, and the testes. Timing studies utilizing transplanted thymidine-labeled worms indicated it takes 12 days for oogonial cells to become labeled, migrate through the ovary, and be incorporated into eggs. It took 96 hr for labeled vitelline cells to reach the vitelline reservoir. Spermatogonia labeled in the initial 6-hr exposure took 48 hr to become primary spermatocytes, 72 hr to become secondary spermatocytes, 96 hr to become spermatids, 120 hr to become mature sperm in bundles, and 156 hr to reach the seminal vesicle. Studies with tyrosine-labeled worms transplanted to the host for 5 days indicated adults taken from multiple infections will not self-inseminate when isolated. Similar worms transplanted with unlabeled worms exclusively cross-inseminate. In a study on an eye fluke of birds, Philophthalmus megalurus, utilizing techniques of radioisotopic labeling, autoradiography, and transplantation, Nollen (1968a) was able to study several aspects of reproductive physiology and behavior. Timing of stages of gametogenesis and migration of vitelline cells as well as patterns of sperm transfer in single and multiple infections were determined in this study.Nollen found these flukes can selfinseminate in isolation but preferentially crossinseminate in multiple infections. Philophthalmus hegeneri, an eye fluke of shore birds in Florida, differs in several ways from P. megalurus. Among these are the apparent lack of self-insemination when adult P. hegeneri are grown in isolation (Fried, 1962). The ability of the snail intermediate host, Batillaria minima, to survive long periods out of the water makes it ideal for shipping. Thus we have been able to raise adult worms in chicks from larval stages shed by snails collected in Florida. The techniques utilized by Nollen (1968b) were applied to a study of P. hegeneri to see if species differences are apparent in reproductive physiology and behavior when compared to P. megalurus. MATERIALS AND METHODS Philophthalmus hegeneri was obtained from naturally infected Batillaria minima collected from Received for publication 2 March 1973. * Supported by the United States-Japan Cooperative Medical Sciences Program administered by NIAID: Grant Al 09330. Clearwater Harbor, Largo, Florida. Cercariae shed from snails encysted in finger bowls and were stored in aerated seawater not longer than 4 days before use. Metacercariae were induced to excyst by replacing seawater with warm (40 C) saline (0.85%), and day-old chicks (Big H Foods, Fairbury, Ill.) were infected by pipetting the metacercariae directly into the orbit. Worms were removed from the conjunctival sacs of chicks after 35 to 45 days of growth and exposed at 38 to 39 C in small stender dishes to tritiated compounds (Amersham/Searle Inc.) diluted with HedonFleigs saline. The radioisotopes used were thymidine (methyl-3H; 22.4 Ci/mmole) at 75 ,uCi/ml and L-tyrosine (-3,5-T; 1 Ci/mmole) at 100 iCi/ml. Worms were exposed to 3H-thymidine for periods of 6 hr or more to determine the time needed for optimum labeling of the cells of the reproductive system. Similar exposures were carried out with 3H-tyrosine to determine areas of incorporation in the tissues of P. hegeneri. After exposure, worms were either killed immediately in alcohol-formalin-acetic acid (AFA) or returned to the conjunctival sacs of uninfected chicks for varying periods of time. Techniques for autoradiography and transplantation of the exposed worms are described by Nollen (1968b).

A comparison of the adult and miracidial stages of Echinostoma paraensei and E. caproni

Adult Echinostoma paraensei and Echinostoma caproni were grown in outbred mice and golden hamsters to compare size, growth rates, infectivity, and habitat selection. Antagonistic responses between the 2 species were investigated by concurrent infections in mice. Miracidial stages were compared for developmental stages, hatching responses, and behaviour to light and gravity. Size differences and growth rates were significantly different in both mice and hamsters. Mice proved to be better hosts for E. caproni and hamsters for E. paraensei. In mature infections, E. paraensei adults localized in the duodenum and E. caproni in the ileum of both mice and hamsters. In concurrent infections of mice, E. paraensei adults were significantly smaller than in single species infections beyond 14 days post-infection, while E. caproni adults were either equal to or larger than those in single species infections. On the other hand, E. paraensei were recovered in larger numbers in concurrent infections than in single species infections, while the reverse was found for infectivity of E. caproni adults. Miracidia of E. paraensei developed at the same rate as those of E. caproni in both light and dark cultures, but E. paraensei hatched much sooner when exposed to light. No miracidia hatched from cultures kept in the dark, indicating light is needed to stimulate the hatching process. All light-stimulated cultures exhibited a circadian hatching pattern from 1100 to 1600 hours. Cultures maintained in the dark past 11 days did not hatch when exposed to light. Miracidia of E. paraensei showed a positive phototaxis but no response to gravity. This comparison of life cycle stages leads us to conclude that E. paraensei and E. caproni are distinct species.

Gorgoderina attenuata: Uptake and incorporation of tyrosine, thymidine, and adenosine☆

Abstract In autoradiographic and scintillation counting studies, tritiated tyrosine, thymidine, and adenosine were found to be absorbed and incorporated by Gorgoderina attenuata in in vitro exposures of up to 12 hr. Tyrosine was incorporated into all tissues with the highest levels found in the intestinal epithelium. No evidence for selective incorporation of tyrosine into vitellaria was found. Thymidine was incorporated mainly into nuclei of gonial cells of the reproductive system and developing miracidia within eggs. Autoradiograms indicated assimilation of tyrosine and thymidine was highly variable among individual worms of a treatment group. Adenosine was absorbed and incorporated by worm tissues in large amounts within 3 hr. Variability of labeling on autoradiograms was not as great as with tyrosine and thymidine. Results of scintillation counting and grain counts of autoradiograms on ligated worms indicated all three compounds can enter the worm through the tegument. Adenosine was considered the best possible compound of the three to use for labeling cells for studies on the reproductive system.