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

Abstract

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 HedonFleig's 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).