Scanning electron micrographs showed damage to the tegument adjacent to ligatures of adult Philophthalmus megalurus, Gorgoderina attenuata, and Megalodiscus temperatus. Leakage of tritiated tyrosine, leucine, and thymidine was detected around the ligatures on adult P. megalurus by an autoradiographic method that localizes soluble compounds. Grain counts were significantly higher adjacent to ligatures than elsewhere. Autoradiography did not indicate leakage in some ligated worms, but all observed with the scanning electron microscope exhibited tissue damage. The technique of ligation is thus considered unreliable for absorption studies involving digenetic trematodes. Investigations studying the uptake and incorporation of metabolites by various digenetic trematodes have utilized ligation to close off the gut from the external environment (Mansour, 1959; Kuralec and Erhlich, 1963; Isseroff and Read, 1969; Isseroff and Walczak, 1971; Pappas, 1971; Nollen et al., 1973; Nollen et al., 1974). In a monogenean, Diclidophora merlangi, Halton and Arme (1971) found such ligation to increase significantly the uptake of a labeled compound as determined by a counting technique. They also observed by transmission electron microscopy (TEM) damage to tissue near ligatures, used Evan's blue to detect such injury in living worms, and stressed the importance of intact specimens for investigating the uptake of metabolites through the tegument. Their findings suggested this study which deals with three species of digenetic trematodes previously used for ligation experiments, and utilized scanning electron microscopy (SEM) and autoradiography to detect tissue leakage near ligatures and its effect on uptake of labeled amino acids and thymidine. MATERIALS AND METHODS Adult Philophthalmus megalurus were obtained from the conjunctival sac of chicks infected for 25 to 32 days. Gorgoderina attenuata and Megalodiscus temperatus were removed from the bladder and rectum of Rana pipiens from commercial sources. For SEM worms were ligated just beReceived for publication 21 February 1974. hind the oral sucker with 6-0 surgical silk and fixed in phosphate-buffered 5% glutaraldehyde (pH 7.3) for 30 min, rinsed in the buffer, and postfixed in phosphate-buffered 1% osmium tetroxide (pH 7.3) for 30 min. They were dehydrated through a graded series of ethyl alcohol, mounted on stubs using conductive silver paint, coated with gold, and examined in a Cambridge Stereoscan SEM Model II-A. For autoradiography, 5 ligated P. megalurus adults were exposed to each of 3 tritiated compounds (Amersham/Searle) (L-tyrosine 3,5-T, sp. act. 1 Ci/mmole; L-leucineT-G, sp. act. 200 mCi/mmole; and thymidine methyl-T, sp. act. 3.0 Ci/mmole) diluted in 2 ml of Hedon Fleig's saline and processed for localization of water-soluble compounds by the method of Wilske and Ross (1965) as modified by Nollen (1968). Grain counts were made with a Whipple eyepiece micrometer grid at 1,500x. For each worm at least 25 areas 100 u' were counted for tissues adjacent to the ligature and the tegument posterior to the ligature. Student's t test was used to determine the significance of differences in mean grain counts.