Cuticular Fine Structure and Molting of Neoaplectana glaseri (Nematoda), after Prolonged Contact with Rat Peritoneal Exudate

Abstract

Third-stage Neoaplectana glaseri in a saline suspension with glycogen were injected into the peritoneal cavity of rats. Worms recovered during the following 3 days were heavily coated with rat peritoneal exudate cells but showed no cuticular alterations; if placed into a favorable culture environment, the worms had a limited ability to molt. Live worms recovered 4 and 5 days following injection also showed heavy cell coats and, in addition, two distinct cuticular changes: a swollen surface sheet and a disrupted striation pattern. These worms survived in culture but did not molt to a more mature stage. No recognizable worms were recovered subsequently. The cuticle that covers and supports nematodes is remarkably resistant to destruction by chemical and physical means. In autolysis or in attack by the digestive processes of other organisms, whether cellular or extracellular, it is most often the cuticle that persists structurally after a nematode's soft tissues have begun to change. However, when this work was begun, possible changes in cuticular fine structure caused by adhering, presumably phagocytic cells had not been looked for with electron microscopy. Neoaplectana glaseri Steiner, 1929, not naturally a parasite of mammals, is a convenient nematode for such studies. Large numbers of worms, uniform as to stage, are available from species isolation cultures (Glaser, 1940); certain effects of mammalian temperature, antibody, and cells on the worms have been described and distinguished in culture (Jackson, 1961, 1962, 1966). MATERIALS AND METHODS The natural and laboratory histories of Neoaplectana glaseri and methods for culturing this nematode in species isolation have been detailed (Stoll, 1959, 1961; Jackson, 1969). To obtain a maximal exudate, 5,000 to 6,000 living third-stage nematode larvae contained in 8 ml of Abbott Isotonic Sodium Chloride Solution (900 mg NaCl/ 100 ml H20) with 0.9% glycogen were injected Received for publication 9 May 1969. * Supported in part by the U. S. Public Health Service through grants AI-04842, K3-AI-9522, and TO1-AI-00192 from the NIAID. t Present address: Department of Zoology, North Carolina State University, Raleigh, North Carolina 27607. into the peritoneal cavity of 100-g female SpragueDawley rats. On one occasion 25-g rats were used. Glycogen increased the number of cells in the peritoneal exudate compared to injections of living worms suspended in saline. Injections of dead worms in saline stimulate slight exudates of short duration but seldom can initially dead worms be recovered from the rat after 24 hr. From previous unpublished results it was known that, in contrast to Turbatrix aceti cultures, many N. glaseri survived at least 24 hr in the rat peritoneal cavity. Consequently, the host population was sampled at daily intervals for 7 days after the initial injection. For these daily samples, 3 anesthetized rats were each injected with 6 ml of the Abbott saline, the rib cage area was massaged, and, after a rat had been killed with ether, the saline was withdrawn with a Pasteur S-C pipette (Stoll, 1961) through a small incinsion into the peritoneal cavity. Worms in the withdrawn samples were allowed to settle without centrifugation in pointed tubes under sterile conditions. Some of the worms from pooled samples were examined by light microscopy; some were transferred to an optimal liquid medium (Stoll, 1961) for culturing; others were fixed for electron microscopy in cold (ca. 0 C) 3% glutaraldehyde, 0.1 M cacodylate. In this mixture the worms were cut in half with a stainless steel razor blade or processed whole until the final sectioning. After 1 hr, both whole and halved worms were washed for at least an hour with several changes of cold 0.1 M cacodylate, 4% sucrose. For postfixation the worms were transferred for 1 hr to cold 2% osmium in 0.2 M sucrose buffered at pH 7.3 with 0.1 M cacodylate before dehydration and embedding in the following series: 70%, 95%, 3 X 100% ethanol; 3 X propylene oxide (10 min for all preceding steps); 1/1, 1/2 propylene oxide and Epon resin (with DMP-30 catalyst to 1.5%), (45 min each); repeated changes in fresh EponDMP over several days at room temperature to aid penetration of the resin. The worms were then oriented for convenient sectioning in Beem capsule covers and cured at 52 C for 3 to 5 days. The