The molting pattern in Trichinella spiralis. I. A light microscope study.

Development of Trichinella spiralis in rats was studied in detail to determine where and how many times the larvae molt. Four molts were observed during the intestinal phase of development, none earlier. In synchronously developing populations obtained by injecting excysted larvae directly into the duodenum, male worms molted at approximately 9, 13, 18, and 25 hr, females at approximately 10, 15, 21, and 28 hr, and inseminated females were first observed at 30 hr after inoculation. Each successive larval stage could be recognized by the thickness of the cast cuticle and the presence or absence of copulatory appendages, tuberculate papillae, and sperm in the male, and by the state of development of the vagina and uterus and the thickness and cross striations of the cuticle in the female. The development of T. spiralis can be divided into two periods, one in the tissues where, although there is no molt, the level of sexual differentiation reaches that of the fourth or early fifth-stage larva of phasmid nematodes, the other in the intestine where there are 5 stages, separated by 4 successive molts. The high level of sexual differentiation reached in the tissue phase of development together with the rapid succession of molts occurring in the intestine renders the Trichinella life cycle atypical among the trichuroids. Although more than a century has elapsed since the general life cycle of Trichinella spiralis (Owen, 1835) Railliet, 1895, was described by Leuckart and Virchow (Gould, 1945), its molting pattern is still disputed. It has been claimed that the first molt occurs within the egg in the uterus of the female, that there are two subsequent molts during the intramuscular phase, and one during the intestinal phase of development (Berntzen, 1965). It has also been reported that only two molts occur, both in the intestine (Podhajecky, 1964; Thomas, 1965; Shanta and Meerovitch, 1967a). One report (Kreis, 1937) suggested that males molt three times, females four times, but some observers have found that both males and females molt four times (Villella, 1958; Ali Khan, 1966). In vitro studies on the development of T. spiralis by Weller (1943) and by Tarakanov (1964) also suggested that the larvae of both Received for publication 2 March 1971. * Portion of a dissertation submitted to the Graduate School of Tulane University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. This study was supported by grants AI-04919 and AI-00002 from the NIH, U. S. Public Health Service. Presented at the 44th Annual Meeting of American Society of Parasitologists, 6 November 1969, Washington, D.C. t Present address: Department of Microbiology, University of Chicago, 939 East 57th Street, Chicago, Illinois 60637. xes molt four times. Kim (1961, 1962) likew se proposed that four, or more, molts occurred in his in vitro system, but Meerovitch (1965) reported only two molts, and Berntz (1965) only one in their respective culture systems. Microand ultrastructural studies were conducted on stages of T. spiralis larvae from the female worms, and from the muscles and intestine of the host, in an attempt to determine how many molts occur during the development of this trichuroid, during which phases of the life cycle they occur, and what morphological characteristics can be used to differentiate the various larval stages. The present report is limited to the results obtained from the light microscopic studies. The ultrastruct-ural studies are presented separately (Kozek, 1971). MATERIALS AND METHODS Larvae were obtained from rats whose infections were 3 to 6 months old. Following a brief mincing in a Waring Blendor, the muscles were digested in 1% pepsin solution containing 1% HC1, for 2 to 6 hr at temperatures ranging between 37 and 40 C. Larvae were subsequently separated from tissue debris by sieving through a double layer of cheesecloth followed by sedimentation. The inoculating dose was administered to experimental[ animals in a 0.4or 0.5-ml volume of pepsin-muscle-digest fluid. The larvae and adult worms were observed alive, or after fixation with either Bles' fluid, AFA, 10% f rmalin, or 3% glutaraldehyde solution. The fixed worms were cleared in 70% ethanol + 5% glycerin mixture and mounted in glycerin.