Developmental Physiology of Cestodes. IX. Cytological Characteristics of the Germinative Region of Hymenolepis diminuta

Abstract

The germinative region of developing Hymenolepis diminuta is defined based on cytological characteristics and DNA synthetic activity as beginning 200 ,u posterior to the apex of the scolex, and extending posteriorly to the point of germinal primordia formation. The structure of the germinative type in this region is described. The kinetics of division in the germinative region are studied, and estimates of the duration of G1, S, and G2 of prophase are 3.0, 2.3, and 3.2 hr, respectively. The time for a complete mitotic division is estimated to be 8.5 + hr. Although proglottisation in most cyclophyllidean cestodes has been attributed to budding from the unsegmented region immediately posterior to the scolex (Wardle and McLeod, 1952; Roberts, 1961), no cytological definition or exact location has been given to the so-called germinative area. Roberts (1961) indirectly defined the germinative area of Hymenolepis diminuta as the area behind the scolex in which genital primordia are not observed. Sandeman (1959) defined the germinative area of Capsulata edenesis, a parasite of Limosa lapponica, as a posteriorly located diffuse area of the strobila from which the rest of the strobila originates. If the germinative region is indeed responsible for proglottid production, the cellular activity in this region should be significantly different from the rest of the strobila, and division indices and an index of cells involved in DNA synthesis could be used as criteria of definition. This region should also contain a cytologically identifiable type which could act as a stem cell for production of proglottids. Bell and Smyth (1958) and Wikgren (1964) have used the criterion of a mitotic index to estimate growth in trematodes and cestodes. The use of the incorporation of tritiated thymidine into DNA as an index of DNA synthesis and, therefore, as an index of replication was discussed by Clever (1967). Received for publication 30 July 1970. * This investigation was supported by Research Grant AI-06153 and Training Grant 5 TOI AI226 from the NIH, U. S. Public Health Service, Bethesda, Maryland 20014. tPresent address: Department of Biology, Notre Dame University, Notre Dame, Indiana 46556. In the present study we have investigated the cytological characteristics of the germinative region of H. diminuta and a possible germinative type within this region. The duration of the cycle of the proposed germinative has been estimated. MATERIALS AND METHODS Recovery and incubation of H. diminuta Male Holtzman rats (120 to 180 g) were inoculated with either 100 or 10 cysticercoids and worms were recovered 2, 4, and 6 to 14 days postinfection, respectively. Worms were recovered 2 and 4 days postinfection from the hosts' small intestine by scraping the intestinal mucosa with a glass microscope slide into phosphate-buffered (pH 7.4) balanced salt solution (BSS) (Roberts and Fairbairn, 1965) or into tris-maleate-buffered Ringer's solution (Read et al., 1963) with 0.1% added glucose (TMR + G; pH 7.4). The gut scrapings were examined under a dissecting microscope, and the worms were separated from the debris with a Pasteur pipette. Older worms, 6 to 14 days postinfection, were flushed from the rat small intestine with a small amount of salt solution. The worms were washed, incubated 15 min in BSS or TMR + G containing 0.4 mg streptomycin and 500 units potassium penicillin-G per ml, and then incubated in the salt solution containing an experimental substrate. All incubations were done in 25-ml Erlenmeyer flasks with a tissue-to-medium ratio of 60 mg worm fresh weight/ml incubation fluid. Incubations were at 37 C in an atmosphere of N2:CO2 (95:5). Following the incubation, the worms were washed with salt solution and fixed for cytological studies. Preparation for determination of mitotic index Worms were incubated in a concentration of colchicine for a length of time necessary to obtain the maximum number of arrested metaphase figures. Entire 2and 4-day-old worms and the ant rior 2 cm of 6to 14-day-old worms were fixed immediately in 3:1 (v:v) 70% ethanol:neutral