Developmental Physiology of Cestodes. VIII. Inhibition of Ribonucleic Acid Synthesis by Actinomycin-D in Developing Hymenolepis diminuta

Abstract

The uptake and incorporation of actinomycin-D by Hymenolepis diminuta (Cestoda:Cyclophyllidea) was measured. Treatment of very young worms (4 days after infection of the host) with concentrations of this drug of 9.5 ILM to 15.9 /M inhibited incorporation of uridine into RNA by greater than 90%. Synthesis of DNA-dependent RNA was 100% inhibited. Inhibition was greater in the germinative and immature regions than in the mature proglottids of the strobila of 8-day-old worms. There was no effect of the drug on incorporation of thymidine into DNA. The data support the use of actinomycin-D as a tool in studying gene activity during development of H. diminuta. Actinomycin-D (AM-D) causes a block in protein synthesis by binding to the guanine bases of DNA, preventing transcription of genetic information by RNA polymerase, and, therefore, inhibiting the synthesis of DNA-dependent RNA (Carter and McCarty, 1966; Muller and Crothers, 1968). Actinomycin-D has been useful in studying control of development in various systems, e.g., developing embryos of sea urchins, amphibians, mice, and chicks (Gross and Cousineau, 1963a, b, 1964; Gross et al., 1964; Brachet et al., 1964; Brachet, 1968; Lane, 1969; Mintz, 1964; Piper and Klein, 1969). It has also been used by Bovarnick et al. (1969) to study the development of chloroplasts in Euglena and by James (1964) and Burchill (1968) to investigate regeneration in Stentor. Recently, the uptake and incorporation of this drug by sea urchin eggs and embryos and by mealy bug tissues in culture has been studied (Thaler et al., 1969; Berlowitz et al., 1969). Harris, Fodge, and Talent (pers. comm.) have used AM-D in an investigation of RNA synthesis in Hymenolepis diminuta. The strobila of H. diminuta, like most cyclophyllidean cestodes, develops by the budding of proglottids from the unsegmented region immediately posterior to the scolex (see discussion by Roberts, 1961, p. 363). Because of the central importance of this germinative region in Received for publication 2 April 1970. * This investigation was supported by Research Grant AI-06153 and Training Grant 5 TOI AI226, from the NIH, U. S. Public Health Service. t Present address: Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556. cestode development, the relation of DNAdependent RNA synthesis to protein synthesis in the region is of considerable interest The present report concerns the use of actinomycin-D as a tool in this investigation. The experiments reported here were performed on young worms recovered from the rat host at 4 days and at 8 days after infection At 4 days postinfection, H. diminuta consists of scolex, germinative area, and a few immature proglottids; organogeny (but not segmentation) has just begun, and the worms are about 5 mm long (Roberts, 1961). By comparison, H. diminuta at 8 days postinfection has 600 to 700 immature and 100 to 300 mature proglottids; gametogenesis is actively proceeding in the terminal proglottids, but embryogenesis has yet to start (Roberts, 1961). MATERIALS AND METHODS Recovery and incubation of H. diminuta Male Holtzman rats (120 to 180 g) were inoculated with either 500 cysticercoids or 10 cysticercoids per rat for recovery and incubation of the worms at 4 days and 8 days postinfection, respectively. The worms were removed from the rat small intestines into Krebs-Ringer's salt solution (phosphate buffer, pH 7.4), then washed and sterilized according to the method described for in vitro cultivation (Roberts and Mong, 1969). The basal incubation medium to which experimental substrates were added was a Hanks' solution with added glucose (as used by Roberts and Mong, 1969, for the cultivation fluid overlay). The worms were incubated for 2, 4, or 8 hr in 25-ml Erlenmeyer flasks at 37 C, under a gas phase of nitrogen/carbon dioxide (95/5), and a shaking rate of 160 excursions/min. One milliliter of medium was provided per 60 mg worm fresh weight. After incubation, the worms were washed 8 times in clean Hanks' solution and either fixed for autoradio-