Hemin as a Requirement in the Development In vitro of Hymenolepis microstoma (Cestoda: Cyclophyllidea)

Abstract

Hymenolepis microstoma developed in continuous axenic culture from juvenile to prepatent adult in a diphasic culture system. Strobilization and maturation occurred only when hemin was added to the overlay of Triple Eagle's Medium or NCTC 135, or when blood was present in the agar base. After 20 days in culture, the average length of the organisms varied from 37 to 52 mm. At this time they contained mature segments often containing preoncospheres. Organisms cultured in the absence of hemin or blood attained a maximum length of 1.6 mm after 4 days in culture and did not progress beyond this point. Preliminary investigations using 14C-labeled hemin suggest that small quantities of this tetrapyrrole are incorporated after 3 days in culture. Hemin and other porphyrin derivatives are commonly found in plants and animals. In most instances, they are synthesized de novo from simple precursors. It has long been noted, however, that some microorganisms and protozoa have strict requirements for hemin or related tetrapyrrole compounds for growth and development. The discovery by Thjotta and Avery that Hemophilus influenzae required a substance associated with blood pigment was one of the earliest reports of a defined bacterial nutrient (see Lascelles, 1962). Other microorganisms, including strains of Staphylococcus aureus, Escherichia coli, Physarum polycephalum, and Bacteroides spp. require hemin or a hemeprotein for growth (Lascelles, 1962). Certain Trypanosomatidae also have a hemin requirement (Lwoff, 1951). Hemin and hemeproteins may play an important role in the development of helminths. Hemoglobin and other porphyrin-containing proteins have been demonstrated in trematodes, nematodes, and in a few cestodes. Recent investigations by Hieb and Stokstad (1970) demonstrated a heme requirement for the in vitro reproduction of the nematode Caenorhabditis briggsae. Complete development of the tetrathyridia of Mesocestoides occurs when hemoglobin is substituted for whole blood in the culture media (Voge and Seidel, 1968). Similar results may be obtained when hemin is Received for publication 17 September 1970. * Supported by Research Grant Number AI 07 332-05, U. S. Public Health Service, NIH, Bethesda, Maryland. added to the media (Seidel and Voge, unpublished). The effect of hemin on the in vitro development of Hymenolepis microstoma will be discussed below. MATERIALS AND METHODS Cysticercoids were dissected from the grain beetle, Tribolium confusum, and transferred into 2-inch petri dishes containing 10 ml of sterile Earle's saline, 900 tLg dihydrostreptomycin sulfate, and 900 units of penicillin. They were transferred into fresh solution every 10 min for a total of 3 washes. Sterile procedures were followed throughout. Cysticercoids were then excysted by Rothman's technique (1959), washed once in culture medium, and inoculated into 150by 25-mm screw cap tubes.