In Vitro Developmental Biology and Spore Production in Nosema furnacalis (Microspora: Nosematidae)

In vitro cell culture systems are important tools for the experimental and genetic manipulation of obligate intracellular entomopathogens, such as the microsporidia. We introduced Nosema furnacalis, a microsporidium of Ostrinia furnacalis, into continuous culture using a Helicoverpa zea cell line, BCIRLHZAM1 clone G5. Infection of the cell line was initiated by germinating alkali (pH 12)-activated spores in the presence of cells at pH 8. Initial infection levels were low but the percentage of infected cells increased as parasites spread from infected to uninfected cells. Infected cultures were first transferred after 1 to 2 weeks. Subsequent transfers were made every 5 to 6 days by mixing infected (1 part) with uninfected (5 parts) cells. In this manner, a line of N. furnacalis was maintained for more than 70 transfers with continued formation of spores. The majority of parasites was undergoing merogony during the first 2 days after subculturing; sporulating stages predominated after 4 days. Six to 7 days were needed for maximum spore yield. Efficient cross infection occurred in the subcultures when transfer were made with cells containing mostly spores, but was retarded when cells harbored mainly meronts. The spread of infection was apparently due to the formation of spores that germinated spontaneously in vitro. N. furnacalis maintained in continuous culture for over 70 transfers produced spores that were infective for O. nubilalis. However, after 40 transfers infectivity and virulence of cultured spores for O. nubilalis declined.