Oosporogenesis by Lagenidium giganteum in liquid culture

The sexual stage of Lagenidium giganteum can be cultured in liquid shake or fermentation culture by modifying techniques used for in vitro oosporogenesis on solid media. In addition to a basal component of yeast extract, consistent yields of viable oospores in liquid culture required exogenous sterols and unsaturated fatty acids, calcium, and magnesium. Fatty acids can be provided by vegetable oils enriched in oleic, linoleic, or linolenic acids. Levels of sterols present in vegetable oils are insufficient for optimum oosporogenesis in liquid media; crude preparations of sitosterol or cholesterol in combination with suitable vegetable oils yielded over 6 × 10 7 oospores/liter in liquid shake culture and 5 × 10 7 oospores/liter in 12-liter fermentation tanks. Sterols apparently enhance fatty acid uptake from culture media. Germination of oospores was regulated by dehydration, maturation protocols, and methods of activation. Following rehydration oospores begin germinating within 2 to 25 days, and asynchronous activation as monitored by sentinel mosquito infection continued for over 2 months under laboratory conditions. Seven-year-old oospores grown in vivo and stored at room temperature in soil germinated beginning 25 days after rehydration and continued infecting mosquitoes for several months.