James L. Kerwin

Sterol induction of sexual reproduction inLagenidium giganteum

Abstract Lagenidium giganteum, an Oomycete facultatively parasitic on mosquito larvae, will produce oosporesin vitro when supplied with exogenous sterols. Following prolonged maintenance on sterol-free medium, the fungus retains its ability for zoosporogenesis, but oosporogenesis does not occur. Oospore yield is affected by changes in the sterol side chain, especially at C-24, and by the degree and position of unsaturation in the sterol ring. Oospore yield and viability can be enhanced by incorporating fatty acids in the growth medium, especially in the form of triglycerides. Oosporogenesis induced by fatty acid sterol esters is much reduced relative to that obtained using free sterols.

Eicosanoid regulation of oosporogenesis by Lagenidium giganteum

Cell proliferation is inhibited in many biological systems by lipid peroxides and related products derived from polyunsaturated fatty acids. Using developmentally synchronized cultures of Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, it has been documented that oxidative lipid metabolism is necessary for the induction and subsequent maturation of its sexual stage, the oospore. Addition of lipoxygenase inhibitors to liquid cultures of the fungus results in the stage-specific disruption of antheridial induction, gametangial fusion, induction of meiosis and spore cell wall formation. Oosporogenesis is inhibited by these compounds at concentrations which have no discernible effect on mycelial viability or asexual reproduction. Cyclooxygenase inhibition had comparable effects using ibuprofen and to a lesser extent with indomethacin. Phenylbutazone and the salicylates affected oosporogenesis only at concentrations which decreased asexual reproduction or mycelial viability. The inhibitory effects of NDGA on oosporogenesis could be reversed using partly purified eicosanoid extracts from growth media.

Oosporogenesis by Lagenidium giganteum in liquid culture

Abstract 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.

Cyclic nucleotide regulation of oosporogenesis byLagenidium giganteum and related fungi

Abstract Incorporation of phosphodiesterase inhibitors into basal growth media limits the inducation of oosporogenesis by Lagenidium giganteum and related pythiaceous fungi. Cyclic AMP enhanced while cyclic GMP antagonized the effects of these compounds. Adenosine, guanosine, and their 5′-monophosphate derivatives acted primarily by reducing oospore maturation rather than induction. Asexual reproduction was generally promoted by cyclic AMP and phosphodiesterase inhibitors. It is suggested that cyclic nucleotides and sterols play a pivotal role in the induction of meiosis and that sterols and other factors affecting reproductive development by these fungi may act in part by regulating cellular cyclic nucleotide levels.

Effects of exogenous phospholipids on lipid composition and sporulation by three strains of Lagenidium giganteum

The California (LGCA) and Butte Sink (LGBS) strains of the sterol auxotrophic fungus Lagenidium giganteum (Oomycetes: Lagenidiales) enter the sexual cycle on media supplemented with sterols. A third isolate of this mosquito pathogen, the North Carolina strain (LGNC), requires sterols plus phospholipids to produce oospores in vitro. Enrichment of the polar and neutral lipid fractions of the LGCA and LGBS strains with unsaturated fatty acids promoted oospore induction, and increased oospore viability. With the exception of the LGCA strain, there was no consistent relationship between phospholipid supplementation in growth media and mycelial phospholipid content.