Taxonomic note on a rare fish infecting freshwater mould Achlya ambisexualis Raper 1939 (Achlyaceae) isolated from Chandraprabha dam, Uttar Pradesh, India

Achlya spp. are oomycetous water moulds, responsible for freshwater fish diseases causing great economic losses. An Achlya sp. implicated in significant fungal infections of both live and dead fish as well as their eggs, has been isolated from the water and soil samples collected from Chandraprabha dam (Chandauli District, Uttar Pradesh, India) employing standard baiting method. Based on morphological characterisation, the strain was identified as Achlya ambisexualis Raper 1939 (Saprolegniales, Oomycetes). It is a dioecious species, characterised by the presence of an achlyoid type of spore dehiscence from both primary and secondary sporangia, differentiated by its oospheres predominantly maturing into eccentric oospores, generally 1-18 per oogonium and gemmae cylindrical in both antheridial and oogonial mycelia. In India, this species was recorded from a single collection in the past but lack proper description and illustrations. The present study describes and illustrates this species for the first time in India and hoped to be beneficial for ichthyopathologists and researchers as A. ambisexualis is known as a necrotroph or parasite of fishes and their eggs.

Regulation of extracellular proteases in Achlya ambisexualis

The use of proteins as a nutrient source by Achlya ambisexualis was investigated, using media containing defined and undefined sources of carbon, nitrogen, and (or) sulfur. Release of extracellular proteases occurred during growth on all proteins and protein hydrolysates tested, but not during growth on yeast extract or in defined medium. In gelatin-containing media, three major bands of extracellular protease activity were detected by electrophoresis, with estimated molecular mass of 26, 48, and 58 kDa. Growth on gelatin was stimulated to a much greater degree by the addition of glucose to the medium than by additions of glutamic acid or methionine. This and the release of ammonia during growth indicate that gelatin is less effective in meeting metabolic needs for carbon than it is in meeting the needs for nitrogen and sulfur. Protease secretion is only partially regulated by glucose, whereas glucose, methionine, and glutamic acid in combination cause almost complete repression. The pattern of regulation indicated by these results is most consistent with one of induction + derepression. Key words: oomycetes, proteinases, regulation, secreted enzymes.

Electrophoretic characterization of endo-(1,4)-β-glucanases secreted during assimilative growth and antheridiol-induced branching inAchlya ambisexualis

Secreted endo-(1,4)-β-glucanases ("cellulases") of Achlya ambisexualis were analyzed by a technique that permits visualization of enzyme activity in situ after electrophoresis in gels containing sodium dodecyl sulfate. Catalytic polypeptides with molecular masses of about 97, 74, 36, 29, and 25 kDa were observed in media from young cultures, though progressively fewer bands were observed as cultures aged. Based on size estimations of native enzymes with gel exclusion chromatography, the 97- and 36-kDa polypeptides were concluded to be subunits of a 245-kDa holoenzyme and the 25-kDa polypeptides were concluded to be subunits of a second holoenzyme of about 92 kDa. The data were insufficient to allow similar assignments for the more ephemeral 74- and 29-kDa polypeptides. The endoglucanases secreted during branch induction by antheridiol or 0.2% peptone comigrated in electrophoretic gels with enzymes secreted during normal assimilative growth. No endoglucanases specific to induced branching were observed.Key words: oomycetes, cell walls, endoglucanases, cellulases, antheridiol.

Steroid hormone regulation of the Achlya ambisexualis 85-kilodalton heat shock protein, a component of the Achlya steroid receptor complex.

The steroid hormone antheridiol regulates sexual development in the fungus Achlya ambisexualis. Analyses of in vivo-labeled proteins from hormone-treated cells revealed that one of the characteristic antheridiol-induced proteins appeared to be very similar to the Achyla 85-kilodalton (kDa) heat shock protein. Analysis of in vitro translation products of RNA isolated from control, heat-shocked, or hormone-treated cells demonstrated an increased accumulation of mRNA encoding a similar 85-kDa protein in both the heat-shocked and hormone-treated cells. Northern (RNA) blot analyses with a Drosophila melanogaster hsp83 probe indicated that a mRNA species of approximately 2.8 kilobases was substantially enriched in both heat-shocked and hormone-treated cells. The monoclonal antibody AC88, which recognizes the non-hormone-binding component of the Achyla steroid receptor, cross-reacted with Achlya hsp85 in cytosols from heat-shocked cells. This monoclonal antibody also recognized both the hormone-induced and heat shock-induced 85-kDa in vitro translation products. Taken together, these data suggest that similar or identical 85-kDa proteins are independently regulated by the steroid hormone antheridiol and by heat shock and that this protein is part of the Achyla steroid receptor complex. Our results demonstrate that the association of hsp90 family proteins with steroid receptors observed in mammals and birds extends also to the eucaryotic microbes and suggest that this association may have evolved early in steroid-responsive systems.

Steroid hormone regulation of the Achlya ambisexualis 85-kilodalton heat shock protein, a component of the Achlya steroid receptor complex

The steroid hormone antheridiol regulates sexual development in the fungus Achlya ambisexualis. Analyses of in vivo-labeled proteins from hormone-treated cells revealed that one of the characteristic antheridiol-induced proteins appeared to be very similar to the Achyla 85-kilodalton (kDa) heat shock protein. Analysis of in vitro translation products of RNA isolated from control, heat-shocked, or hormone-treated cells demonstrated an increased accumulation of mRNA encoding a similar 85-kDa protein in both the heat-shocked and hormone-treated cells. Northern (RNA) blot analyses with a Drosophila melanogaster hsp83 probe indicated that a mRNA species of approximately 2.8 kilobases was substantially enriched in both heat-shocked and hormone-treated cells. The monoclonal antibody AC88, which recognizes the non-hormone-binding component of the Achyla steroid receptor, cross-reacted with Achlya hsp85 in cytosols from heat-shocked cells. This monoclonal antibody also recognized both the hormone-induced and heat shock-induced 85-kDa in vitro translation products. Taken together, these data suggest that similar or identical 85-kDa proteins are independently regulated by the steroid hormone antheridiol and by heat shock and that this protein is part of the Achyla steroid receptor complex. Our results demonstrate that the association of hsp90 family proteins with steroid receptors observed in mammals and birds extends also to the eucaryotic microbes and suggest that this association may have evolved early in steroid-responsive systems.