Toxicity and Pathology of Crude Broth Filtrate Produced by Hirsutella thompsonii var. thompsonii in Shake Culture

Abstract

Metabolic products secreted by the fungal mycelia of Hirsutella thompsonii var. thompsonii (CBS 556.77D) in a defined culture broth in shake culture were tested for toxicity to Galleria mellonella larvae and Drosophila melanogaster adults via injection and per os application, respectively. In addition, the toxic effect of broth filtrate was observed in vitro in a cell line of Bombyx mori. Czapek-Dox broth fortified with 1% yeast extract stimulated more rapid mycelial growth and correspondingly more toxin production in time. At 25-30°C, metabolic toxin(s) was detected in broth via bioassay at about 4-5 days postinoculation when mycelial biomass reached 5 mg/ml (dry wt). At these temperatures, biological activity of the filtrate peaked at about 8-10 days when mycelial growth reached a maximum (10 mg/ml, dry wt). This suggests a positive relationship between toxic metabolite and mycelial production. After 10 days, the toxicity of the filtrate appeared to decline gradually. Pathogenicity symptoms of the metabolites developed slowly in both G. mellonella and D. melanogaster . Early signs of lethargy appeared at 4 days postinjection and cumulative mortality of G. mellonella larvae was low after 1 week; however, the percentage of mortality reached 98-100% after 14 days. At death, G. mellonella larvae displayed small dark spots on a brownish cuticle. Histopathological effects were observed in the larval midgut, malpighian tubules, hypodermis, fat body, hemocytes, muscle, and silk glands. Cellular change consisted of pycnosis of the nucleus and a reduction in cytoplasm density. Highest mortality (78.8%) to adult D. melanogaster occurred after 10 days post-treatment. A strong cytotoxic effect was detected in B. mori cells after 24 hr.