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From our experiments it appears that parasitic fungus, Conidiobolus coronatus, infected 12 insect species we have tested, while another 8 species did not suffer after contact with this pathogen. The culture filtrates and mycelial homogenates of C. coronatus contain toxic proteins. By means of two steps FPLC: gel filtration followed by ion-exchange chromatography, we isolated a 30 kDa toxic protein from the culture filtrate. The 30 kDa protein appeared to be very potent on Galleria mellonella larvae: LD₅₀ was less than 5 ng/larva. Rabbit polyclonal anti-30 kDa immunoglobulins specifically recognized this fungal toxin. Using antibodies we showed that 30 kDa mycotoxin is present in C. coronatus conidia, hyphae and is released by the fungus into the incubation liquid medium.
The histopathology of Conidiobolus coronatus infected Galleria mellonella larvae was investigated. In order to distinguish between morphological changes accompanying the dying process and changes connected with the activation of host-defense mechanisms, two fungal strains were used: A(+) killing 90-100% of Galleria larvae and A(-) causing 20-35% mortality in infected hosts. C. coronatus infection resulted in the appearance of black spots on the insect cuticle. Cross-sections revealed callosity and melanization of the cuticle at the infection site. Melanized epidermal cells together with the haemocytes accumulating beneath the place of infection formed a haemocytic capsule. Characteristic changes were observed in the Malpighian tubule morphology of insects surviving infection. Up to 60% of tubules inside survivor become brown, thick and fragile. Histological studies of the deformed tubules revealed the disappearance of the brush border and pycnosis of epithelial cell nuclei taking place at the time of termination of the exposure to the fungal pathogen. Twenty four hours later lumens of deformed Malpighian tubules were filled to capacity with a brown substance and surrounded by numerous layers of haemocytes forming a non-melanized nodule. All other organs of the infected Galleria larvae remained unchanged. No fungal structures were found inside the haemocoel and organs of the host larvae. The fat bodies of the survivors as well as of the dying insects were not changed and contained lipid droplets, suggesting that larval death was due to mycotoxin(s) produced by the pathogenic fungus Saprophytic development of C. coronatus was observed in the case of only one cadaver out of the 53 investigated.
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