Opportunistically pathogenic fungi have varying potential to cause disease in animals. Factors contributing to their virulence include specialized metabolites, which is some cases evolved in contexts unrelated to pathogenesis.  Specialized metabolites that increase fungal virulence in the model insect Galleria mellonella include the ergot alkaloids fumigaclavine C in Aspergillus fumigatus (syn. Neosartorya fumigata) and lysergic acid α-hydroxyethylamide (LAH) in the entomopathogen Metarhizium brunneum. Three species of Aspergillus recently found to accumulate high concentrations of LAH were investigated for their pathogenic potential in G. mellonella.  Aspergillus leporis was most virulent, A. hancockii was intermediate, and A. homomorphus had very little pathogenic potential. Aspergillus leporis and A. hancockii emerged from and sporulated on dead insects, thus completing their asexual life cycles. Inoculation by injection resulted in more lethal infections than did topical inoculation, indicating A. leporis and A. hancockii were pre-adapted for insect pathogenesis but lacked an effective means to breach the insect’s cuticle. All three species accumulated LAH in infected insects, with A. leporis accumulating the most.  Concentrations of LAH in A. leporis were similar to those observed in the entomopathogen M. brunneum. LAH was eliminated from A. leporis through a CRISPR/Cas9-based gene knockout, and the resulting strain had reduced virulence to G. mellonella. The data indicate A. leporis and A. hancockii have considerable pathogenic potential and that LAH increases the virulence of A. leporis.

Larvae of the model insect Galleria mellonella were purchased from Josh’s Frogs (Owosso, MI) and injected with conidia (20 µL at 3,000 conidia/µL, or in one experiment 250 conidia/µL) of Aspergillus leporis, A. hancockii, and A. homomorphus with the aid of a 29-gauge insulin syringe. Control groups of 12 to 15 larvae per trial were injected with 20 µL of PBS solution (phosphate buffered saline) lacking Aspergillus spores. Injected larvae were incubated at room temperature (or in one trial at 37 °C) and observed for one to two weeks following inoculation; any dead larvae were removed throughout this period. Insects also were inoculated topically by placing larvae on sporulating cultures on sucrose-yeast extract agar (20 g sucrose, 10 g yeast extract, 1 g magnesium sulfate heptahydrate, and 15 g agar per liter) for 24 hours before removing them to empty Petri dishes.  Insects inoculated through this topical, or natural, approach were observed for 16 days following inoculation. 

Alkaloids were extracted from individual larvae eight days post-inoculation by bead-beating in 1 mL of methanol. Alkaloids also were extracted from fungi cultured on sucrose-yeast extract agar medium for at least six days at room temperature, 30°C, or 37°C.  Samples of ~400 µL volume (containing the hyphae and spores of the fungus and the supporting agar medium) were combined with 400 µL of methanol, rotated end-over-end (~40 rpm) for 30 minutes, and then centrifuged for 10 minutes for clarification. Extracts (20 µL) were analyzed by high performance liquid chromatography (HPLC) with fluorescence detection. Fluorescence of lysergic acid derivatives was detected by excitation at 310 nm and measuring the emission at 410 nm; chanoclavine-I, which has a different double bond structure in its ring system, was detected with fluorescence settings of 272 nm/372 nm. Concentrations of lysergic acid amides were estimated by comparison of areas under HPLC peaks to a standard curve prepared from dilutions of ergonovine, which contains the same fluorophore as the other lysergic acid amides. Because of this comparison to ergonovine, the concentrations of LAH and ergine should be considered as ‘relative to ergonovine’ rather than as absolute.

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