DOI: 10.5061/dryad.r7sqv9shm
title: Virulence of three Aspergillus species to the model insect Galleria mellonella and the contribution of ergot alkaloids to the pathogenic potential of Aspergillus leporis

description: 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.
description: Larvae of the model insect <em>Galleria
 mellonella</em> 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 <em>Aspergillus leporis</em>, <em>A.
 hancockii</em>, 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
 <em>Aspergillus </em>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.
description: Data are in a single MS Excel file with six separate tabs.  Data
 are ready to be copied and pasted into a JMP worksheet.