Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. cerevisiae, with IC 50 of 12 mM, but had no detectable activity against bacteria. Purified recombinant diapausin-1 was active against S. Diapausin-1 was produced as a recombinant protein in Escherichia coli. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. sexta transcriptome and genome databases. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Most antimicrobial pep-tides from insects have antibacterial but not antifungal activity. sexta caterpillars combine a suite of parameters that make them suitable for the study of fungal virulence.Insects secrete antimicrobial peptides as part of the innate immune response.
Notably, these animals are large enough for weight to provide a reliable and reproducible measure of fungal disease and to facilitate host tissue-specific expression analyses. Infected caterpillars can be rescued by treatment with antifungal drugs. Importantly, fungal burden can be assessed daily throughout the course of infection in a single caterpillar’s feces and hemolymph. These caterpillars can be maintained at 37☌, are suitable for injections with defined amounts of yeast cells, and are susceptible to the most threatening yeast pathogens, including C. To complement existing invertebrate host models, we developed fifth instar caterpillars of the Tobacco Hornworm moth Manduca sexta as a novel host model. While this is commonly done in mammalian models, which are afflicted by ethical and practical concerns, invertebrate models, such as wax moth larvae and nematodes have been introduced over the last two decades. It is thus imperative to study fungal virulence mechanisms, efficacy of antifungal drugs, and host response pathways. The two leading yeast pathogens of humans, Candida albicans and Cryptococcus neoformans, cause systemic infections in >1.4 million patients worldwide with mortality rates approaching 75%.
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