Tobacco Hornworm (Manduca sexta) caterpillars as a novel host model for the study of fungal virulence and drug efficacy

Abstract

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%. It is thus imperative to study fungal virulence mechanisms, efficacy of antifungal drugs, and host response pathways. 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. To complement existing invertebrate host models, we developed fifth instar caterpillars of the Tobacco Hornworm moth Manduca sexta as a novel host model. These caterpillars can be maintained at 37°C, are suitable for injections with defined amounts of yeast cells, and are susceptible to the most threatening yeast pathogens, including C. albicans, C. neoformans, C. auris, and C. glabrata. Importantly, fungal burden can be assessed daily throughout the course of infection in a single caterpillar's feces and hemolymph. Infected caterpillars can be rescued by treatment with antifungal drugs. 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. M. sexta caterpillars combine a suite of parameters that make them suitable for the study of fungal virulence.

De novo genome assembly of the tobacco hornworm moth (Manduca sexta)

“…The size of M. sexta makes it an optimal system for studying the biochemical pathways associated with programmed cell death, but the system is limited by the quality of the reference genome. M. sexta’ s innate immune pathways, many of which share mammalian homologs, have been characterized in detail and used as a model system for studying fungal virulence and drug efficacy ( Kanost et al 2004 ; Lyons et al 2020 ). Finally, M. sexta is useful for understanding serine proteases, a repertoire of proteins involved in mediating defence responses such as hemolymph clotting, melanotic encapsulation, food digestion, antimicrobial peptide induction, and cytokine activation ( Jiang et al 2010 ; Cao et al 2015 ).…”

A novel Hsp90 phospho-switch modulates virulence in the major human fungal pathogenCandida albicans

“…Once we established the importance of phosphorylation of S530 and T25 in virulence-relevant traits using different in vitro assays, we aimed to determine if and how Hsp90 phosphorylation affects fungal virulence. To test this, Manduca sexta caterpillars, an invertebrate model host of fungal disease 38 , were infected with 10 7 yeast cells. Caterpillar survival was then assessed in groups of ten after 16 hours of incubation at the physiologically relevant temperature of 37°C.…”

“…To assess if Hsp90 phosphorylation affects virulence, we infected caterpillars of the Tobacco Hornworm Manduca sexta 38 , a novel invertebrate model of fungal disease. In preparation for virulence assays, C. albicans strains were grown overnight in YPD at 30°C and stationary phase cultures were washed twice with 1x PBS.…”

Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms

“…Here, we focused on the silkworm infection model due to the easy handling and low costs, a published genome [ 53 ] and large body size facilitating injections and manipulation. In particular, when compared to the larvae of G. mellonella and M. sexta , both of which have the ability to grow further even after fungal infections into the next developmental stages (larvae → pupae → moths) [ 15 , 54 ], the growth of silkworms is generally inhibited by an incubation temperature higher than 30 °C [ 35 ], and the silk moths already lost their ability to fly due to domestication [ 18 ]. Thus, the silkworm infection model has a clearly much lower risk of biohazard.…”

“…To overcome these challenges, several invertebrate infection models were proposed as a cost- and time-effective replacement for mammalian hosts with respect to large-scale, preliminary in vivo studies. Present major invertebrate models include the fruit fly ( Drosophila melanogaster ), a nematode ( Caenorhabditis elegans ), larvae of the greater wax moth ( Galleria mellonella ), the domestic silk moth ( Bombyx mori ), or the tobacco hornworm moth ( Manduca sexta ) [ 13 , 14 , 15 ].…”