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.

29Pathogenic yeast species can cause life-threatening infections in humans. The two leading 30 yeast pathogens, Candida albicans and Cryptococcus neoformans, cause systemic infections 31 in >1.4 million patients world-wide with mortality rates approaching 75%. It is thus 32 imperative to study fungal virulence mechanisms, stress response pathways, and the efficacy 33 of antifungal drugs. This is commonly done using mammalian models. To address ethical and 34 practical concerns, invertebrate models, such as wax moth larvae, nematodes, or flies, have 35 been introduced over the last two decades. To address short-comings in existing invertebrate 36 host models, we developed fifth instar caterpillars of the Tobacco Hornworm moth Manduca 37 sexta as a novel host model for the study of fungal virulence and drug efficacy. These 38 caterpillars can be raised at standardised conditions, maintained at 37˚C, can be injected with 39 defined amounts of yeast cells, and are susceptible to the most threatening yeast pathogens, 40 including C. albicans, C. neoformans, C. auris, and C. glabrata. Infected caterpillars can be 41 rescued by treatment with commonly deployed antifungal drugs and importantly, fungal 42 burden can be assessed daily throughout the course of infection in a single caterpillar's faeces 43 and hemolymph. Notably, these animals are large enough so that weight provides a reliable 44 and reproducible measure of fungal virulence. This model combines a suite of parameters that 45 recommend it for the study of fungal virulence. 46 47 50 tuberculosis 1 . The leading yeast pathogens, Candida albicans and Cryptococcus neoformans, 51 together account for >1,400,000 life-threatening infections world-wide with mortality rates 52 approaching 75% 1 . Candidemia, most commonly caused by C. albicans, is the fourth most 53 common cause of nosocomial blood stream infections, only surpassed by infections with 54Staphylococci and Enterococcus spp. Disturbingly, candidemia incidence rates are on the 55 rise. Within less than ten years, incidence rates increased by 36% 2 . Although cryptococcosis 56 incidence rates are on the decline in North America, this AIDS-defining illness is responsible 57 for 15% of all AIDS-related deaths world-wide 2,3 . This already dire situation is further 58 confounded by the emergence of drug resistant yeast species. Patients at risk of developing 59 invasive candidemia are often prophylactically treated with fluconazole, while the 60 echinocandins are considered a first line defence strategy 4 . Yet, C. glabrata, the most 61 3 common non-albicans Candida species associated with nosocomial blood stream infections 5 , 62 is intrinsically less susceptible to azole drugs and acquires resistance to echinocandins 63 rapidly 6 . The rapid global spread of multi-drug resistant C. auris has further exacerbated the 64 threat posed by fungi. C. auris was first reported in 2009 in Japan 7 . In 2015, C. auris arrived 65 in Europe causing an outbreak involving 72 patients in a cardio-thoracic hospital i...