Galleria mellonella immune melanization is fungicidal during infection

AbstractA key component of the insect immune response is melanin production, including within nodules, or aggregations of immune cells surrounding microbes. Melanization produces oxidative and toxic intermediates that limit microbial infections. However, a direct fungicidal role of melanin during infection has not been demonstrated. We previously reported that the fungusCryptococcus neoformansis encapsulated with melanin within nodules ofGalleria mellonellahosts. Here we developed techniques to study melanin’s role duringC. neoformansinfection inG. mellonella. We provided evidence that in vivo melanin-encapsulation was fungicidal. To further study immune melanization, we applied tissue-clearing techniques to visualize melanized nodules in situ throughout the larvae. Further, we developed a time-lapse microscopy protocol to visualize the melanization kinetics in extracted hemolymph following fungal exposure. Using this technique, we found that cryptococcal melanin and laccase enhance immune melanization. We extended this approach to study the fungal pathogensCandida albicansandCandida auris. We find that the yeast morphologies of these fungi elicited robust melanization responses, while hyphal and pseudohyphal morphologies were melanin-evasive. Approximately 23% of melanin-encapsulatedC. albicansyeast can survive and breakthrough the encapsulation. Overall, our results provide direct evidence that immune melanization functions as a direct antifungal mechanism inG. mellonella.