Metarhizium robertsii microsclerotia are fungal aggregates composed of compacted, pigmented hyphae. As they are highly tolerant to desiccation and produce infective conidia, they are promising candidates to be formulated as bioinsecticides. Despite this potential, the nature of the pigments within these structures remains unclear. In this study, routine culture media used for the differentiation of M. robertsii microsclerotia were supplemented with four melanin inhibitors, and the resulting propagules were characterized. Inhibitors of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthetic pathway such as tricyclazole and guaiacol induced significant phenotypic and molecular modifications in the obtained M. robertsii propagules, which exhibited a more spherical shape, reduced size, and increased susceptibility to desiccation, heat, and oxidative stress than microsclerotia obtained without inhibitors. Additionally, genes encoding for a polyketide synthase (Mrpks2) and a putative 1,3,6,8-tetrahydroxynaphthalene reductase (Mrthnr), potentially involved in the DHN-melanin biosynthetic pathway, were upregulated in fungi grown in the inhibitor-added media. In conclusion, M. robertsii microsclerotia contain melanins of type DHN that might play a role in both microsclerotia differentiation and environmental stress tolerance.

The ability of entomopathogenic fungi, such as Beauveria bassiana, to infect insects by penetrating their cuticle is well documented. However, some insects have evolved mechanisms to combat fungal infections. The red flour beetle (Tribolium castaneum), a major pest causing significant economic losses in stored product environments globally, embeds antifungal compounds within its cuticle as a protective barrier. Previous reports have addressed the contributions of non-cuticular infection routes, noting an increase in mortality in beetles fed with conidia. In this study, we further explore the progression and dynamics of oral exposure in the gut of T. castaneum after feeding with an encapsulated B. bassiana conidia formulation. First, we characterized the formulation surface using atomic force microscopy, observing no significant topological differences between capsules containing and not containing conidia. Confocal microscopy confirmed uniform conidia distribution within the hydrogel matrix. Then, larvae and adult insects fed with the conidia-encapsulated formulation exhibited B. bassiana distributed throughout the alimentary canal, with a higher presence of conidia before the pyloric chamber. More conidia were found in the larval midgut and hindgut compared to adults, but no germinated conidia were observed in the epithelium. These results suggest that the presence of conidia obstructs the gut, impairing the insect’s ability to ingest, process, and absorb nutrients. This disruption may weaken the host, increasing its susceptibility to infections and, ultimately, leading to death. By providing the first direct observation of fungal conidia within the alimentary canal of T. castaneum, this study highlights a novel aspect of fungal–host interaction and opens new avenues for advancing fungal-based pest control strategies by exploiting stage-specific vulnerabilities.