Black yeast biota in the mangrove, in search of the origin of the Lethargic Crab Disease (LCD)

Knowledge of natural ecology is essential for a better understanding of pathogenicity and opportunism in black yeast-like fungi. Although etiological agents of diseases caused by these fungi are supposed to originate from the environment, their isolation from nature is difficult. This is probably due to their oligotrophic nature, low competitive ability, and, overall, insufficient data on their natural habitat. We obtained environmental samples from mangrove areas where mortalities by lethargic crab disease (LCD) are reported and areas without disease recorded. Isolation of chaetothyrialean black yeasts and relatives was performed using a highly selective protocol. Species-specific primers were used to determine if these isolates represented Exophiala cancerae or Fonsecaea brasiliensis, two proven agents of LCD, in order to test hypotheses about the origin of the disease. Isolates, identified by morphology as Fonsecaea- or Exophiala-like, were tested specific diagnostic markers for the fungi associated with LCD. Although several black fungi were isolated, the main causative agent of the LCD, E. cancerae, was not found. Molecular markers for F. brasiliensis revealed 10 positive bands for isolates from biofilms on mangrove leaves, branches, and aerial roots, of which four were confirmed by ITS sequencing. The absence of E. cancerae in environmental samples suggests that the species is dependent on the crab, as a genuine pathogen, different from F. brasiliensis, which is probably not dependent on the host species, U. cordatus. However, we did not attempt isolation from the marine water, which may represent the pathway of dispersion of the black yeast species between neighbor mangroves