Chaverri, P.; Escudero-Leyva, E.; Mora-Rojas, D.; Calvo-Obando, A.; Gonzalez, M.; Escalante-Campos, E.; Mesen-Porras, E.; Wicki-Emmenegger, D.; Rojas-Gatjens, D.; Avey-Arroyo, J.; Campos-Hernandez, M.; Castellon, E.; Moreira-Soto, A.; Drexler, J. F.; Chavarria, M.

Sloths, with their ruminant-like digestive systems, possess the slowest digestion among mammals due to their low metabolic rate, minimal food intake, and extremely low-energy diet. However, no comprehensive studies have characterized the sloth\'s gut microbiota, including fungi, and their role in digestion. This study hypothesized that effective plant fiber-degrading fungi (e.g., Neocallimastigomycota) would be scarce in the sloth\'s gut. The aim was to describe the gut microbiota of three-toed (Bradypus variegatus) and two-toed (Choloepus hoffmanni) sloths to understand their link to slow digestion. Microbial composition and functionality were analyzed using shotgun metagenomics, metatranscriptomics, fungal metabarcoding (ITS 1 and 2 nrDNA), and cellulose degradation analysis. Microbial communities were dominated by bacteria (92-97%), followed by viruses (1-7%). Fungi accounted for only 0.06-0.5% of metagenomic reads and 0.1% of transcripts. Functional analysis revealed minimal CAZy abundance (1.7-1.9% in metagenomes, 0.2% in metatranscriptomes), with no fungal CAZys or glycoside hydrolases detected. Neocallimastigomycota had negligible abundance in metagenomic data and was absent in metatranscriptomic or ITS metabarcoding data. Bradypus variegatus showed overall lower CAZy abundance and fungal presence compared to Choloepus hoffmanni. Lastly, cellulose degradation analyses revealed that only ~5-35% of the intake was digested. This study highlights the unique microbial ecosystem in sloths\' guts, showing minimal presence of plant fiber-degrading anaerobic fungi and limited microbial CAZys, aligning with their slow digestion and low metabolic rate, thus enhancing our understanding of their digestive efficiency and metabolic adaptations.