Exhaled Breath Condensate of G.macrorhynchus and D.delphis

Cetaceans play a critical role in marine ecosystems and function as sentinel species for detecting environmental perturbations, underscoring the importance of assessing their health for effective marine conservation. This study employed 16S rRNA gene sequencing to characterize the prokaryotic communities present in exhaled breath condensate (EBC) samples from cetaceans, utilizing both short-read (Illumina) and long-read (PacBio) sequencing platforms. Putative pathogenic taxa were identified using the Multiple Bacterial Pathogen Detection (MBPD) database. Substantial differences in microbial community composition were observed between sequencing approaches. The PacBio platform yielded 2,647 amplicon sequence variants (ASVs) spanning 30 bacterial phyla, with 614 ASVs identified as potential pathogens. In contrast, the Illumina dataset generated 409 ASVs across 17 phyla, of which 46 were flagged as potentially pathogenic. Discrepancies were also evident in diversity metrics: PacBio-derived profiles exhibited higher alpha diversity and produced beta diversity clustering patterns that corresponded with sample metadata, while Illumina-based profiles did not reveal meaningful clustering. Distinct EBC microbial signatures were identified for Globicephala macrorhynchus and Delphinus delphis, and with clear differences from the surrounding seawater microbiota. These findings support the use of EBC as a non-invasive and informative tool for respiratory microbiome analysis in marine mammals. Notably, this study provides the first comprehensive characterization of the respiratory microbiota in D. delphis, offering a valuable methodological baseline for future research into host–microbiome interactions, health assessment, and pathogen surveillance in free-ranging cetacean populations, using non-invasive approaches.