Cetaceans are key sentinel species of marine ecosystems and ocean health, being a strategic taxonomic group to evaluate the well-being of aquatic habitats and to detect harmful environmental trends. Respiratory diseases are among the main causes of death in these animals, so the identification of the microbiome community existent in their exhaled breath condensates (EBCs), i.e. blows, has been proposed as a key biomarker for assessing respiratory health. Yet, to characterise microbiomes related to these animals’ respiratory tract and use them as a proxy for health status, it is necessary to develop baseline data on the microorganisms associated with cetaceans. Here, the short-finned pilot whale (SFPW, Globicephala macrorhynchus) was used as a model species to validate the most suitable primer set to explore the prokaryotic diversity of the cetaceans’ respiratory tract. DNA extracted from blow samples (n = 12) of island-associated animals off Madeira Island was sequenced to amplify both V3-V4 and V4-V5 hypervariable regions of the 16S rRNA gene, using the same sequencing platform (Illumina MiSeq). Independently of the primer set used, all blows shared Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla in their composition. V3-V4 resulted in higher diversity of taxa with relative abundance above 1%, whereas the V4-V5 primers captured a higher number of microbial Amplicon Sequence Variants, detecting the microbial rare biosphere with pathogen potential. Additionally, it captured more efficiently the core microbiome. Thus, this study provides a detailed characterization of SFPW respiratory-associated microbial communities, also strengthening the idea of sociality influencing microbiome composition in the respiratory tract. Moreover, it supports the use of EBCs as a relevant biomarker for the physiological state of the airways in free-ranging cetaceans.

blow sampling, exhale breath condensate, health status, metabarcoding, respiratory disease, short-finned pilot whale