Optimizing cetacean blow collection methods under controlled conditions: implications for further non-invasive UAV-based sampling of the cetacean respiratory microbiome in the wild

Background: Unmanned aerial vehicles (UAVs) are gaining attention as non-invasive tools for wildlife research, offering the ability to collect biological samples without physically handling animals. In cetaceans, drone-based sampling of exhaled breath condensate—or “blow”—has emerged as a promising method for assessing health, particularly through analysis of the respiratory microbiome. However, standardized protocols for effective and reliable blow collection are still lacking. Results: This study aimed to evaluate and compare four different blow collection tools—DNA/RNA Shield, dry and pre-moistened biocide-free cellulose sponges, and indicating FTA cards—using controlled trials conducted 1 m above five bottlenose dolphins (Tursiops truncatus) under human care. Metabarcoding analyses of the captured microbial communities revealed that the type of sampling collection tool had a stronger influence on microbiome composition and structure than the individual dolphin or sample origin (blow, air, or water). FTA cards and DNA/RNA Shield consistently yielded the most informative and high-quality results. In contrast, both types of sponges showed evidence of overgrowth by copiotroph opportunistic bacteria, limiting their reliability. While DNA/RNA Shield offered superior DNA yield and storage conditions, FTA cards proved to be highly practical due to their ease of use, portability, and cost-effectiveness. Conclusions: The results underscore the critical role of sampling tool selection in shaping respiratory microbiome profiles obtained via UAV-based blow collection in dolphins. Both DNA/RNA Shield and FTA cards are valid options, with the final choice depending on specific logistical considerations, resource availability, and research priorities. This study provides a foundational step toward standardizing drone-assisted protocols for respiratory microbiome sampling in wild cetaceans.