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

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. 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. 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.