The research, published in the journal Metabarcoding and Metagenomics, focused on the short-finned pilot whale (Globicephala macrorhynchus), a highly social species with a global distribution and a well-documented propensity for mass strandings. Respiratory tract infections rank among the leading causes of death in cetaceans, making the microbial community of the airways a potentially powerful biomarker of respiratory health. Yet most existing data on cetacean-associated microbes come from captive, stranded, sick or injured individuals, which cannot be considered representative of healthy free-ranging populations. The new study set out to fill that gap by sampling wild whales in their natural environment.

Between September and October 2018, researchers conducted at-sea monitoring campaigns in the southern waters of Madeira Island, Portugal. Using a PERFORMAgene PG-100 swab collection kit attached to a five-metre extendable aluminium pole, the team positioned the sampling device roughly 40 to 50 centimetres above the blowhole of surfacing whales to capture droplets from the exhaled plume. The animals were approached slowly from an eight-metre rigid inflatable boat under permits from the Instituto de Florestas e Conservação da Natureza IP-RAM. In total, twelve blow samples were collected across six sampling events, nine from individual animals and three from pools of individuals within the same group. Photographic identification against Madeira’s catalogue of the species confirmed that the nine individual samples corresponded to seven distinct animals, two of which were sampled on separate occasions.

A notable methodological first accompanied the sampling: the PERFORMAgene kit, previously used for livestock and pets, had never been reported in the literature for collecting blow from free-ranging cetaceans. The kit offers practical advantages for fieldwork, stabilising DNA samples for up to a year at room temperature, which provides safe flexibility during transportation and storage. Although DNA concentrations extracted from the swabs were low, the method proved suitable for downstream metabarcoding analysis. The researchers also observed no evident changes in the whales’ swimming patterns or behaviour during sampling, suggesting the procedure causes minimal distress, though they recommend future studies formally monitor behavioural responses alongside collection.

The core of the study was a rigorous comparison of two primer sets targeting different hypervariable regions of the 16S rRNA gene, the standard molecular marker for characterising bacterial and archaeal communities. The V3-V4 region, amplified with primers 341F and 806R, had been used in previous cetacean blow studies, while the V4-V5 region, amplified with primers 515F-Y/926R, is currently recommended by the Earth Microbiome Project for targeting marine microbes. By applying both primer sets to the same twelve samples, using identical DNA extraction, library preparation, sequencing on an Illumina MiSeq platform and bioinformatic processing through the DADA2 pipeline, the researchers eliminated technical biases that typically confound such comparisons.

Sequencing yielded a total of 685,692 reads for the V3-V4 dataset and 862,763 reads for the V4-V5 dataset. After quality filtering, the final output comprised 415,340 sequences assigned to 2,764 Amplicon Sequence Variants (ASVs) in the V3-V4 dataset and 417,733 sequences assigned to 3,665 ASVs in the V4-V5 dataset. ASVs resolve sequences to the level of a single nucleotide difference, offering finer taxonomic resolution than traditional operational taxonomic units. Taxonomic classification was performed with a Naïve Bayes classifier against the curated SILVA database, and undesirable lineages such as chloroplast, eukaryote and mitochondrial sequences were excluded from further analysis.

The comparison revealed striking differences between the two primer sets. Alpha diversity metrics varied significantly: the V4-V5 dataset captured roughly 32 percent more bacterial ASVs, with higher values for the Observed and Chao1 richness measures, while the V3-V4 dataset produced higher Shannon and Inverse Simpson diversity values. A statistical test using the Wilcoxon Signed-Rank procedure confirmed significant differences between primer sets for most measures. Beta diversity analysis, based on Bray-Curtis dissimilarity and visualised through non-metric multidimensional scaling, showed that samples clustered according to the primer set used. A PERMANOVA test found that the primer choice explained approximately 25 percent of the variation in prokaryotic community distribution, a substantial technical effect that researchers designing future cetacean microbiome studies cannot afford to ignore.

Despite these differences, all blow samples shared a core microbiota composed of four dominant phyla: Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. This aligns with previous findings in bottlenose dolphins, humpback whales and other cetacean species, where Proteobacteria is consistently highly abundant. At the genus level, the V3-V4 dataset was dominated by Pseudomonas, while the V4-V5 dataset was dominated by Cutibacterium, a genus typical of nasal microbiota. The oceanic bacterium Prochlorococcus, one of the most abundant bacteria in the sea, was detected in both datasets and is probably a seawater-associated organism within the blow. The researchers noted that control water samples were not taken in this study but consider them extremely important for future work to disentangle the influence of ambient seawater on the detected microbial composition.

Perhaps the most intriguing finding concerns sociality. In the V4-V5 dataset, individuals that were travelling together when sampled, such as the pairs Gma_2 and Gma_3 or Gma_4 and Gma_5, clustered together in the community analyses, a pattern not observed with the V3-V4 primers. This supports the hypothesis that social behaviour shapes the respiratory microbiome in cetaceans. Short-finned pilot whales live in matrilineal groups with long-lasting relationships, and behaviours such as surfacing and breathing near each other or feeding cooperatively could facilitate microbial transfer between individuals. Previous work on whale species has detected a positive correlation between sociality and microbial diversity in blow, and the new results strengthen the idea that social complexity influences microbiome composition in the respiratory tract of these marine mammals.

The study also carries implications for disease surveillance. Four of the most abundant genera detected in both datasets, Pseudomonas, Flavobacterium, Rhodococcus and Sphingomonas, are potential pathogenic genera commonly related to pulmonary infections, although their presence does not necessarily indicate disease since not all members of these taxa are pathogenic. The V4-V5 primers proved particularly valuable for detecting the rare microbial biosphere, the low-abundance taxa that could include organisms with pathogenic potential, and for capturing the core microbiome more consistently. The authors conclude that primer selection should depend on the analytical goal: V3-V4 performs better for capturing diversity present at higher relative abundance, while V4-V5 is preferable for identifying unique ASVs and rare taxa. A combination of both primer sets may ultimately provide the most robust characterisation of the blow microbiome. With further optimisation of sampling protocols and integration with complementary health measures such as photogrammetry-based body condition assessment, whale breath could become a routine biomarker for monitoring the physiological state of airways in free-ranging cetacean populations worldwide.

Subject of Research: Respiratory microbiome characterisation of free-ranging short-finned pilot whales using blow samples and 16S rRNA metabarcoding

Article Title: Hidden in the blow – a matrix to characterise cetaceans’ respiratory microbiome: short-finned pilot whale as case study

Article References: Santos, B., Afonso, L., Alves, F., Dinis, A., Ferreira, R., Correia, A. M., Valente, R., Gil, Á., Castro, L. F. C., Sousa-Pinto, I., Rosso, M., Centelleghe, C., Mazzariol, S., Magalhães, C., & Tomasino, M. P. (2024). Hidden in the blow – a matrix to characterise cetaceans’ respiratory microbiome: short-finned pilot whale as case study. Metabarcoding and Metagenomics, 8, Article e121060. https://doi.org/10.3897/mbmg.8.121060

Image Credits: AI Generated

DOI: 10.3897/mbmg.8.121060

Keywords: cetaceans, short-finned pilot whale, blow microbiome, respiratory health, 16S rRNA metabarcoding, Madeira Island, exhaled breath condensate, marine mammals, microbiome, non-invasive sampling, rare biosphere, sociality