Humpback whale populations share a core skin bacterial community: towards a health index for marine mammals?

Abstract

Microbes are now well regarded for their important role in mammalian health. The microbiology of skin--a unique interface between the host and environment--is a major research focus in human health and skin disorders, but is less explored in other mammals. Here, we report on a cross-population study of the skin-associated bacterial community of humpback whales (Megaptera novaeangliae), and examine the potential for a core bacterial community and its variability with host (endogenous) or geographic/environmental (exogenous) specific factors. Skin biopsies or freshly sloughed skin from 56 individuals were sampled from populations in the North Atlantic, North Pacific and South Pacific oceans and bacteria were characterized using 454 pyrosequencing of SSU rRNA genes. Phylogenetic and statistical analyses revealed the ubiquity and abundance of bacteria belonging to the Flavobacteria genus Tenacibaculum and the Gammaproteobacteria genus Psychrobacter across the whale populations. Scanning electron microscopy of skin indicated that microbial cells colonize the skin surface. Despite the ubiquity of Tenacibaculum and Psychrobater spp., the relative composition of the skin-bacterial community differed significantly by geographic area as well as metabolic state of the animals (feeding versus starving during migration and breeding), suggesting that both exogenous and endogenous factors may play a role in influencing the skin-bacteria. Further, characteristics of the skin bacterial community from these free-swimming individuals were assembled and compared to two entangled and three dead individuals, revealing a decrease in the central or core bacterial community members (Tenacibaculum and Psychrobater spp.), as well as the emergence of potential pathogens in the latter cases. This is the first discovery of a cross-population, shared skin bacterial community. This research suggests that the skin bacteria may be connected to humpback health and immunity and could possibly serve as a useful index for health and skin disorder monitoring of threatened and endangered marine mammals.

Figure 1. Rarefaction analysis of bacterial SSU rRNA gene sequences.

This analysis represents data subsampled at 3,662 sequences per sample. Samples with OTU richness greater than 130 are listed by name.

Figure 2. Similarity analysis of whale skin-bacteria.

(A) Clustering dendogram, and (B) nonmetric multidimensional scaling analysis of 97% similarity grouped OTUs of bacterial communities associated with whale skin sampled from different populations and locations, and seawater.

Figure 3. Taxonomic composition whale skin-bacteria.

The 97% similarity grouped OTU bacterial communities were associated with humpback whale skin sampled from different populations and locations. Graphs represent total composition (A), and abundances of skin-bacterial sequences specifically belonging to the Tenacibaculum and Psychrobacter lineages (B). Each bar graph represents the relative abundance of each major taxonomic group, and sums to 100%.

Figure 4. Abundances of Tenacibaculum and Psychrobacter-affiliated sequences.

Presence of skin-bacterial OTUs (97% similarity grouped) classified as Tenacibaculum (A) and Psychrobacter (B), in relation to the geographic region of the whales.

Figure 5. Phylogenetic relationship of Tenacibaculum-affiliated sequences.

Represented SSU rRNA gene sequences include Tenacibaculum-affiliated sequences recovered from whale skin, cultivated Tenacibaculum isolates, and relatives within the Tenacibaculum lineage. Red sequences are short-reads from this study and blue are full-length sequences from a previous study on humpback whale skin . The scale bar corresponds to 0.10 substitutions per nucleotide position, and only bootstrap values >70 are listed. Sequences from Caldilinea aerophila (AB067647), Roseiflexus castenholzii (CP000804) and Actinomyces oris (GQ421308), were used to form the outgroup. Full accession numbers for the dolphin respiratory-bacteria sequences are available in .

Figure 6. Oligotyping analysis of major humpback skin-bacterial groups.

MDS analysis of the healthy animals from four regional groups based on Tenacibaculum (A) and Psychrobacter (B) oligotypes. Centroid and shape of each ellipse is defined by the distribution and standard deviation of points in the defined regional groups.

Figure 7. Phylogenetic relationship of Psychrobacter-affiliated sequences.

Represented SSU rRNA gene sequences include Psychrobacter-affiliated sequences recovered from whale-skin, cultivated Psychrobacter isolates, and relatives within the Gammaproteobacteria. Red colored sequences are short-reads from this study, and blue full-length sequences are from a previous study . The scale bar corresponds to 0.10 substitutions per nucleotide position, and only bootstrap values >70 are listed. Sequences from Hymenobacter soli (AB251884), Salegentibacter flavus (AY682200) and Ureaplasma urealyticum were used to form the outgroup.

Figure 8. Scanning electron micrographs of whale skin-bacteria.

Overview (A, C, E, G) and detailed (B, D, F, H) scanning electron micrographs of the surface of humpback whale skin from individuals CCS2010-100 (A, B), CCS2010-99 (C,D), CCS2010-96 (E,F) and CCS2010-98 (G,H). Diatoms, and microbes residing on these cells, were present on the surfaces of CCS2010-100 (I) and CCS2010-97 (J). Arrows indicate possible unique microbial cell morphotypes.