Metagenomic Insights Into the Role of Gut Microbes in the Defensive Ink "Tsunabi" of Physeteroid Whales

Abstract

Whales of the superfamily Physeteroidea, which includes the genera Physeter and Kogia, exhibit a unique visual defense mechanism involving the release of dark reddish-brown feces (locally called "tsunabi-ink" in Japan) into the water to obscure themselves from predators and other threats. However, the mechanism underlying pigmentation remains unknown. Because physeteroids possess an enlarged distal colon that retains fecal material, a possible explanation is that symbiont microbial metabolism contributes to the feces pigmentation. To investigate this, we provided a shotgun metagenomic catalog of gut microbiomes from the intestinal tracts of eight cetacean species, including two physeteroids: a sperm whale (Physeter macrocephalus) and a pygmy sperm whale (Kogia breviceps). The colonic microbiome of physeteroids exhibited relatively high abundances of tryptophan metabolism genes, particularly indolepyruvate ferredoxin oxidoreductases (iorA and iorB), suggesting that physeteroids accumulate indole-3-pyruvate-derived pigments in their colons. Furthermore, bacterial members of the phyla Bacillota and Bacteroidota were identified in the physeteroid colon as primary taxa conferring heavy-metal resistance, which may be related to the primary predation of physeteroids on cephalopods, which bioaccumulate high levels of heavy metals. Prolonged fecal retention can expose gut microbes to chronic heavy-metal stress and colonize them as heavy metal-tolerant microbial communities, some of which may produce pigments to reduce their toxicity. Thus, we propose that tsunabi-ink is a metabolic byproduct of shifts in the gut microbial community, influenced by the host's digestive physiology and foraging behavior through sustained ecological interactions with gut symbionts. Moreover, we believe that further empirical investigation would validate this hypothesis.

Keywords: heavy metals; pigmentation; sperm whale; stranding; symbiosis; tryptophan metabolism.

FIGURE 1

The appearance of tsunabi‐ink in Physeter macrocephalus . (A) A photograph of tsunabi‐ink released by P. macrocephalus , obtained at 42.26667° N, 152.12333° E. The whale's back and blowhole are visible to the left of the image, surfacing above the water. A plume of dark reddish‐brown feces (tsunabi‐ink) disperses into the seawater toward the right side of the frame. 2025 Taiga Mukai. (B) The color of the solutions prepared by dissolving whale feces in 20 mL of distilled water. From left to right, the tubes contain (1) 20 mL of distilled water only, (2) 3 mg of P. macrocephalus feces in 20 mL of distilled water, (3) 10 mg of P. macrocephalus feces in 20 mL of distilled water, and (4) 10 mg of L. obliquidens feces in 20 mL of distilled water. The solution containing 3 mg of P. macrocephalus feces showed that tsunabi‐ink exhibited a reddish‐brown hue. In the solution containing 10 mg of P. macrocephalus feces, the intense coloration indicates that tsunabi‐ink effectively serves as a smoke‐screen‐like visual deterrent against potential threats.

FIGURE 2

A diagram of the sampling locations. (A) Balaenoptera acutorostrata possesses a cecum. The intestinal contents were sampled from the duodenum, jejunum, and ileum (proximal to the cecum) and the colon and rectum (distal to the cecum). (B) In Physeter macrocephalus and Kogia breviceps , the diameter of the distal intestine significantly increases. Samples were collected from the duodenum, jejunum, and ileum (proximal to the enlarged region) and from the colon and colonic ampulla within the enlarged distal section. (C) In the remaining species, five equidistant sampling points (G_1–G_5) were established along the intestinal tract from the gastric to the anal end, and the contents were collected accordingly. Each symbol corresponds to a distinct region: Duodenum (DUO), jejunum (JEJ), ileum (ILE), cecum (CEC), colon (COL), rectum (REC), and colonic ampulla (COA).

FIGURE 3

Accumulation curves of metagenomic open reading frames (ORFs). (Left) Accumulation curves of ORFs obtained from each cetacean species at a 95% local sequence identity clustering threshold. (Right) Accumulation curves of ORFs obtained from all cetacean species at 95%, 75%, and 50% local sequence identity clustering thresholds. B. acut: Minke whale, P. macr: Sperm whale, K. brev: Pygmy sperm whale, Z. cavi: Cuvier's beaked whale, M. stej: Stejneger's beaked whale, L. obli: Pacific white‐sided dolphin, P. phoc: Harbor porpoise, and P. dall: Dall's porpoise.

FIGURE 4

The phylogenetic tree of the reconstructed metagenome‐assembled genomes (MAGs) from the intestinal microbiome of eight cetacean species. The tree depicts the estimated genome size, quality scores, taxonomic classification, and host species for each MAG.

FIGURE 5

Microbial diversity and community composition in the gut of eight cetacean species. (A) The phylum‐level composition of the gut microbiome across eight cetacean species. Relative abundances are presented as percentages. (B) Alpha diversity indices (observed richness and Pielou's evenness) calculated for each gut region of each species. Values are based on order‐level relative abundance data. (C) NMDS plot based on Aitchison distance, illustrating beta diversity among gut regions and species. NMDS was performed using order‐level relative abundance data. B. acut: Minke whale, P. macr: Sperm whale, K. brev: Pygmy sperm whale, Z. cavi: Cuvier's beaked whale, M. stej: Stejneger's beaked whale, L. obli: Pacific white‐sided dolphin, P. phoc: Harbor porpoise, and P. dall: Dall's porpoise.

FIGURE 6

Heatmaps showing the normalized mean TPM values of genes annotated to KEGG amino acid metabolism pathways and indolepyruvate ferredoxin oxidoreductase alpha and beta subunits (iorA and iorB, respectively) across various intestinal regions and cetacean species. (A) A heatmap of all intestinal regions across all cetacean species. (B) A heatmap of only the distal‐most intestinal regions in each species (rectum, colonic ampulla, or G_5).

FIGURE 7

The phylogenetic tree of the indolepyruvate ferredoxin oxidoreductase alpha and beta subunits (iorA and iorB), which are encoded in tandem. This tree includes the taxonomic classification of the iorA and iorB sets, along with information on the host species.

FIGURE 8

An alluvial plot illustrating the core microbiome in the colonic regions of Physeter macrocephalus (top) and Kogia breviceps (bottom) encoding genes for tryptophan metabolism (ko00380), cadmium resistance, copper resistance, and iron resistance. The thickness of the alluvium lines represents the mean TPM values observed in the colon and colonic ampulla of P. macrocephalus and K. breviceps .