Sampling
The whales were harvested in the offshore waters of Hokkaido, Japan, and transported to fishing facilities for processing. In order to obtain the nasal mucosa, we meticulously dissected the occipital bone of common minke whales and carefully extracted their brain, subsequently identifying the entrances of the left and right olfactory tract tunnels. The head was then trimmed using a chain saw to create a bony block that encompassed the olfactory bulb tract and the nasal chamber. Upon observing the medial view of the sections, we noted three prominent nasal turbinals, namely the lamina semicircularis and the ethmoturbinals I and II, arranged dorsal to ventral position (Klima, 1999), providing important indications for orientation.
The bony block, measuring 12 cm anteroposterior direction, 7 cm dorsoventrally, and 5 cm transversally, was obtained from the left side of 16NPCK-M009. Subsequently, it was fixed in 10% formalin at the collection site and utilized for gross examination of the nasal chamber. A 5 mm square piece of mucosa was extracted from the posterior end of the frontoturbinal from the block, which was then labeled as H-009 for histological analysis.
We obtained two mucosal samples from 18NPCK-M001. Both samples were collected from the left side nasal mucosa on the ethmoturbinal II, situated in front of the cribriform plate (Fig. 1a and b). One sample was designated as R-001 and was preserved by freezing in RNA-later (Thermo Fisher Scientific Inc., Waltham, MA, USA) for subsequent RNA-seq analysis. The other sample, labeled as H-001, was fixed in 10% formalin for microscopic examination.
Another mucosal sample was acquired for RNA-seq analysis from 18NPCK-M006. The nasal chamber on the left side was carefully trimmed off the head (Fig. 1c and d), promptly frozen, and transported to the laboratory. Subsequently, a 5 mm square mucosal sample was excised from the posterior end of the frontoturbinal region and designated as R-006.
The bony block derived from specimen 18NPCO-M046 was sectioned in a transverse manner, yielding two mucosal pieces extracted from the anterior portion of the right nasal chamber. The mucosal piece intended for RNA-seq analysis was labeled as R-046 and enclosed in a vinyl bag containing RNA-later, ensuring preservation in a freezer. Additionally, an adjacent mucosal piece, designated as H-046, was collected and preserved in 10% formalin for subsequent microscopic examination.
To facilitate comparisons of gene expressions across different organs, the samples were also obtained from the olfactory bulb and the external skin. 18NPCK-M008 was dissected, and the anterior 5 mm tip of the right olfactory bulb was excised and stored in a freezer with RNA-later. This particular piece was labeled as R-008. Furthermore, external skin samples were obtained from specimens 19SK214 and 19SK215, identified as R-214 and R-215 respectively, and preserved in a freezer.
The sampling procedures were recorded through both handwritten and digital macrophotography (Tough TG-5; Olympus Corporation, Tokyo, Japan). We quantified the post-mortem interval as the duration between the animal’s capture into the boats above the sea and the preservation of the collected sample in either a freezer or formalin. The maximum recorded post-mortem interval was 8.5 hours.