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.