EXISTENCE OF OLFACTORY EPITHELIUM IN BALEEN WHALES?

Short note

NASAL MUCOSA RESEMBLING AN OLFACTORYSYSTEM IN THE COMMON MINKE WHALE(BALAENOPTERA ACUTOROSTRATA)
Ayumi HIROSE1, Takushi KISHIDA2 and Gen NAKAMURA1*
1
Laboratory of Cetacean Biology, Tokyo University of Marine Science and Technology,
4 – 5 – 7 Konan, Minato-ku, Tokyo 108–0075, Japan *(Corresponding author: gnakam1@kaiyodai.ac.jp)
2
Wildlife Research Center, Kyoto university, 2 – 24 Tanaka Sekiden-cho, Sakyo, Kyoto 606 – 8203, JapanAbstract
Although it has long been assumed that modern cetaceans lack nervous system structures thatmediate olfaction, recent studies suggest that mysticetes still maintain olfactory nerves. We
collected samples of the mucous membrane covering the cribriform plate at the bottom of thedorsal nasal meatus from a mature female common minke whale. The samples were then thinsectioned and stained with hematoxylin and eosin. Microscopic observations revealed that themucosa was covered with a pseudostratified columnar epithelium with vessels, glands, andnerve plexuses in its lamina propria. These histological characteristics resembled those of theolfactory epithelium in terrestrial mammals, suggesting that mysticetes do indeed possess asense of smell.
Key words

Baleen whale, Olfaction, Histology, Nasal cavity, Olfactory epithelium.

In terrestrial mammals, the external nares and nasal passages function not only as a respiratory organ
but also as an olfactory organ. Meanwhile, cetaceans use their blowhole as a respiratory tract, but theirolfactory system seems to be highly degenerated. Extremely, odontocetes are reported to possess nonervous systems that mediate olfaction (Glezer, 2002; Oelschläger and Oelschläger, 2009).
In contrast, recent studies suggest that mysticetes can smell in air partly due to the presence of olfactory bulbs in their brain (Thewissen, George, Rosa and Kishida, 2011; Kishida, Thewissen, Usip,
Suydam and George, 2015). The bony nasal passages of mysticetes branch into two sections: theventral nasal meatus, which is broad and connects the blowhole and larynx, and the dorsal nasal
meatus, a narrower passage the posterior part of which leads to the brain case (Godfrey, Geisler andFitzgerald, 2013; Fig. 1). Because these structures resemble those of terrestrial mammals, previousstudies support the presence of an olfactory system in mysticetes (Cozzi, Huggenberger andOelschläger, 2016). Analyses of the olfactory marker protein gene further supports this, suggesting theexistence of a functional olfactory system in mysticetes, even albeit much reduced compared withterrestrial mammals (Kishida and Thewissen, 2012; Springer and Gatesy, 2017).
Of the various functions and morphologies of the nasal epithelium, the portion known as the olfactory region affects an animal’s olfactory ability (Kato and Yamauchi, 2003). The olfactory epithelium
is a pseudostratified columnar epithelium composed of three types of cells: basal cells, sustentacularcells and olfactory receptor cells. Olfactory receptor cells have olfactory cilia projecting into the nasal
cavity which react to odoriferous substances, stimulating the part of the brain known as olfactory bulbs(Wheater, Burkitt and Daniels, 1979) and subsequently the sense of smell will arise. The olfactory
region is located near the cribriform plate of the ethmoid bone with great inter-species variations
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Fig. 1. The positions of mucosa sampling on the cribriform plate. Lateral view of the headshowing structure of the ventral nasal meatus (dark gray), the dorsal nasal meatus (lightgray), and brain case (stripes). There were a series of deep folds at the bottom of dorsal
nasal meatus.

among mammals (Kato and Yamauchi, 2003). There are small serous glands below the olfactory epithelium known as Bowman’s glands, the ducts of which penetrate into the epithelium and opening of
the nasal cavity. Olfactory nerve fascicles are also found in the lamina propria.
Thus, since the presence of an olfactory epithelium indicates the existence of olfaction, it is a goodstarting point in discussing the feasibility of olfaction in mysticetes. In this study, we carried out histological observations (light microscopy) of nasal mucosa samples from the common minke whale todescribe the olfactory anatomy in this species.
We analyzed a female common minke whale (Balaenoptera acutorostrata) with a body length of
7.68 m. This individual was captured in 2016 during the second phase of the Japanese Whale Research
Program under the special permit in the Western North Pacific (JARPNII), and was appeared to besexually matured based on analysis of its ovaries.
Samples were prepared immediately after the animal’s death (approx. within 4.5 h). Since the locationof the olfactory epithelium was thought to be inside the bony nasal passages, the head was separated into
two halves along the mid-sagittal plane using a chainsaw. After carefully locating the right dorsal nasal
meatus, two transverse sections were cut off to remove anterior part of the dorsal nasal meatus and posterior part of the ethmoid bone. Lateral bony parts were also removed, leaving the entire dorsal nasal
meatus inside the specimen. Finally, the specimen was trimmed into a cube of approximately 10×5×20
cm using a hand saw, and the tissues were fixed in 10% formalin and preserved in 70% ethanol.
Three epithelium samples were collected from the mucosa on the cribriform plate. The location of
the cribriform plate was assumed to be the posterior wall of the recess at the bottom of the dorsal nasal
meatus (Fig. 1). Three square pieces of mucosa with 2 – 3 mm thick including the tissue beneath theepithelium were collected from the preserved specimen using a surgical knife. Two samples were collected from the dorsal side of the wall and one from the ventral side.
The epithelial samples were then processed according to standard histological techniques and stainedwith hematoxylin and eosin (H/E). The epithelium was observed under an optical microscope withmagnification of 10 × and 40 × , paying particular attention to whether the epithelial cells consisted ofa pseudostratified columnar epithelium typical of olfactory organs. The thickness of the epitheliumand structure of the lamina propria were also determined.
Dorsal epithelial samples of mucosa, which constituted the posterior wall of the recess, showedfeatures of a pseudostratified columnar epithelium 70 – 80 µm thick (Fig. 2). A similar epithelium wasalso observed on the ventral side; however, the thickness varied. On the dorsal portion of this ventral
sample, the thickness was 80 µm or more, while the ventral portion was approximately 50 µm thick.
Nerve plexuses and glandular cells were observed in the lamina propria in all three samples, and in
one dorsal sample, some of these serum ducts appeared to open into the nasal cavity.
Thickness of the epithelium exceeds 100 µm in some areas, but less than 100 µm in other areas. In26

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EXISTENCE OF OLFACTORY EPITHELIUM IN BALEEN WHALES?

Fig. 2. Sections showing the epithelium samples from the cribriform plate. Sections were 6 µm
thick and stained with H/E. Scale bars = 50 µm. The lumen (nasal cavity) is located at the topof the pictures. Close-up of epithelial cells (a, b; 40×) and epithelial tissue showing the lamina
propria (c; 10 × ) from the dorsal side of the cribriform plate. Similar images from the ventral
side (d, e, f).

terrestrial mammals, the olfactory epithelium is typically composed of a pseudostratified columnar
epithelium approximately 100 – 120 µm thick (Kato and Yamauchi, 2003). The epithelial samples ofnasal mucosa observed here show the morphological characteristics similar to those in terrestrial mammals.
The lamina propria of the thick pseudostratified columnar epithelium contained numerous glands,
and these glands opened into the lumen in the dorsal sample. Although more detailed examination isrequired to conclude, we assume that these are Bowman’s glands. Numerous blood vessels were alsoobserved, suggesting a role in helping warm inhaled air. Near these vessels, peripheral nerve fascicles
were also observed. Overall, the distribution of these tissues in the lamina propria resembled the olfactory region of the nasal mucosa in other mammals (Kato and Yamauchi, 2003; Harkema, Carey andWagner, 2006; Chamanza and Wright, 2015).
Areas of pseudostratified columnar epithelium were located near the olfactory bulbs. Meanwhile, inmegascopic observations of parasagittal sections of the animal’s head, the bony canal representing theolfactory tract, or anterior elongation of the brain case, faced the olfactory recess. This elongated area
is occupied by the olfactory bulb, and the bone dividing the brain case and bony nasal passage is thecribriform plate of the ethmoidal bone (Thewissen et al., 2011; Godfrey et al., 2013; Ichishima, 2016).
Epithelium samples of nasal mucosa from the cribriform plate therefore resembled the olfactory epithelium in terrestrial mammals, supporting the feasibility of olfaction in mysticetes.
In general, mysticetes have left and right nasal passages completely isolated from each other (Berta,
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Ekdale and Cranford, 2014), suggesting that left and right nasal passages could serve as independentolfactory organs. That is, since the left and right auditory organs are able to locate a sound source,these two olfactory organs may be capable of locating the origin of an odoriferous substance via different stimuli of chemical substances between the left and right nasal cavities (Kikuta, Sato, Kashiwadani,
Tsunoda, Yamasoba and Mori, 2010). We therefore hypothesize that mysticetes have two nasal openings
with symmetrical nasal passages, which they use in olfaction (Kishida, 2016).
The microscopic structure of the epithelium samples observed in this study strongly supports thepossibility of an olfactory epithelium. However, it should be noted that similar morphologies do notalways suggest an identical function. Further analyses of the role of this epithelium are required toconfirm the existence of olfaction in the common minke whales.
Acknowledgements

We are grateful to Dr. Hideyoshi Yoshida, National Research Institute of Far Seas Fisheries, and Mr.
Yoshiichi Shimomichi, Shimomichi Suisan for the opportunity to collect specimens. We would like toexpress our gratitude to Dr. Yasuhiro Uekusa, the Senshu-Futaba Kai Medical Corporation for hisadvice based on medical knowledge. We also thank Dr. Katsumi Isono, Professor Emeritus of Kobe
University for his detailed comments on the manuscript, and Dr. Masahide Hasobe, Tokyo Universityof Marine Science and Technology for his helping in our experiments.

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Received: April 13, 2018
Accepted: April 25, 201828

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