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\begin{document}

\title{Localized expression of the olfactory receptor genes in the olfactory
organ of common minke whales}



\author[1]{Ayumi Hirose}%
\author[2]{Masato Nikaido}%
\author[3]{Gen Nakamura}%
\author[4]{Yoshihiro Fujise}%
\author[3]{Hidehiro Kato}%
\author[5]{Takushi Kishida}%
\affil[1]{Tokyo Institute of Technology - Ookayama Campus}%
\affil[2]{Tokyo Kogyo Daigaku - Ookayama Campus}%
\affil[3]{Tokyo University of Marine Science and Technology}%
\affil[4]{Institute of Cetacean Research}%
\affil[5]{Nihon University College of Bioresource Sciences Graduate School of Bioresource Sciences}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Baleen whales (Mysticeti) possess the necessary anatomical structures
and genetic elements for olfaction. Nevertheless, the olfactory receptor
gene (OR) repertoire has undergone substantial degeneration in the
cetacean lineage following the divergence of Artiodactyla and Cetacea.
The functionality of the highly degenerated mysticete ORs within their
olfactory epithelium remains unknown. In this study, we extracted total
RNA from the nasal mucosa of common minke whales (\emph{Balaenoptera
acutorostrata}) to investigate the localized expression of ORs. All
three sections of the mucosae examined in the nasal chamber displayed
comparable histological structure, whereas the posterior portion of the
frontoturbinal region exhibit notably high expression of ORs and another
gene specific to the olfactory mucosa. Neither the olfactory bulb nor
the external skin exhibited expression of these genes. Although this
species possesses four intact class-1 ORs, all the ORs expressed in the
nasal mucosa belong to class-2, implying the loss of aversion to
specific odorants. These anatomical and genomic analyses suggest that
ORs are still responsible for olfaction within the nasal region of
baleen whales, enabling them to detect desirable scents such as prey and
potential mating partners.%
\end{abstract}%



\sloppy


\textbf{Localized expression of the olfactory receptor genes}

\textbf{in the olfactory organ of common minke whales}

Ayumi Hirose\textsuperscript{1,2}, Masato Nikaido\textsuperscript{1},
Gen Nakamura\textsuperscript{2}, Yoshihiro Fujise\textsuperscript{3},
Hidehiro Kato\textsuperscript{2,3}, Takushi Kishida\textsuperscript{4,5}

(1) School of Life Science and Technology, Tokyo Institute of
Technology, Tokyo, Japan

(2) Tokyo University of Marine Science and Technology, Tokyo, Japan

(3) Institute of Cetacean Research, Tokyo, Japan

(4) Museum of Natural and Environmental History, Shizuoka, Japan

(5) College of Bioresource Sciences, Nihon University, Fujisawa, Japan

MAIL TO: hirose.a.ad@m.titech.ac.jp

KEYWORDS

Mysticeti / olfaction / RNA-seq / histology / epithelium /
secondary-aquatic

ABSTRACT

Baleen whales (Mysticeti) possess the necessary anatomical structures
and genetic elements for olfaction. Nevertheless, the olfactory receptor
gene (OR) repertoire has undergone substantial degeneration in the
cetacean lineage following the divergence of Artiodactyla and Cetacea.
The functionality of the highly degenerated mysticete ORs within their
olfactory epithelium remains unknown. In this study, we extracted total
RNA from the nasal mucosa of common minke whales (\emph{Balaenoptera
acutorostrata} ) to investigate the localized expression of ORs. All
three sections of the mucosae examined in the nasal chamber displayed
comparable histological structure, whereas the posterior portion of the
frontoturbinal region exhibit notably high expression of ORs and another
gene specific to the olfactory mucosa. Neither the olfactory bulb nor
the external skin exhibited expression of these genes. Although this
species possesses four intact class-1 ORs, all the ORs expressed in the
nasal mucosa belong to class-2, implying the loss of aversion to
specific odorants. These anatomical and genomic analyses suggest that
ORs are still responsible for olfaction within the nasal region of
baleen whales, enabling them to detect desirable scents such as prey and
potential mating partners.

\section*{1. INTRODUCTION}

{\label{introduction}}

Olfaction, the sense of smell, represents one of the sensory modalities
encompassing biologically important behaviors such as foraging, predator
avoidance, mother-calf relationships, mating and territorial display
(Doty, 1986; Nei et al., 2008; Corona and L\selectlanguage{ngerman}évy, 2015). The sense of
smell arises when olfactory receptor proteins within the nasal cavity
capture volatile chemical substances. These proteins are localized on
the membranes of olfactory cells. Stimulation is subsequently
transmitted from the receptor protein through the cribriform plate into
the main olfactory bulb by olfactory nerves, extending from the base of
olfactory cells. Olfactory receptor genes (ORs) are responsible for
encoding these olfactory receptor proteins (Buck and Axel, 1991). ORs
comprise the largest gene family in mammals and are broadly classified
into two categories, class-1 and class-2, based on their nucleotide
sequences (Niimura and Nei, 2005; Niimura, 2009b). Each individual OR
encodes a specific olfactory receptor protein that interacts with
particular ligands, thereby enabling the discrimination of different
odors (Malnic et al., 1999; Saito et al., 2009).

The observed variation in mammalian olfaction is recognized as a result
of anatomical and genomic factors, as expounded upon by the
aforementioned mechanism. Anatomical features, such as the dimensions of
the cribriform plate (Pihlström et al., 2005), generally align with
olfactory abilities, exhibiting interspecies variations. Furthermore, an
augmented number of OR copies within a species denotes an elevated
discriminatory capacity and enhanced olfactory significance (Niimura,
2009a; Zhou et al., 2021). The comparatively diminished size of the main
olfactory organ in primates, incontrast to other mammals, indicates a
reduced olfactory capacity within the human species (\emph{Homo
sapience} ) (Moran et al., 1982; Bird et al., 2018). Consistently,
humans possess approximately 400 copies of ORs (Niimura and Nei, 2003),
which is significantly fewer than the approximate 1000 copies found in
mice (\emph{Mus musculus} ) or rats (\emph{Rattus norvegicus} ) (Niimura
and Nei, 2007). Both morphological investigations and genomic studies
provide evidence supporting the diminished importance of olfaction in
humans.

The main olfactory organ in mammals is positioned within the respiratory
passage, enabling the detection of odors with each inhalation. At this
juncture, a query arises: can this sensory system suffice for fully
aquatic mammals? Cetaceans, having transitioned aquatic environment over
50 million years ago (Roe et al., 1998; Clementz et al., 2006; Gatesy et
al., 2013), encompass two distinct monophyletic lineages known as baleen
whales (Mysticeti) and toothed whales (Odontoceti) (Nikaido et al.,
2001). They breathe air sorely when they ascend to the water's surface.
While the frequency of breaths may vary based on activity levels,
consistent breathing patterns have been observed across numerous
species. For instance, small toothed whales generally take breaths every
1-2 minutes, killer whales (\emph{Orcinus rca} ) breathe no more
frequently than every 8 minutes, and deep-diving species such as sperm
whales (\emph{Physeter macrocephalus} ) and beaked whales (Ziphiidae)
can remain submerged for approximately 1 hour (Miller and Roos, 2018).
In the case of baleen whales, blue whales (\emph{Balaenoptera musculus}
) typically exhibit breathing intervals of approximately 4 minutes
(Miller and Roos, 2018). Humpback whales (\emph{Megaptera novaeangliae}
) during the breeding season demonstrate an average interdive breathing
interval of 6 minutes and 45 seconds, and in some instances,
particularly among singers, this interval can extend to approximately 13
minutes (Chu, 1988; Hedley et al., 2011). Consequently, cetaceans
experience periods of interrupted respiration during dives, leading to
intermittent reception of sensory information through the olfactory
modality.

Recently, the olfactory capabilities in baleen whales have been
investigated through morphological and genomic studies. The skeletal
components of the main olfactory organ, such as cribriform plate and
turbinals, have been observed in common minke whales (\emph{Blaenoptera
acutorostrata} ) (Godfrey et al., 2013; Ichishima, 2016). In this
species, the nasal mucosa covering the cribriform plate demonstrates
similarities to the olfactory mucosa found in terrestrial mammals, as it
is lined with pseudostratified columnar epithelium and glandular organs
like Bowman's gland within the lamina propria (Hirose et al., 2018).
Gross and microscopic examinations have provided evidence for the
presence of the main olfactory organ in bowhead whales (\emph{Balaena
mysticetus} ) (Thewissen et al., 2011; Kishida et al., 2015b), and
subsequent immunohistochemical staining has identified olfactory nerves
in the nasal mucosa of this species (Farnkopf et al., 2022). Han et al.
(2022) conducted a search for ORs in seven baleen whale species using
publicly available whole genomes, annotating between 54 and 95 intact
copies of ORs. The number of ORs identified in baleen whales is lower
compared to other mammals, which corresponding to the reduced anatomical
complexity of their main olfactory organ.

Both morphological and genomic investigations postulate the hyposmia of
cetaceans, and this diminished olfactory capability is discernible
subsequent to the divergence of Artiodactyla and Cetacea (Kishida,
2021). However, baleen and toothed whales exhibits this reduction in
distinct manners. While the aforementioned genomic studies suggest a
relatively less efficient sense of smell in baleen whales compared to
terrestrial mammals, they possess a larger repertoire of ORs compared to
toothed whales (Kishida et al., 2015a; Kishida, 2021; Han et al., 2022;
Christmas et al., 2023). Analysis of the olfactory marker protein gene
(OMP), which exhibits high expression in the olfactory epithelium and
plays a crucial role in olfaction (Danciger et al., 1989; Buiakova et
al., 1996), indicates that the sense of smell in baleen whales is
subjected to purifying selection pressures, whereas toothed whales
experience more relaxed selective pressures (Kishida and Thewissen,
2012; Springer and Gatesy, 2017). Furthermore, baleen whales exhibit
anatomical features essential for olfaction that are reminiscent of
those found in terrestrial mammals. In contrast, the nasal cavity
morphology of extant toothed whales has undergone significant
modifications for biosonar signal generation, and it is widely accepted
that olfactory structures are absent in this lineage (Cranford et al.,
1996; Berta et al., 2014; Hirose et al., 2022). Airborne odorants have
been proposed to serve as a locating cue for krill, attracting baleen
whales through olfactory modality rather than toothed whales (Thewissen
et al., 2011; Kishida, 2021). Behavioral experiments targeting humpback
whales, long-finned pilot whales (\emph{Globicephala melas} ) and
bottlenose dolphins (\emph{Tursiops truncatus} ) have provided support
for this hypothesis (Bouchard et al., 2019; Bouchard et al., 2022). The
sense of olfaction provides a captivating illustration of how cetaceans
interact with their aquatic environment, primarily due to the
accelerated evolutionary rate observed in ORs in placental mammals
(Christmas et al., 2023). The remarkable diversification of ORs
highlights their significant role in shaping species diversity through
the influence of olfactory perception.

While exploring ORs provides a powerful methodology for evaluating
olfactory capabilities, it has certain limitations in comprehending the
sense of smell (Go and Niimura, 2008). It is important that not all ORs
are exclusively expressed within the olfactory mucosa, which is
intricately linked to olfactory reception (Kishida et al., 2019). The
existence of ectopic ORs, which are expressed in various non-olfactory
tissues, have been documented (Chen et al., 2018). Notably, specific ORs
in humans and mice exhibit expression in the testis and are involved in
sperm chemotaxis (Parmentier et al., 1992; Spehr et al., 2003; Fukuda et
al., 2004; Rouquier and Giorgi, 2007). Furthermore, a gene known as
OR51E2, classified as a class-1 OR, is present in nearly all mammalian
species including both baleen and toothed whales (Han et al., 2022) and
has been identified in the prostate (Neuhaus et al., 2009). Hence, the
mere presence of ORs does not unequivocally signify the capability of
odor detection.

Prior investigations have established that baleen whales possess the
essential anatomical structures and genetic elements for olfaction;
however, these findings alone do not guarantee the existence of a sense
of smell based on the same mechanism as observed in other mammals.
Therefore, the objective of the present study is to determine whether
intact ORs are exclusively expressed in the mucosa of the putative main
olfactory organ in baleen whales or not. To address this objective, we
utilized the common minke whale, Balaenopteridae, as our research
subjects and extracted total RNA from nasal mucosa to examine the
localized expression of ORs.

\section*{2. MATERIALS}

{\label{materials}}

The present study examined common minke whales obtained from the coastal
regions of Japan. The specimens were acquired from seven animals (Table
1). 16NPCK-M009 and 16NPCK-M012 were procured in 2016 during the second
phase of the Japanese Whale Research Program under the special permit in
the Western North Pacific (JARPNII). 18NPCK-M001, 18NPCK-M006 and
18NPCK-M008 were obtained in 2018 during the New Scientific Whale
Research Program in the western North Pacific (NEWREP-NP). 19SK214 and
19SK215 were acquired in 2019 during Japanese commercial whaling
operations. The research programs adhered to the regulations outlined in
Article VIII of the International Convention for the Regulation of
Whaling (ICRW). All samples were collected in accordance with legal
procedures.

Table 1 List of specimens.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Location\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
year\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
ID\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Sex\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Body length (m)\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
PI* (h)\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Techniques used\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Description of specimen\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Side\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Sample\#\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Kushiro, Hokkaido\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2016\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
16NPCK-M009\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
7.01\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Histology\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Nasal mucosa\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
L\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
H-009\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Kushiro, Hokkaido\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2018\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
18NPCK-M001\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5.50\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
RNA-seq Histology\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Nasal mucosa (Ethmoturbinal II)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
L\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
R-001, H-001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
18NPCK-M006\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.09\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
RNA-seq\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Nasal mucosa (Frontoturbinal)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
L\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
R-006\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18NPCK-M008\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4.62\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
RNA-seq\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Olfactory bulb\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
R\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
R-008\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Abashiri, Hokkaido\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2018\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
18NPCO-M046\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
F\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.30\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
RNA-seq Histology\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Nasal mucosa (Anterior portion)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
R\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
R-046, H-046\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Kushiro, Hokkaido\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2019\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
19SK214\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
7.33\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
8.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
RNA-seq\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
External skin\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
R-214\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
19SK215\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
M\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
7.56\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5.5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
RNA-seq\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
External skin\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
R-215\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

* PI means the post-mortem interval.

\section*{3. METHODS}

{\label{methods}}

\emph{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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} Portions where samples R-001, H-001 and R-006 were
harvested. (a) A lateral view of a parasagittal section of the cranium
from 18NPCK-M001, illustrating the localization of the nasal turbinals
(marked by a white circle) within left nasal chamber. (b) An enlarged
view of the ethmoturbinals I and II, positioned anterior to the
cribriform plate. The highlighted region, delineated by a white line,
represents the posterior end of the ethmoturbinal II. To expose its
medial side, this segment is folded over. The red arrowhead denotes the
site of mucosal sample R-001 extraction (enclosed by a dashed line).
Additionally, in proximity to this area, histological sample H-001 was
obtained. (c) A parasagittal section of the left nasal chamber from
18NPCK-M006. This section, depicted in a medial block, provides a
lateral view. (d) The corresponding lateral side (in medial view) of the
section shown in (c). This photograph displays two frontoturbinals.
Mucosal sample R-006 was collected from the posteriormost region of the
dorsal frontoturbinal (indicated by the red arrowhead). Abbr: Ca.,
cartilage; ET I and II, ethmoturbinal I and II, respectively; Fr.,
frontal bone; FT, frontoturbinal; LSC, lamina semicircularis; Na., nasal
bone; Pa., palatine bone; Sp., presphenoid bone; Vo., vomer bone.

\emph{RNA expression}

The RNA expression analysis in the present study employed the same
methods as described in Kishida et al. (2019). The OR genes were queried
against the common minke whale genome assembly (GenBank accession
GCA\_000493695.1) (Yim et al., 2014) using the TBLASTN program in the
BLAST+ v. 2.6.0 package (Camacho et al., 2009) with a cut-off E-value of
1 \selectlanguage{ngerman}× 10\selectlanguage{english}\textsuperscript{-5}. Deduced amino acid sequences of all intact
ORs of green anole (\emph{Anolis carolinensis} ) and western clawed frog
(\emph{Xenopus tropicalis} ), cow (\emph{Bos tauros} ), and mouse
identified by Niimura (2009b) and Niimura et al. (2014) were used as
queries. Each obtained sequence was searched against the GenBank protein
database using the BLASTX program and if its best hit did not correspond
to an OR, it was discarded. A sequence was deemed a non-functional
pseudogene if it contained premature stop codons and/or frame shifts, or
it lacked five or more consecutive amino acids, including a
transmembrane domain. Sequences interrupted by contig-gaps, though not
classified as pseudogenes, were labelled as `truncated'.

To search for the OMP gene, FATE
(\emph{https://github.com/Hikoyu/FATE/blob/master/fate.pl}) was employed
to search the common minke whale genome assembly (GenBank accession
GCA\_000493695.1) using the annotated query sequence NW\_006728793.1,
identified as OMP, from GenBank Refseq GCF\_000493695.1. The resulting
single sequence found in GCA\_000493695.1 was used for OMP gene mapping.

Total RNA was extracted from the nasal chamber mucosa (R-001, R-006,
R-046), olfactory bulb (R-008) and external skin (R-214, R-215) using
the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the
manufacturer's guidelines. The olfactory bulb and external skin samples
served as negative controls. The extracted RNA was used to construct
paired-end sequencing libraries using the TruSeq Stranded mRNA LT Sample
Prep Kit (Illumina Inc., San Diego, CA, USA). Subsequently, Illumina
NovaSeq platform (2x101 bp) was employed for sequencing, generating
RNA-seq reads with the following sizes: R-001, 5.89 G bp; R-006, 4.57 G
bp; R-046, 5.54 G bp; R-008, 5.37 G bp; R-214, 5.35 G bp; R-215, 6.03 G
bp. Low-quality sequences and adapters were removed using Trimmomatic v.
0.38 (Bolger et al., 2014) with the following parameters:
ILLUMINACLIP:TruSeq3-PE-2.fa: 2:30:10, LEADING:20, TRAILING: 20,
SLIDINGWINDOW: 4:20, and MINLEN: 36. HISAT2 (Kim et al., 2015) v. 2.1.0
with default parameters was used to map trimmed RNA-seq reads to the
conspecific genome assembly. The expression levels of genes were
quantified using fragments per kilobase of exon per million mapped
fragments (FPKM) values with Cufflinks (Trapnell et al., 2010; Roberts
et al., 2011) v. 2.2.1 after removing duplicated reads. The expression
level of ORs was calculated by dividing it by that of \selectlanguage{greek}β-\selectlanguage{english}actin and
multiplying it by 100, giving expression-percentage.

The annotated intact ORs of common minke whales were incorporated into a
phylogenetic tree. The nucleotide sequences were aligned using MAFFT
(Kuraku et al., 2013; Katoh et al., 2019) v. 7, and a suitable model was
determined by Modeltest, IQtree (Trifinopoulos et al., 2016) v. 1.6.12.
Subsequently, the TIM3+F+I+G4 model was selected, and the phylogenetic
tree was constructed using RAxML-ng (Kozlov et al., 2019) v. 0.9.0 with
the root set as Class-1 ORs.

\emph{Gross observation}

The bony block obtained from 16NPCK-M009 was bisected along the medial
wall to reveal the inner side of the nasal chamber. The cross-section of
the lamina semicircularis and ethmoturbinals I and II were observed, as
shown in Fig. 2. Upon removal of the skeletal tissues of the medial
nasal turbinals (ethmoturbinal I and II), the laterally positioned
turbinals became visible. The anatomical nomenclature used to identify
the nasal turbinals was based on Ito et al. (2022) and supplemented by
relevant references (Klima, 1999; Maier and Ruf, 2014).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2} The parasagittal section of the left nasal chamber of
16NPCK-M009. (a) Medial view of the nasal chamber and the posteriorly
adjoining olfactory bulb chamber. (b) A closer medial view of the nasal
chamber following the removal of ethmoturbinals I and II. The lateral
region of the nasal chamber becomes visible. The regions previously
occupied by the ethmoturbinals I and II are indicated by circles
outlined with white dashed lines. The left and bottom sides correspond
to the posterior and ventral directions, respectively. Abbr: ET I and
II, ethmoturbinal I and II, respectively; ET I p, ethmoturbinal I
posterior part; FT, frontoturbinal; LSC, lamina semicircularis; OB,
olfactory bulb.

\emph{Histology staining}

The epithelial specimens underwent standard histological techniques. The
samples were dehydrated using a series of ethanol concentrations and
then cleared with xylene. Following infiltration and embedding in
paraffin wax (melting point 56-58), they were sectioned using a rotary
microtome (PR-50; Yamato Kohki Industrial Co., Ltd, Saitama, Japan) into
slices measuring 4-6 \selectlanguage{greek}μ\selectlanguage{english}m. These sections were spread out on warm water,
carefully transferred onto glass slides, and subsequently dried in an
incubator at 60 for 30 minutes. During the staining process, they were
sequentially immersed in deparaffinization solution, hydration medium,
and stain solution. Following mounting, the epithelial samples were
examined and photographed using a digital microscope (VHX-7000; Keyence,
Osaka, Japan). We evaluated whether these epithelial tissues qualified
as olfactory epithelium based on the criteria proposed by Farnkopf et
al. (2022): epithelium constructed of basal cells, supporting cells, and
olfactory sensory neurons; the presence of Bowman's glands; the absence
of goblet cells; and the distance between the apical surface and the
nuclei of the supporting cells.

\section*{4. RESULTS}

{\label{results}}

\emph{Expression of the olfactory receptor genes}

Transcriptome sequencing using RNA-seq was performed on the nasal mucosa
of the putative olfactory organ (R-001, R-006, R-046), the olfactory
bulb (R-008), and the external skin (R-214 and R-215) of common minke
whales. The FPKM values of \selectlanguage{greek}β-\selectlanguage{english}actin were as follows: R-001, 983; R-006,
285; R-046, 549; R-008, 371; R-214, 161; R-215, 143 (Table 2),
indicating successful RNA extraction from all the samples. In this
study, 81 intact, 12 pseudo, and 266 truncated genes were annotated. The
maximum expression level (as a percentage of FPKM for \selectlanguage{greek}β-\selectlanguage{english}actin) of intact
ORs in negative controls (the olfactory bulb, R-008, and external skin,
R-214 and R-215) was 0.75 in R-008 (Fig. 3). The average expression of
intact ORs with non-zero expression across all samples was 1.014. This
expression level was used as the criterion for significant expression in
this study.

Among the nasal mucosa samples, R-006 exhibit the presence of 22
significantly expressed intact ORs, while five of them were also
expressed in R-001. OMP, on the other hand, was exclusively expressed in
R-006, with an expression level greater than 1.014. In contrast, R-046,
obtained from the anterior portion of the nasal chamber, did not
demonstrate significant expression of ORs or OMP. Out of the 81
annotated intact ORs, all the genes expressed in the nasal mucosa
samples belonged to Class-2 OR. Although four copies of intact Class-1
OR were annotated, none of them were expressed in the six samples
investigated. The significantly expressed intact ORs did not form a
cluster in the phylogenetic tree (Fig. 4). It is worth noting that some
pseudogenes were expressed in the nasal mucosa, and one pseudogene was
exclusively expressed in the external skin, as per our investigation.

\emph{Gross observation}

Three distinct nasal turbinals were observed in the medial aspect of the
nasal chamber in all the investigated animals (Figs. 1 and 2). These
structures were identified as the lamina semicircularis, ethmoturbinals
I and II. The configuration of these nasal turbinals closely resembled
the 'ethmoturbinates/olfactory recess' described in a common minke whale
(Figs. 6 and 7, Godfrey et al., 2013), as well as in bowhead whales
(Fig. 7, Farnkopf et al. 2022).

The cranial bony block from 16NPCK-M009 exhibited a complicated
morphology of nasal turbinals situated laterally to the ethmoturbinals I
and II (Fig. 2). the ethmoturbinal I was positioned just anterior to the
olfactory bulb chamber and was accompanied ventrolaterally by the
posterior part of ethmoturbinal I (Fig. 2b, ET I p). Laterally to the
ethmoturbinal I, the dorsal region of the nasal chamber was occupied by
two slender frontoturbinals (Fig. 2b, FT), which corresponded to the
same area from which R-006 was obtained (Fig. 1c and d). A delicate
turbinal structure, known as the interterbinal, was located lateral to
the ethmoturbinal II (Fig. 2b, IT).

\textbf{Table 2 FPKM and calculated expression percentage.}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{R-001}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{R-006}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{R-046}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{R-008}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{R-214}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{R-215}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{β actin (FPKM)}\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
983\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
285\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
549\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
371\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
161\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
143\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{OMP}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
FPKM\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9.510\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
21.101\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Expression*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.966\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
7.378\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Average of expressing intact OR genes}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Average of expressing intact OR genes}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Average of expressing intact OR genes}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Average of expressing intact OR genes}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
FPKM\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3.640 \selectlanguage{ngerman}±4.946\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.537 \selectlanguage{ngerman}±8.735\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.244 \selectlanguage{ngerman}±0.372\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.543 \selectlanguage{ngerman}±0.876\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.171 \selectlanguage{ngerman}±0.098\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.112 \selectlanguage{ngerman}±0.054\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Expression*\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.370 \selectlanguage{ngerman}±0.503\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
2.286 \selectlanguage{ngerman}±3.054\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.045 \selectlanguage{ngerman}±0.068\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.146 \selectlanguage{ngerman}±0.236\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.106 \selectlanguage{ngerman}±0.061\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.078 \selectlanguage{ngerman}±0.038\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

* Expression (\%) was calculated as: FPKM of a gene was divided by that
of \selectlanguage{greek}β \selectlanguage{english}actin, then multiplied by 100.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} Expression of ORs and OMP. R-001, R-006, and R-046
correspond to the nasal mucosae of the ethmoturbinal II, frontoturbinal,
and anterior nasal chamber, respectively. R-008 originated from the
olfactory bulb, and both R-214 and R-215 were obtained from the external
skin. The average expressions of intact ORs exhibiting non-zero
expression were as follows: R-001, 0.370\selectlanguage{ngerman}±0.503; R-006, 2.286±3.054;
R-046, 0.045±0.068; R-008, 0.146±0.236; R-214, 0.106±0.061; R-215,
0.078±0.038.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4} Phylogenetic tree constructed of intact ORs. Genes
labeled in blue were determined to have significant expression in R-006.
The root of the tree established based on class-1 ORs. Nodes displaying
bootstrap values of 100, 90-99, 70-80 are denoted by blue squares,
yellow circles, and open circles outlined in purple, respectively, while
nodes with bootstrap values below 70 are not marked.

\emph{Histological staining}

Histological examination of the mucosae (H-001, H-009, H-046) revealed
that they were all covered with pseudostratified columnar epithelium and
contained glands with visible orifices (Table 3, and Fig. 5). These
glands appeared to be serous glands, and the presence of goblet cells
was not observed. In all three mucosae, a nucleus-free zone was observed
between the apical surface of the epithelium and the nuclei of
supporting cells; however, this phenomenon was particularly evident in
H-009. Additionally, peripheral nerves were distributed beneath the
lamina propria in H-009 (Fig. 5).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5} Mucosal samples. The photographs in the far-left,
center, and far-right columns were taken at magnifications of \selectlanguage{ngerman}×100
(scale bars indicating 500 \selectlanguage{greek}µ\selectlanguage{english}m), \selectlanguage{ngerman}×200 (100 \selectlanguage{greek}µ\selectlanguage{english}m), and \selectlanguage{ngerman}×700 (100 \selectlanguage{greek}µ\selectlanguage{english}m),
respectively. The nasal cavity is located at the top of each image.
Abbr: G, glands; N, peripheral nerve; V, blood vessel.

\begin{CJK}{UTF8}{gbsn}\textbf{Table 3　Results of RNA-seq and histological observation of
mucosal samples.}\end{CJK}\selectlanguage{english}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Description of Specimen\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Specimen number\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
OR gene expression\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
OMP gene expression\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Pseudostratified columnar epithelium\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Bowman's grands\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Absence of goblet cells\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Nucleus free zone\strut
\end{minipage} & \begin{minipage}[b]{0.08\columnwidth}\raggedright\strut
Peripheral nerve\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Ethmoturbinal II\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
H-001 R-001\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
low\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
low\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
N\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Frontoturbinal\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
H-009 R-006\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Anterior portion\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
H-046 R-046\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
No\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
N\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Y\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
N\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\section*{5. DISCUSSION}

{\label{discussion}}

This study has confirmed the specific expression of 22 copies of ORs in
the bottom region of the nasal chamber in common minke whales. The ORs
were predominantly expressed in the posterior portion of the nasal
chamber, facing towards the olfactory bulb, with higher expression
levels observed in the frontoturbinal region (R-006) and moderately in
ethmoturbinal II (R-001). It has been previously reported that these
nasal turbinals in typical mammals are covered with the olfactory
epithelium (Van Valkenburgh et al., 2014a). Furthermore, the expression
level of OMP was found to be highest in sample R-006, indicating that
the posterior portion of the frontoturbinal region can be identified as
olfactory region. Although the OMP expression in R-001 (0.966) did not
meet the criteria of 1.014, it was higher than expression levels
observed in R-046, R-008, R-214 and R-215 which showed no expression of
OMP. Moreover, the expression of ORs in R-001 was distinctly higher than
in R-046, R-008, R-214 and R-215, although lower than R-006 (Table 3).
These findings lead us to hypothesize that the sampled region R-001,
specifically the posterior medial surface of ethmoturbinal II (Fig. 1b),
encompasses both respiratory and olfactory areas. It is commonly
reported that respiratory and olfactory mucosae are distributed in a
mosaic-like pattern, and this distribution may hold for cetaceans as
well.

In this study, we conducted microscopic examination of the ethmoturbinal
II (H-001), frontoturbinal (H-009), and anterior region of the nasal
chamber (H-046). These samples were assessed based on the histological
criteria proposed by Farnkopf et al. (2022), and were likely identified
as olfactory mucosa. However, it was the proximal region of the
frontoturbinal (R-006) that strongly suggested being the olfactory
epithelium based on RNA-seq data. Notably, the same region (H-009)
exhibited a rich abundance of peripheral nerves, indicating its high
sensitivity. On the other hand, within H-046, which was obtained from a
more anterior region of the nasal cavity, dense clusters of vessels with
thick walls were observed (Fig. 5). This region may serve as a
respiratory area where the vascular epithelium plays a role in
thermoregulation.

Although the expressed ORs did not form a distinct cluster in the
phylogenetic tree (Fig. 4), it should be noted that the present study
does not exclude the possibility of a concealed cluster consisting of
ORs expressed in unexplored regions. This is because the distribution of
ORs, which mediate odoriferous stimuli to the olfactory bulb, is not
uniform across the olfactory epithelium (Ressler et al., 1993; Vassar et
al., 1993; Marchand et al., 2004). Furthermore, the distribution pattern
of the olfactory mucosa within the nasal chamber varies among lineages;
(Smith et al., 2011; Ruf, 2014; Smith et al., 2014)(Smith et al., 2011;
Ruf, 2014; Smith et al., 2014; Ito et al., 2021). It is plausible that
common minke whales possess additional ORs that contribute to their
olfactory modality, and identifying such receptors would enhance our
understanding of the molecular mechanisms underlying cetacean olfaction.

To comprehend the expression pattern of ORs, a thorough anatomical
examination of the cetacean nasal chamber is indispensable. Our
observations unveiled the presence of additional nasal turbinals
positioned laterally to ethmoturbinals I and II (Fig. 2b, FT and IT),
which have been scarcely documented in cetaceans. However, due to the
dearth of comprehensive anatomical data encompassing the entire nasal
chamber, precise determination of the exact locations from which H-046
and R-046 were obtained remained elusive. Nasal turbinals can be broadly
categorized into olfactory and respiratory turbinals, both of which play
a pivotal role in unraveling the aquatic adaptation of these creatures
(Van Valkenburgh et al., 2011; Van Valkenburgh et al., 2014a; Martinez
et al., 2020). In the nasal chamber of common minke whales, the anterior
segment from which H-046 and R-046 were harvested is inferred to
represent the respiratory region. It is conceivable that baleen whales
also possess respiratory turbinals, and investigating this structure is
warranted in future studies. Although the present study primarily
focused on the posterior region adjacent to the cribriform plate, it is
crucial to obtain a comprehensive understanding of the entire
architecture of this intricate labyrinth (Van Valkenburgh et al.,
2014b).

Fundamental anatomical data can function as an Atlas during dissection
process. As highlighted by Farnkopf et al. (2022), the extraction of the
nasal chamber from the cranial bones of large whales presents a
formidable challenge due to its remarkable dimensions, thickness, and
robust structure. The identification of the nasal turbinals from
sectional images can prove challenging, as their appearance exhibits
variations with slight deviations in cutting angles. Moreover, the
intricate nature of this structure impedes the efficient penetration of
fixation solutions into the tissues. To surmount these sampling
difficulties, the comprehensive description of the entire nasal chamber
using CT imaging becomes imperative. Subsequently, the determination of
olfactory epithelium distribution emerges as the next crucial step. The
locations of the olfactory epithelium might be predicted based on the
surface coloration of the nasal mucosae. While the majority of the nasal
mucosa in common minke whales displayed a pale pink hue, R-006, which
exhibited distinct olfactory characteristics, was obtained from an
epithelium displaying a yellowish appearance. A previous study
tentatively proposed the possibility of yellow pigmentation in the
olfactory epithelium of bowhead whales (Farnkopf et al., 2022).

The entirety of the expressed ORs identified in this study exclusively
belong to class-2 (Fig.4). While there remained significant room for
exploration, the probability of class-1 OR expression in common minke
whales is presumed to be minimal even when thoroughly screening the
entire lining mucosa of the nasal chamber. This presumption is rooted in
the observed morphology, which unveiled the absence of the dorsal domain
of the olfactory bulb in common minke whales. Class-1 olfactory
receptors typically transmit input to the dorsal domain of the olfactory
bulb. Consequently, it is anticipated that class-1 ORs would not be
expressed in the nasal mucosa of an animal lacking this specific region
of the olfactory bulb. It has been documented that bowhead whales have
also lost the dorsal domain of the olfactory bulb (Thewissen et al.,
2011; Kishida et al., 2015b), and the investigated common minke whales
exhibit a dorsoventrally flattened olfactory bulb, akin to that of
bowhead whales.

This study strongly suggests that the class-1 ORs do not partake in
olfactory reception in baleen whales. In mice, class-1 ORs receive
stimuli that trigger avoidance behaviors and project them into the
dorsal domain of the olfactory bulb (Kobayakawa et al., 2007). Hence,
our findings imply the loss of the typical avoidance response to
specific odorants, such as predators or putrefying substances, in
whales.

Considering that Sirenians, another lineage of fully aquatic mammals,
still retain their olfactory organ and possess a large repertoire of ORs
(Barboza and Larkin, 2020; Han et al., 2022; Christmas et al., 2023),
the diminished olfactory ability of baleen whales cannot be sorely
attributed to their aquatic lifestyle, which restricts continuous
respiration. One possible explanation for this lies in the necessity for
discerning ingested foods. Anatomically, the esophagus of cetaceans is
separated from the airway (Tyack and Miller, 2002), preventing them from
detecting smells emanating from the oral cavity. Genomic research has
revealed a degeneration of sense of taste, the other form of
chemoreception, in cetaceans (Feng et al., 2014; Zhu et al., 2014;
Kishida et al., 2015a; Policalpo et al., 2023). Furthermore, there is no
anatomical description of taste buds in the tongues of baleen whales
(Sonntag, 1922; Ogawa and Shida, 1950; Tarpley, 1985). Consequently, it
follows that baleen whales do not rely on chemosensory modalities to
evaluate food within their mouths, which may have contributed to the
reduction of their olfactory capabilities.

Our histological and genomic investigations suggest that residual
class-2 ORs are responsible for olfaction in baleen whales.
Specifically, the present study indicates that baleen whales are able to
receive preferred odors, such as those associated with prey and
potential mating partners. This research establishes a foundational link
between the anatomy and genome of baleen whales, and further
investigations hold the potential to illuminate the natural social and
behavioral biology of these animals, thereby aiding in their
conservation.

\subsection*{Acknowledgements}

{\label{acknowledgements}}

We are grateful to late Hideyoshi Yoshida for helping a lot during
sample collection. We thank Saeko Kumagai and Hiroto Murase for
collecting the skin samples; Kai Ito for identifying nasal turbinals;
Masahide Hasobe for staining; Takashi Sakamoto for preparing surgical
instruments; Andrew Shedlock for improving the manuscript; and our Lab
members. This study was financially supported by the Moritani
Scholarship Foundation, Grant for Basic Science Research Projects from
The Sumitomo Foundation (grant no. 180641), KAKENHI (Grant-in-Aid for
JSPS fellows, grant no. 23KJ0956). Computations were partially performed
on the NIG supercomputer at ROIS National Institute of Genetics.

\subsection*{DATA AVAILABILITY
STATEMENT}

{\label{data-availability-statement}}

All sequence reads were deposited in the DDBJ Sequence Read Archive
under BioProject accession no. PRJDB16252.

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\includegraphics[width=0.70\columnwidth]{figures/Figure3/Figure3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure4/Figure4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure5-new/Figure5-new}

\end{center}
\end{figure}

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