1. 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 Lévy, 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 (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
(Mus musculus ) or rats (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 (Orcinus rca ) breathe no more
frequently than every 8 minutes, and deep-diving species such as sperm
whales (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 (Balaenoptera musculus )
typically exhibit breathing intervals of approximately 4 minutes (Miller
and Roos, 2018). Humpback whales (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 (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 (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 (Globicephala melas ) and
bottlenose dolphins (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.