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.