5. 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.

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.

DATA AVAILABILITY STATEMENT

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