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. 2022 Apr 18;241(3):809–819. doi: 10.1111/joa.13671

The vomeronasal system in semiaquatic beavers

Jumpei Tomiyasu 1,✉, Anna Korzekwa 1, Yusuke K Kawai 2, Christian A Robstad 3, Frank Rosell 3, Daisuke Kondoh 2
PMCID: PMC9358757  PMID: 35437747

Abstract

In contrast to the main olfactory system that detects volatile chemicals in the nasal air, the vomeronasal system can detect nonvolatile chemicals as well as volatiles. In the vomeronasal system, chemicals are perceived by the vomeronasal organ (VNO) projecting axons to the accessory olfactory bulb (AOB). Beavers (Castor spp.) are semiaquatic mammals that have developed chemical communication. It is possible that the beaver's anal gland secretions, nonvolatile and insoluble substances, may work as a messenger in the water and that beavers may detect the nonvolatile chemicals floating on the water surface via the VNO. The present study aimed to clarify the specificities of the beaver vomeronasal system by histologically and immunohistochemically analyzing the VNO and AOB of 12 Eurasian beavers (C. fiber). The VNO directly opened to the nasal cavity and was independent of a narrow nasopalatine duct connecting the oral and nasal cavities. The VNO comprised soft tissues including sensory and nonsensory epithelium, glands, a venous sinus, an artery, as well as cartilage inner, and bone outer enclosures. The AOB had distinct six layers, and anti‐G protein α‐i2 and α‐o subunits were, respectively, immunoreactive in rostral and caudal glomeruli layers indicating expressions of V1Rs and V2Rs. According to gene repertories analysis, the beavers had 23 and six intact V1R and V2R genes respectively. These findings suggested that beavers recognize volatile odorants and nonvolatile substances using the vomeronasal system. The beaver VNO was developed as well as in other rodents, and it had two specific morphological features, namely, disadvantaged contact with the oral cavity because of a tiny nasopalatine duct, and a double bone and cartilage envelope. Our results highlight the importance of the vomeronasal system in beaver chemical communication and support the possibility that beavers can detect chemicals floating on the water surface via the VNO.

Keywords: Castor, nonvolatile chemicals, rodents, semiaquatic mammals, vomeronasal system, water adaptation

The present study clarified morphological specificities of the vomeronasal system of beavers, semiaquatic mammals. The beaver VNO was developed as well as in other rodents, and it had two specific morphological features, namely, disadvantaged contact with the oral cavity because of a tiny nasopalatine duct, and a double bone and cartilage envelope. Our results highlight the importance of the vomeronasal system in beaver chemical communication.

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1. INTRODUCTION

Olfactory communication among mammals living in aquatic environments was generally regarded as less important (Meisami & Bhatnagar, 1998). This might be true for some aquatic mammals. For example, dwarf sperm whale (Kogia sima) and common dolphin (Delphinus delphis) lack the olfactory nervous system (Oelschläger et al., 2010), and sea lion (Zalophus californianus) has a much smaller size of the olfactory bulb relative to the brain size than general terrestrial mammals (Montie et al., 2009). In contrast, other aquatic mammals, such as the capybara (Hydrochoerus hydrochaeris) and the hippopotamus (Hexaprotodon liberiensis and Hippopotamus amphibius), have large, well‐developed olfactory bulbs, both main and accessory ones (Butti et al., 2014; Kondoh, Watanabe, et al., 2017; Torres et al., 2020), indicating the importance of olfactory communication. Thus, the olfactory ability might differ among aquatic mammals.

Some aquatic mammals seem to detect chemicals in their water environment in a unique way. For example, star‐nosed moles (Condylura cristata) and water shrews (Sorex palustris) perceive volatile chemicals even in the water (Catania, 2006). Volatile substances are transferred from the water to the nasal cavity via the air bubble that is emitted from the nose (Catania, 2006). On the other hand, the way of detection of nonvolatile chemicals, important as pheromones in laboratory rodents (Kimoto et al., 2005), in the water environment is still unknown.

The vomeronasal and main olfactory systems mediate the detection of chemicals (Wysocki, 1979). In contrast to the main olfactory system that detects volatile chemicals in the nasal air, the vomeronasal system can recognize nonvolatile pheromonal chemicals, in addition to volatiles, by direct contact (Brennan & Zufall, 2006). In the vomeronasal system, chemicals are received by peripheral sensory vomeronasal organs (VNOs) (McCotter, 1912) that are found in all mammals except some species in apes, bats, and aquatic mammals (Silva & Antunes, 2017). The mammalian VNO attached to the nasal septum is a blind‐ended tubular structure comprising a cartilage or bone enclosure and soft tissues including sensory and nonsensory epithelial, blood vessels, and glands (Halpern, 1987). The VNO in rodents directly opens into the nasal cavity (Vaccarezza et al., 1981), and chemical signals are pumped into the vomeronasal lumen by expansion and contraction of venous structures (Meredith, 1994; Meredith et al., 1980). The information received by the VNO is transmitted via vomeronasal nerves to the accessory olfactory bulbs (AOBs) (McCotter, 1912). The mammalian AOB is hemispherical and located on the dorsocaudal side of the main olfactory bulb (Meisami & Bhatnagar, 1998).

Chemical signals are detected in the rodent VNO by vomeronasal type 1 (V1R) and type 2 (V2R) receptors that are, respectively, coupled with the G protein subunits α‐i2 (Gαi2) and α‐o (Gαo) (Dulac & Axel, 1995; Herrada & Dulac, 1997; Matsunami & Buck, 1997; Ryba & Tirindelli, 1997). The properties of detectable chemicals and neuronal projection sites in the AOB differ between V1R and V2R. The sensory neuron expressing V1R, with high affinity for small volatile molecules, projects into the glomeruli of the rostral AOB, whereas the sensory neuron expressing V2R, with high affinity for large nonvolatile molecules, projects into the glomeruli of the caudal AOB (Jia & Halpern, 1996; Kimoto et al., 2005; Leinders‐Zufall et al., 2004; Peele et al., 2003; Shinohara et al., 1992; Sugai et al., 2006). Laboratory rodents have >100 and >50 intact V1R and V2R genes, respectively (Shi & Zhang, 2007), indicating that they detect many types of chemicals via V1R and V2R.

Beavers (Castor spp.; family Castoridae) are myomorph rodents that have become ecological specialists in semiaquatic environments (Mortensen et al., 2021) and they have uniquely adapted to aquatic life. For example, they have webbed hind feet, a flat tail that functions as a rudder in the water, and combing claws responsible for maintaining nonwettable fur (Bailey, 1923; Müller‐Schwarze & Sun, 2003). Beavers are social animals that have developed chemical communication using castoreum (an exudate from the castor sacs) and anal gland secretions (Campbell‐Palmer & Rosell, 2010; Rosell & Sundsdal, 2001). Some researchers pointed out the possibility that the anal gland secretions may work as a messenger in the water (Grønneberg & Lie, 1984; Rosell & Sundsdal, 2001). Because anal gland secretions are mostly nonvolatiles (Rosell & Sundsdal, 2001) and insoluble in water (Svendsen, 1978), it is possible that these chemicals may float on the water surface and spread to downstream areas (Rosell et al., 1998) and that other beavers may detect floating the nonvolatile chemicals via the VNO (Rosell & Pedersen, 1999). However, the features of the beaver vomeronasal system remain unknown. Morphological and genome analysis of the beaver vomeronasal system would improve our understanding of the mechanisms through which they detect nonvolatile chemicals of the water environment via the VNO. The present study aimed to clarify the characteristics and specificities of the beaver vomeronasal system by histologically and immunohistochemically analyzing the VNO and AOB of Eurasian beavers (C. fiber). We also analyzed the gene repertoires of V1Rs and V2Rs using the genome assemblies of the North American beaver (C. canadensis), as this is the only beaver species with genome information.

2. MATERIALS AND METHODS

2.1. Animals

We collected tissues from 12 wild Eurasian beavers harvested by professional hunters as nuisance control under the permission of the Regional Direction of Environment Protection in Warsaw (permission number: WPN‐I.6401.72.2020.AJC). Although approval from the Ethics Committee for Animal Experimentation is waived for collecting samples from dead animals in Poland, all procedures in this study followed the Institutional Regulations on the Management and Operation of Animal Experiments. Table 1 shows individual information (sex and age) about the 12 beavers, all of which were free of pathological findings.

TABLE 1.

Topographic, histological, and immunohistochemical analysis of 12 beavers

ID Sex Age a Topography Histology Immunohistochemistry
VNO VNO b AOB VNO AOB
1 Male Adult ✓ ✓
3 Male Adult ✓ ✓
6 Female Adult ✓ ✓
9 Female Adult ✓
10 Female Cub ✓ ✓
12 Female Adult ✓ ✓ ✓ ✓
14 Female Adult ✓ ✓
15 Female Juvenile ✓ ✓
16 Female Adult ✓ ✓
19 Female Juvenile ✓ ✓ ✓ ✓
20 Female Adult ✓ ✓ ✓
21 Female Cub ✓ ✓ ✓ ✓
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a

Age is classified as a cub, juvenile, or adult based on sexual organs, body weight, and total length.

b

All were stained with hematoxylin–eosin, and beavers 1, 3, and 6 were also stained with periodic acid‐Schiff and Alcian blue (pH 2.5). AOB, accessory olfactory bulb; VNO, vomeronasal organ.

2.2. Topography

Bones forming the lateral side of the nasal cavity in three beavers were removed using a saw to generate a lateral view of the VNO. The noses of five beavers were sawed transversely in series, and the VNO was analyzed using an SMZ1500 stereomicroscope (Nikon). The shape of the vomeronasal cartilage and bone was established based on a series of cross‐sections of the VNO.

2.3. Histology

The noses were fixed in Bouin fluid, decalcified in Plank–Rychlo solution, and then embedded in paraffin using standard procedures. The brains were removed from the heads, fixed with Bouin fluid, and embedded in paraffin. The nose and brain specimens were, respectively, cut transversely and sagittally into 5‐μm‐thick sections, deparaffinized and visualized by hematoxylin–eosin (HE), periodic acid‐Schiff (PAS), or Alcian blue (AB; pH 2.5) staining.

2.4. Antibodies

Primary antibodies used in this study were anti‐olfactory marker protein (OMP) (2.0 μg ml−1, sc‐67219; Santa Cruz Biotechnology Inc.), anti‐Gαi2 (2.5 μg ml−1, ab20392; Abcam), and anti‐Gαo (2.0 μg ml−1, sc‐387; Santa Cruz Biotechnology Inc.). sc‐67219 is a rabbit polyclonal antibody raised against full‐length of human OMP that has a high (90.24%) identity with the amino acid sequence of beaver OMP. Ab20392 and sc‐387 are rabbit polyclonal antibodies raised against human Gαi2 and rat Gαo that have 97.75% and 97.00% identity with the amino acid sequences of beaver Gαi2 and Gαo respectively. The secondary antibody was biotinylated goat polyclonal anti‐rabbit IgG (2.0 μg ml−1, BA‐1000; Vector Laboratories Inc.).

2.5. Immunohistochemistry

We immunohistochemically stained OMP, Gαi2, and Gαo to detect projected axons of neurons associated with differentiated olfaction and vomeronasal neurons expressing V1Rs and V2Rs, respectively, in the beaver AOB and VNO as described (Kondoh, Watanabe, et al., 2017). Deparaffinized 5‐μm‐thick sections were incubated with 0.3% H2O2 in methanol, followed by 3% normal goat serum. The sections were incubated at 4°C overnight with a primary antibody, followed by secondary antibody at room temperature for 60 min. Thereafter, the sections were incubated with avidin–biotin–peroxidase complex (PK‐6100; Vector Laboratories Inc.) for 30 min, then visualized by staining with 0.02% 3,3′‐diaminobenzidine tetrahydrochloride in Tris–HCl buffer containing 0.006% H2O2. Sections of the VNO were counterstained with hematoxylin. The sizes of areas stained by anti‐Gαi2 and anti‐Gαo in the AOB were evaluated at the point of maximum rostral–caudal length in sagittal AOB sections.

2.6. Genome assembly analysis

To identify V1R and V2R genes in the North American beaver, we performed TBLASTN searches with an e‐value of <1e‐6 as the identity threshold on RNA and pseudogene sequence database based on the beaver genome assembly (GCA_001984765.1). Query amino acid sequences (Data S1 and Data S2) were obtained by for searching “vomeronasal” or “Vmn” in the Ensembl database (release 104) for mice and humans (Homo sapiens). We excluded genes with different annotations from BLAST results and confirmed the gene classification by phylogenetic analysis. In this study, the genes were classified as intact genes including partial and full‐length pseudogenes, according to the classification in the gene database at the National Center for Biotechnology information (NCBI) (Brown et al., 2015).

3. RESULTS

3.1. Morphological features of the vomeronasal organ

The bilateral VNO was located around the inferior part of the nasal septum at the rostral area of the nasal cavity (Figure 1a, b). The vomeronasal ducts directly opened into the nasal cavity by a tiny hole at the rostral end (Figure 1b, c). The nasal and oral cavities were connected via a tiny nasopalatine duct (diameter ~ 150 μm), with a significantly widened nasal opening on the rostral–caudal axis that did not connect to the VNO (Figure 2).

FIGURE 1.

FIGURE 1

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Location of the vomeronasal organ (VNO) in beavers. (a) Right lateral view of head. (b) Right lateral view of the rostral nasal septum (dotted square in a). Bones covering the lateral region and right incisor were removed. Arrows, VNO at the basal region of the nasal septum. (c) Serial histological cross‐sections of an area in VNO that directly opens into the nasal cavity (dotted square in b), corresponding to the rostral end of Figure 3a. Arrowhead, the opening of VNO. C, vomeronasal cartilage; L, vomeronasal lumen; NC, nasal cavity. Scale bars: (a, b), 10 mm; (c) 1 mm

FIGURE 2.

FIGURE 2

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Location of beaver nasopalatine duct and VNO. (a) Schema of nasopalatine duct (red) and VNO (blue). Dotted area, incisor. (b–g) Serial stereomicroscopic images of the cross‐sectioned nasal region. Lines in panel (a) correspond to images b–g. (h–k) Representative, serial histological sections of the transected nasal region between d and g. Nasopalatine ducts that communicate between oral and nasal cavities, run for ~150 μm along the rostral–caudal axis (arrows in panel j). Scale bars: (a) 10 mm, (b, h) 0.5 mm

The soft tissue of the VNO was enclosed internally by cartilage and externally by bone (Figure 3). The vomeronasal cartilage covered the lateral, ventral, and medial parts of the soft tissue over essentially the entire length from the rostral to the caudal regions and the ventral and medial parts of the caudal end (Figure 3a, b). The left and right vomeronasal cartilages were partly fused (Figures 3d, 4a). A cross‐section of the middle region of the VNO revealed U‐shaped cartilage (Figure 3a, c and d). A bony capsule surrounded the lateral and ventral parts of the soft tissue and cartilage from the middle to the caudal regions (Figure 3a, b). An artery and a venous sinus ran laterally throughout the VNO (Figure 3a). The vomeronasal lumen was round at the rostral end, crescent at the rostral and middle regions, planiform at the caudal regions, and culminated in a blind end (Figure 3a).

FIGURE 3.

FIGURE 3

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Morphological features of the envelope, lumen, and blood vessels in beaver VNO. (a) Transverse sections of cartilage (gray), bone (brown), lumen (black), artery (red), and vein (blue) based on stereomicroscope findings (blood vessels were discriminated by microscopy). Left side is the lateral and upper dorsal regions. (b) Schema of cartilage (gray) and bone (brown) envelopes. The left side of the panel shows caudal and upper dorsal regions. (c) Stereomicroscopy image of VNO cross‐section in the middle region. (d) Histological image of dotted square in panel (c). A, artery; B, bone; C, cartilage; L, lumen; VS, venous sinus. Scale bars: (a, d) 1 mm, (b) 5 mm

FIGURE 4.

FIGURE 4

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Histological features of beaver VNO. (a) Cross‐sections of VNO. (b, c) High magnification of nonsensory epithelium (NSE) and sensory epithelium (SE) images. Arrowhead and arrow indicate basal cell and intraepithelial blood vessels respectively. A, artery; B, bone; C, cartilage; G, vomeronasal glands; RC, receptor cells; SC, supporting cells; V, vessels; VS, venous sinus. Scale bars: (a) 500 μm, (b, c) 20 μm

3.2. Histological features of the vomeronasal organ

The medial and lateral walls of the vomeronasal lumen were, respectively, covered by sensory and nonsensory epithelia (Figure 4a). A distinct venous sinus was located in the lateral part of the lamina propria and several vessels were located in the medial and ventral regions (Figure 4a, and Figure 5d and f). An artery was located dorsal to the venous sinus (Figure 4a and Figure 5e). Vomeronasal glands were located mainly and partially in the dorsal and lateral ventral regions respectively (Figures 4a and 5a). Secretory cells in the vomeronasal glands were stained positive for PAS and negative for AB (Figure 5a–c). The sensory and nonsensory epithelia in the VNO were pseudostratified (Figure 4b and c). The sensory epithelium consisted of supporting, receptor, and basal cells (Figure 4c). Receptor cells contained round nuclei arranged in four to five layers of cells in the middle region, supporting cells contained oval nuclei that were arranged in the apical region, and basal cells with irregular nuclei were scattered in the basal region (Figure 4c). In the sensory epithelium, intraepithelial blood vessels are located (Figure 4c). The nonsensory epithelium mainly comprised columnar and basal cells and a few goblet cells (Figure 4b).

FIGURE 5.

FIGURE 5

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Histological features of vomeronasal glands (a–c), venous sinus (d), artery (e), and vessels (f) in beaver VNO. Hematoxylin–eosin (a, d–f), periodic acid Schiff (b), and Alcian blue pH 2.5 (c) stains. Scale bars: (a–c) 200 μm, (d–f) 100 μm

3.3. Morphological and histological features of the accessory olfactory bulb

A large olfactory portion of the beaver brain included mature olfactory bulbs (Figure 6). A hemispheric AOB was located at the dorsocaudal area in the olfactory bulb (Figures 6b and 7a). A prominent structure in the AOB consisted of a vomeronasal nerve, glomerular, external plexiform, mitral/tufted cell, internal plexiform, and granular layers (Figure 7b).

FIGURE 6.

FIGURE 6

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Beaver olfactory bulbs (OBs). (a) Ventral view of the brain shows the olfactory pathway. (b) Lateral view of the OB. * Accessory olfactory bulb (AOB). (c) Medial view of the OB. LOT, lateral the olfactory tract; OB, olfactory bulb; pi, piriform lobe. Scale bars: (a), 10 mm; (b), 0.5 mm

FIGURE 7.

FIGURE 7

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Histological features of beaver AOB. (a) Sagittal section of OB. Square corresponds to the magnified image of AOB in panel b. (b) Prominent structure in AOB consists of the vomeronasal nerve (VNL), glomerular (GL), external plexiform (EPL), mitral/tufted cell (M/TCL), internal plexiform (IPL), and granular (GrL) layers. LOT, lateral olfactory tract. MOB, main olfactory bulb. Scale bars: (a) 500 μm, (b) 200 μm

3.4. Immunohistochemical features of the accessory olfactory bulb and the vomeronasal organ

Olfactory marker protein was expressed in the vomeronasal nerve and glomerular layers throughout the AOB (Figure 8a), but the AOB areas expressing Gαi2 and Gαo differed (Figure 8b, c). The glomerular layer of the rostral AOB was intensely, and somewhat positively stained for anti‐Gαi2 and anti‐Gαo respectively (Figure 8b, c). A weakly positive reaction to anti‐Gαo has been identified in the anti‐Gαi2‐positive glomerular layer in many mammals, including mice (Shinohara et al., 1992). This might be caused by dendrites derived from underlying anti‐Gαo‐positive layers (Shinohara et al., 1992). On the other hand, the glomerular layer of the caudal AOB was positive for anti‐Gαo but negative for anti‐Gαi2. The areas of the rostral and caudal parts of the AOB, respectively, labeled with anti‐Gαi2 and anti‐Gαo were similar (Figure 8b, c). As an atypical pattern, some glomeruli showed a different immunoreaction; a few glomeruli in the rostral AOB were positive for anti‐Gαo (Figure 8c), and a few other glomeruli in the caudal AOB were positive for anti‐Gαi2 (Figure 8b). Receptor cells in the sensory epithelium of the VNO were positive for anti‐Gαi2 and anti‐Gαo (Figure 8d, e). The staining intensity for anti‐Gαi2 and anti‐Gαo seemed strong in the apical and basal regions respectively (Figure 8d, e).

FIGURE 8.

FIGURE 8

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Immunohistochemical staining of beaver AOB (a–c) and sensory epithelium in the VNO (d, e). Immunoreactivity against anti‐olfactory marker protein (OMP) (a), anti‐G protein subunit α‐i2 (Gi2) (b, d), and anti‐G protein subunit α‐o (Go) (c, e). Anti‐Gi2 and anti‐Go are mainly positive in nerves and glomeruli in rostral and caudal regions, respectively (b, c), and similarly sized rostral and caudal glomerular layers are distinguished by dotted lines (b, c). In contrast, some glomeruli demonstrate a different immunoreactive pattern; a few caudal glomeruli (single arrowheads) are positive for anti‐Gi2 (negative for anti‐Go), and a few other rostral glomeruli (double arrowheads) are positive for anti‐Go (negative for anti‐Gi2) (b, c). Scale bar: (a–c) 200 μm, (d, e) 50 μm

3.5. Individual variations of the vomeronasal organ and the accessory olfactory bulb

Although the VNO and the AOB in individuals of different ages and sexes were checked in the present study (Table 1), no marked difference between them was found.

3.6. Gene repertoires of vomeronasal receptors

North American beavers had 23 intact genes and 44 pseudogenes within the V1R family, and six intact genes and 40 pseudogenes in the V2R family (Tables S1 and S2).

4. DISCUSSION

To our knowledge, this is the first study to reveal the detailed structural characteristics of the beaver VNO and AOB. We also characterized beaver vomeronasal receptor genes, by analyzing G protein α‐subunits using immunohistochemistry and genome assembly.

Beavers had a thick sensory epithelium in the VNO, and its histological features were similar to those in rats (Vaccarezza et al., 1981), mice, and hamsters (Mendoza, 1993). Secretory cells in vomeronasal glands, which secrete fluids that are probably associated with pheromone reception (Khew‐Goodall et al., 1991), were PAS‐positive and AB‐negative, like those of other rodents (Kondoh et al., 2020). Moreover, the beaver AOB had a distinct glomerular layer that expressed OMP, a marker of differentiated olfactory receptor neurons. These results suggested that the vomeronasal system of the beaver is functionally developed for chemical detection as in other rodents.

The beaver VNO opened into the nasal cavity, and a tiny nasopalatine duct opened around the caudal part of the VNO. Mammals have two types of VNO openings, a nasopalatine (incisive) duct or a direct path into the nasal cavity. Whether animals have one or the other might be associated with phylogenic factors. The VNO merges with the incisive duct and opens to the nasal and oral cavities of mammals in the order Artiodactyla, namely sheep (Ovis aries) (Kratzing, 1971) and cows (Bos taurus) (Salazar et al., 2008), and Carnivora such as cats (Felis catus) (Salazar et al., 1996), dogs (Canis lupus familiaris) (Salazar et al., 2013), and brown bears (Ursus arctos) (Tomiyasu et al., 2017). In contrast, the VNO opens to the nasal cavity, and the nasopalatine duct does not connect with the VNO in Rodentia, such as hamsters (Mesocricetus auratus) (Mendoza, 1993), rats (Vaccarezza et al., 1981), and capybaras (Torres et al., 2020), and in Lagomorpha, such as rabbits (Oryctolagus cuniculus) (Villamayor et al., 2018). Thus, the type of the beaver VNO opening is the same as that in rodents and lagomorphs. The nasopalatine duct might channel pheromones from the mouth into the nose of rabbits (Villamayor et al., 2018). However, considering the small, immature nasopalatine duct in the beaver, chemical signals might be received mainly from the nasal cavity.

Soft tissue is surrounded only by cartilage in most of the mammals that have been investigated to date (minks [Mustela lutreola], cats, dogs, pigs [Sus domesticus], cows, horses [Equus ferus caballus] (Salazar et al., 1995), bears (Tomiyasu et al., 2017), giraffes (Giraffa camelopardalis) (Kondoh, Nakamura, et al., 2017), and hedgehogs (Atelerix albiventris) (Kondoh et al., 2021)), or only by bone in rodents like mice and hamsters (Mendoza, 1993). The vomeronasal envelope developmentally changes during the early growing stage in rats (Salazar & Sánchez‐Quinteiro, 2011); the soft tissue is enclosed by cartilage in very young individuals (aged 3 days), by bones and cartilage in young adults (aged 12–18 days), and only by bone in adults. On the other hand, the soft tissue is enclosed by bone and cartilage in rabbits (Villamayor et al., 2018). We found here that beavers of all ages had a bone and cartilage enclosure, and thus the beaver VNO enclosure type is an exception among rodents, as it resembles the rabbit type.

The developed vein structure in beaver VNO was confirmed, as well as in rats (Vaccarezza et al., 1981), mice and hamsters (Mendoza, 1993), and rabbits (Villamayor et al., 2018). The mature veins of rodents contract and relax to pump chemicals into the vomeronasal lumen (Meredith, 1994; Meredith et al., 1980). Thus, the mature vein structure in the beaver VNO supports that beavers may receive nonvolatile chemical signals mainly by directly attaching their noses (Herr, 2005) and invoking the chemicals by the pumping function of vessels.

Our immunohistochemical research demonstrated that receptor cells in the sensory epithelium in VNO and segregated areas in AOB were positive for anti‐Gαi2 and anti‐Gαo, which we interpreted as the expression of both types of vomeronasal receptors in the beaver. Gene analysis also confirmed that beavers express V1R and V2R. Rodents such as mice (Jia & Halpern, 1996) and rats (Shinohara et al., 1992), and lagomorphs such as rabbits (Villamayor et al., 2020) express V1R‐ and V2R‐neurons that are, respectively, positive for anti‐Gαi2 and anti‐Gαo, whereas other mammals such as goats (Capra hircus) (Takigami et al., 2000), dogs (Salazar et al., 2013), cats (Salazar & Sánchez‐Quinteiro, 2011), and hippopotami (Kondoh, Watanabe, et al., 2017) express only V1R‐neurons. Considering the difference in detectable molecular weights between V1R and V2R (Kimoto et al., 2005; Leinders‐Zufall et al., 2004; Peele et al., 2003; Sugai et al., 2006), beavers could receive a variety of properties of chemicals regardless of volatility. Our results might be consistent with the findings that the major chemical compounds of castoreum and anal gland secretions are, respectively, volatile and nonvolatile (Rosell & Sundsdal, 2001). Moreover, the existence of V2R in beaver vomeronasal system may support the possibility that beavers can detect nonvolatile chemicals floating on the water surface using the VNO.

In beavers, areas of the rostral and caudal parts of the AOB, respectively, labeled with anti‐Gαi2 (V1R subdomains) and anti‐Gαo (V2R subdomains) were similar, like those in laboratory myomorph mice (Jia & Halpern, 1996) and rats (Shinohara et al., 1992). In contrast, the terrestrial degus (Octodon degus) have a larger rostral AOB than the caudal AOB (large V1R subdomain) (Fernández‐Aburto et al., 2020; Suárez & Mpodozis, 2009) and the semiaquatic capybaras have the larger caudal AOB than the rostral AOB (large V2R subdomain) (Suárez et al., 2011), probably because this asymmetry of AOB subdomains may reflect ecological traits of animals (Suárez & Mpodozis, 2009). Although we could not establish why beavers had a similar size of AOB subdomains, despite being semiaquatic rodents like capybaras, asymmetric AOB subdomains might be specific to a hystricomorph rodent phylogeny. The similar size of AOB subdomains of beavers may suggest that volatile and nonvolatile odors are equally important in the lives of beavers.

Surprisingly, our genome assembly findings revealed that beavers have fewer intact V1R (23) and V2R (6) genes than mice (V1R, 187; V2R, 70) and rats (V1R, 106; V2R, 59) (Shi & Zhang, 2007). This might mean that the beaver vomeronasal system detects a smaller variety of chemicals than mice and rats. However, as described above, the vomeronasal system of beavers has developed like that in other rodents, indicating that beavers can recognize limited substances with high sensitivity that are essential to communicate with their surroundings and other beavers.

The present study revealed detailed features of the beaver vomeronasal system. The beaver VNO was developed as well as in other rodents, but it had two specific morphological features, namely, disadvantaged contact with the oral cavity because of a tiny nasopalatine duct, and a double bone and cartilage envelope. The AOB receives a similar amount of V1R‐ and V2R‐neuronal projections; thus beavers might use volatile and nonvolatile odors in their aquatic habitats. Our results highlight the importance of the vomeronasal system in beaver chemical communication and support the possibility of detection of nonvolatile chemicals floating on the water surface via the VNO (Rosell & Pedersen, 1999).

AUTHOR CONTRIBUTIONS

J.T., F.R., and D.K. conceptualized the study. J.T., A.K., C.R., and F.R. collected specimens. J.T. and D.K. conducted histological and immunohistochemical analyses. Y.K.K. analyzed gene repertoires. J.T., Y.K.K., and D.K. wrote the initial draft of the manuscript and all authors reviewed, edited, and approved the final version of the manuscript.

Supporting information

Table S1.

Click here for additional data file. (116.2KB, zip)

ACKNOWLEDGMENTS

We thank Mr. Hubert Miszczuk and Mr. Rafał Dubełek for their help with collecting samples in the field. We thank Dr. Katarzyna Jankowska, Dr. Izabela Wocławek‐Potocka, Dr. Krzysztof Witek, and Dr. Marek Bogacki for assistance with histological analyses. We are grateful to Dr. Anna Szóstek‐Mioduchowska, Dr. Dariusz Jan Skarżyński, Dr. Anna D. Kononiuk, and Dr. Angelika Kotlarczyk for continuously supporting ongoing beaver research. This work was partially supported by grants for the internship program of the Society for Reproductive Biology (Towarzystwo Biologii Rozrodu) in Poland and by a MINATURA (2020/04/X/NZ9/01711) grant from the Polish National Science Center. The authors have no conflicts of interest associated with this manuscript to declare.

Tomiyasu, J. , Korzekwa, A. , Kawai, Y.K. , Robstad, C.A. , Rosell, F. & Kondoh, D. (2022) The vomeronasal system in semiaquatic beavers. Journal of Anatomy, 241, 809–819. Available from: 10.1111/joa.13671

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1.

Click here for additional data file. (116.2KB, zip)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Journal of Anatomy are provided here courtesy of Anatomical Society of Great Britain and Ireland

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