A Comparative Anatomical and Histological Study of the Olfactory Organ of Amphidromous and Land-locked Sweet Fish, Plecoglossus altivelis(Osmeriformes, Osmeridae), in Seomjingang River System, Korea

섬진강 수계에 출현하는 양측회유성과 육봉형 은어 Plecoglossus altivelis 후각기관의 해부 및 조직학적 구조 비교 연구

Abstract

This study was carried out the anatomical and histological investigation of the olfactory organ on a land-locked and an amphidromous Plecoglossus altivelis in Deokam-ri, Shinpyeong-myeon, Imsil-gun, Jeollabuk-do and Daeduk-ri, Gojeon-myeon, Hadong-gun, Gyeongsangnam-do, August, 2021, Korea. The external morphology of the two types of P. altivelis was indistinguishable, featuring a pair of the elliptical anterior nostrils, the semicircular posterior nostrils, and the nasal flaps on the snout. The olfactory organ consisted of 20 to 22 olfactory lamellae forming a rosette structure in the nasal chamber. The secondary folds appeared on the lamellae. The histological results were also uniform. The sensory epithelium was a continuous type, consisting of olfactory receptor neurons, supporting cells, basal cells, cilia, and an unidentified cell. The non-sensory epithelium found only in the terminal part of lamellae, consist of stratified epithelial cells, basal cells, and mucous cells. For the olfactory organ between a land-lock type and an amphidromous type, no difference was observed in external and histological structure between amphidromous and land-locked type. However, it is considered that P. altivelis has higher olfactory sensitivity than other anadromous fish due to more number of olfactory lamellae and the presence of the secondary folds.

2021년 8월에 섬진강 수계인 전라북도 임실군 신평면 덕암리와 경상남도 하동군 고정면 대덕리에 서식하는 육봉형과 양측회유성 은어의 후각기관의 형태와 조직학적 특징을 확인하기 위해 실체현미경, 광학현미경, 주사전자현미경을 이용하였다. 그 결과, 두 유형의 은어의 외부 형태는 유사하였으며 타원형의 전비공, 반원형의 전비공과 비경계판이 한 쌍을 이루었다. 20~22개의 후판이 로제트 구조를 형성하였으며 이차 주름이 후판의 표면에서 확인되었다. 조직학적 결과에서도 두 유형은 서로 유사하였다. 감각상피는 연속분포 유형이었으며 후감각뉴런, 지지세포, 기저세포, 섬모, 미확인 세포가 확인되었으며 비감각상피에서는 편평상피세포, 기저세포, 점액세포가 확인되었다. 본 연구에서 육봉형 은어와 양측회유성 은어의 해부·조직학적 차이는 확인할 수 없었으나 다른 소하성 어류보다 많은 후판의 개수와 이차 주름의 존재로 인해 다른 소하성 어류보다 후각 민감도가 높을 것으로 사료된다.

INTRODUCTION

The chemical sensory system in fishes generally includes two senses, gustation (taste) and olfaction (smell) to perceive chemical stimuli surrounding its environment(Olivares and Schmachtenberg, 2019). The olfactory senses in a paired olfactory organs located on its head, with numerous olfactory lamellae having olfactory rosette and epithelium (Døving, 1998; Hansen and Eckart, 1998), and the morphology and location of olfactory organ in fish implies notable variation depending on systematic groups and ecological habitats(Zeiske et al., 1992). Olfaction is essential for performing ecological behaviors, such as discriminating kin (Brown and Brown, 1996), identifying odor-based conspecifics and reproduction (Stacey, 2003), and enabling larval settlement (Bilodeau and Hay, 2022). Studies on olfaction have been extended to other different behavioral patterns: reproduction, schooling, defense, migration, agonistic behavior, and territorial behavior (Hara, 1986; Kasumyan, 2004) from a wide range of fish. In particular, there are more reports on migratory fish that imprinting the amino acid of the natal stream during embryonic and larval stages (Kimmel et al., 2023) or juvenile (Ueda, 2019): anadromous Salmonids (Thommesen, 1983; Kudo et al., 2009), catadromous Anguillidae (Sola et al., 1993; Atta, 2013), and land-locked few fishes Lethenteron reissneri(Kim and Park, 2020) and Oncorhynchus nerka (Rheinsmith et al., 2023).

The ayu sweetfish, Plecoglossus altivelis is an amphidromous fish. The adults spawn in the freshwater streams in the autumn, and then newly hatched larvae migrate to downstream through shore for wintering and feeding zooplankton. The juvenile fish drift upstream and feed phytoplankton during the summer(Kim and Park, 2002). Unlike amphidromous type, the ayu has another type so called a land-locked type. The land-locked ayu lives in freshwater stream which are connected to lakes not estuary (Ko et al., 2007; Shrimpton, 2012). This type fish found in some Far East Asia lakes: Okjeongho Lake, Andongho Lake, Jinyangho Lake, Hapcheonho Lake (Ko et al., 2007; Chae et al., 2019) in Korea and Biwa lake in Japan (Yagishita and Kume, 2021). Based on this unique ecological character, some comparative studies have been conducted on both types of ayu. For example, the amphidromous ayu prefer higher temperatures while land-locked ayu showed aggression at lower temperatures during territorial behavior(Shibuya and Taniguchi, 1995), additionally the amphidromous ayu revealed advanced sperm motility than land-locked ayu when thawed (Yokoi et al., 2009). Also, the number of tooth plates in amphidromous ayu is slightly more than land-locked type (Kaewsank et al., 2001). However, comparative studies between amphidromous and land-locked types on the olfactory organ in P. altivelis have been unperformed. The anatomical and histological investigation of the olfactory organ can be used to understand the fish’s habitats and ecology (Yopak et al., 2015; Ferrando et al., 2016). Therefore, based on the anatomical and histological approaches, the aim is to clarify the difference between the two types of the olfactory organ of P. altivelis with ecological traits.

MATERIALS AND METHODS

1. Specimen collection

The adult amphidromous (n=10) and land-locked (n=10) P. altivelis used in this study were collected in August, 2021 using a casting net (5×5 mm in mesh) in Daeduk-ri, Gojeon-myeon, Hadong-gun, Gyeongsangnam-do, South Korea, 35°00ʹ23ʺN 127°48ʹ49ʺE and in Deokam-ri, Shinpyeong-myeon, Imsil-gun, Jeonllabuk-do, South Korea, 35°37ʹ54ʺN 127°12ʹ53ʺE(Fig. 1). 15 specimens were fixed in 10% formalin for external morphology and light microscopy and 5 specimens were fixed in 2.5% glutaraldehyde solution for scanning electron microscopy.

Fig. 1. The collection sites of two types of Plecoglossus altivelis. A: the site of the land-locked type in Deokam-ri, Shinpyeong-myeon, Imsil-gun, Jeonllabuk-do, South Korea where weir (deviant bar) was constructed. B: the site of the amphidromous type in Daeduk-ri, Gojeon-myeon, Hadong-gun, Gyeongsangnam-do, South Korea. The scale bar indicates 3 km.

2. External and histological study

To investigate the external and anatomical structure of the olfactory organ, the snout fixed in a 10% formalin solution were dissected using a surgical blade under a stereo microscope (Stemi DV4, Carl Zeiss, Germany) and were photographed by a digital camera (TG3, Olympus, Japan).

For conducting light microscopic examine, the snout dissected with surgical blade for took out the olfactory organ. Then the olfactory organ fixed with 10% formalin washed for 24 hours in tap water. The olfactory organ was dehydrated with gradually increasing ethanol concentration, cleared with xylene, and embedded in paraffin wax (Paraplast, Oxford, UK) for 24 hours. The paraffin block was sectioned in 5 μm and then deparaffinized. The sectioned tissues stained with hematoxylin-eosin (H-E) stain, Masson’s trichrome stain (Masson, 1929), and alcian blue-periodic acid Schiff (AB-PAS, pH 2.5) (Yamabayashi, 1987). All of them were observed by light microscope (LE REL 4.4, Carl Zeiss, Germany).

To perform scanning electron microscopy, the snouts were fixed in 2.5% glutaraldehyde solution post-fixed in 1% osmium tetroxide in 0.1 mol/L phosphate buffer, and dehydrated in a graded series of ethanol solutions of increasing concentration, and using tert-butyl alcohol in the final step. They were freeze-dried (VFD-21S, Vacuum Device Co., Ltd., Japan), coated with osmium (HPC-1SW; Vacuum Device Co., Ltd.), and filmed by scanning electron microscope (SEM; SUPRA40VP, Carl Zeiss, Germany).

RESULTS

The anatomical and histological structures of both the land-locked type and amphidromous type in P. altivelis were identical.

1. External and anatomical structure

The olfactory organs of the both types were located on the snout and consisted of two pairs, the elliptical anterior nostril and the semicircular posterior nostril, and the semicircular nasal flap. The nasal flap existed between the anterior nostril and the posterior nostril (Fig. 2A). The bilaterally symmetrical rosette structure was beneath the nasal chamber, consisting of several tongue-like lamellae that radiated from the raphe (Fig. 2B). The number of the lamellae in two types was between 20 and 22.

Fig. 2. The photographs of the external olfactory organ of Plecoglossus altivelis(the land-locked type). A: the elliptical anterior nostril(AN), the semicircular posterior nostril(PN) and nasal flap (NF). The blue arrows show the flow of water; B: the olfactory lamellae consisting of rosettes structure in the nasal chamber (NC). The lamellae (L) are exteneded from raphe (R).

2. Light microscopy

The olfactory lamellae for the two types were separated by the sensory epithelium(SE) and non-sensory epithelium (NSE) (Figs. 3 and 4). They contained the connective tissue, the basement membrane, and the olfactory epithelium from the basal to the apical(Figs. 3C~G, 4C~G).

Fig. 3. The histology of the olfactory organ of the amphidromous Plecoglossus altivelis stained with Hematoxylin-Eosin (A, C, D, F), Masson’s trichrome (B, E), and alcian blue (pH 2.5) - periodic acid Schiff (G). A and B: the olfactory lamellae (L) are extended from raphe (R). The olfactory epithelium consisting of sensory epithelium(SE, blue oval) and non-sensory epithelium(NSE, red square) in the inner wall(IW) of the nasal chamber (NC); C~E: the SE having olfactory receptor neurons(ORNs), supporting cells(SCs), basal cells(BCs), cilia (c), blood capillaries(Ca), blood cells (BlC), fibroblasts(Fbl), and unidentified cells(UCs). The basement membrane (BM) and connective tissue (CT) are below olfactory epithelium. The arrows indicate secondary folds; F and G: the NSE having stratified epithelial cells(SECs), BC, mucous cells(arrowhead), and mucous film (MF). The scale bars indicate 500 μm(A, B) and 50 μm(C~G).

Fig. 4. The histology of the olfactory organ of the land-locked Plecoglossus altivelis stained with Hematoxylin-Eosin (A, C, F), Masson’s trichrome (B, D, E), and alcian blue (pH 2.5) - periodic acid Schiff (G). A and B: the olfactory lamellae are (L) extended from raphe (R). The olfactory epithelium consisting of sensory epithelium(SE, blue oval) and non-sensory epithelium(NSE, red square) in the inner wall(IW) of the nasal chamber(NC). The arrows indicate secondary folds; C~E: the SE having olfactory receptor neurons(ORNs), supporting cells(SCs), basal cells(BCs), blood cells(BlC), cilia (c), blood capillaries(Ca), fibroblasts(Fbl), and unidentified cells(UCs). The basement membrane (BM) and connective tissue (CT) are below olfactory epithelium. The arrows indicate secondary folds; F and G: the NSE having stratified epithelial cells(SECs), BC, mucous cells(arrowhead), and mucous film(MF). The scale bars indicate 500 μm(A, B) and 50 μm(C~G).

The SE had secondary folds and showed a continuous type that was distributed from the raphe to the tip of the lamellae, while the NSE was only positioned on the tip of the lamellae and the inner wall of the nasal chamber(Figs. 3A, B, 4A, B). The SE consisted of a pseudostratified columnar layer having olfactory receptor neurons(ORNs), supporting cells, basal cells, unidentified cells, cilia, blood capillaries, blood cells, and fibroblasts. The cilia mainly covered SE (Figs. 3C, D, 4D, E). The ORNs were bipolar cells, whose dendrite extended to the nasal chamber and the basal membrane. The nuclei of the ORNs were purple in H-E and dark purple in Masson’s trichrome, with cytoplasms of scarlet and pale purple color in each stain (Figs. 3C, D, 4C~E). The basal cells were situated above the basement membrane and those were elliptical spheres. Their nuclei at the basal part were round or oval, and they had cilia on the apical part. The cytoplasms were swollen and purple in H-E staining weaker than those of ORNs (Figs. 3C~E, 4C~E). The unidentified cells were situated in the upper part of the epithelium and typically distributed on the terminal part of lamellae. The cytoplasms were elongated and elliptical, and their nuclei were situated at the basal part. The nuclei were stained dark purple in H-E, and Masson’s trichrome, whereas its cytoplasms were stained red and scarlet in H-E and Masson’s trichrome (Figs. 3D, 4C~E). The cilia existed on the upper part of SE(Figs. 3C, D, 4C~E), not on the NSE. The blood capillaries beneath the secondary folds were seen in the connective tissue (Figs. 3C, D, G, 4C, D, G). The nuclei of blood cells were stained purple in H-E, Masson’s trichrome, and AB-PAS, while their cytoplasms were pink in H-E and AB-PAS, light purple in Masson’s trichrome (Figs. 3E, 4E). The connective tissue beneath the basement membrane included fibroblasts and blood capillaries. The fibroblasts were stained purple in H-E and Masson’s trichrome (Figs. 3C, D, F, 4C, F).

Having no secondary folds, ORNs, and supporting cells, the NSE had a stratified squamous layer unlike SE but showed a similar structure: the stratified epithelial layer cell, basal cell, blood capillary, blood cell, and fibroblast (Figs. 3F, G, 4F, G). Unlike the SE, interestingly, there were ellipsoid mucous cells with granules stained blue in AB-PAS (Figs. 3G, 4G).

3. Scanning electronic microscopy

With the secondary folds, the SE had the olfactory receptor neurons (ORNs), supporting cells, microvilli, and cilia in the two types (Fig. 5). 4 to 6 cilia in an average length of 0.4 μm were arranged along the knob of the ORN. The microvilli were dispersed compactly on the supporting cell (Fig. 5E, G). Meanwhile, only in the region of the NSE, microridge and mucous opening appeared (Fig. 5E, H). The microridge seemed to be a labyrinth structure (Fig. 5E, H), and contained the mucous opening as a tiny hole (Fig. 5H).

Fig. 5. Scanning electron micrographs of the olfactory organ of amphidromous(A, C, E, F) and land-locked (B, D, G, H) Plecoglossus altivelis. A and B: the olfactory lamellae (L) are extended from raphe (R) and they consisting of rosette structures; C and D: the L were uncinated structures that had secondary folds (arrows). The red circle indicates cilia; E: supporting cell (SC), olfactory receptor neuron (ORN), and cilia (red circle) were in the sensory epithelium (SE) and non-sensory epithelium (NSE) showed microridge (MR); F: SC (blue square) and cilia (red circle) near the NSE; G: the ORN had 4 to 6 cilia and its length was 0.4 μm; H: the mucous(arrowhead) and mucous hole (arrow) were on NSE. And microvilli(MV) were on SC. The scale bars indicate 10 μm (A, B), 500 μm (C, D), and 1 μm (E~H).

DISCUSSION

The location, number, diameter, and morphology of the nostrils in fish depend on the habitat characteristics of the species(Zeiske et al., 1992), and the nasal flap served to protect the inflow of water into the posterior nostril (Døving, 1998). The elliptical anterior nostril showed in anadromous Osmeridae Delta smelt Hypomesus transpacificus(Triana-Garcia et al., 2021). The semicircular posterior nostril in Cyprinid species were Labeo rohita (Burne, 1909), Rhodeus uyekii, Coreoleuciscus splendidus, and Microgobio yaluensis(Kim, 2018), while the semicircular anterior nostril with a nasal flap was observed in energetic fishes, it was useful in running water systems and proper to inhabit the rapid stream or the flowing river (Ojha and Kapoor, 1973). Hence, the nostril shape of both types are suitable for torrential flow. The number and arrangement of the olfactory lamellae forming rosettes in a narrow nasal chamber may be related to the olfactory sensitivity that could enhance the surface area of the olfactory epithelium (Kasumyan, 2004). The number of the lamellae was 18 in anadromous Oncorhynchus keta from the terminal part of the rostral basal region (Kudo et al., 2009), 13 to 14 in O. nerka (Rheinsmith et al., 2023), 14 to 16 in other anadromous fish Salmo trutta and 11 to 18 in other anadromous fish Hypomesus transpacificus(Moran et al., 1992; Triana-Garcia, 2021). The secondary folds found only in the SE was seen in other Osteoglossiformes(Dymek et al., 2021) and in some other Salmonid anadromous fish (Pfeiffer, 1963; Bertmar, 1972; Chen and Arratia, 1994), except for H. transpacificus that mentioned above. In Chondrichthyes (Ferrando et al., 2019), the secondary folds amplify the total surface area of olfactory lamellae and the consequently magnified surface area could potentially induce getting higher olfactory sensitivity. Based on the other studies, P. altivelis with 20 to 22 lamellae may have higher olfactory sensitivity than other anadromous fishes.

The olfactory epithelium in fishes was divided by a sensory epithelium (SE) and a non-sensory epithelium (NSE). As seen in our study, Hara (1994) reported that the SE contains olfactory receptor neurons and supporting cells, while the NSE lacks these two types of cells and instead contains stratified epithelial cells and mucous cells. But we saw unidentified cells, which weren’t watched by Hara. This cell had a pear-shaped cell, a rod-shaped granule, and a fibrous capsule then it hypothesized that its cytoplasm is acidic, based on this research. To identifies the cell property, histochemical investigation such as Crossmon trichrome (Crossmon, 1937) Mallory triple trichrome (Mallory, 1936), and Van Gieson (Van Gieson, 1889) would be demanded. Additionally, using a transmission electron microscope could contribute to understanding the ultrastructure (Abd-Elhafeez et al., 2020). The length of cilia on olfactory receptor neurons varies across species(Hansen and Zielinski, 2005) and as the cilia get longer, the chance of contact between the cilia and odorous molecules may increase (Hara, 1975). In our study, the length and number of cilia on olfactory receptor neurons in P. altivelis were 0.4 μm and 4 to 6 cilia, which it was shorter and lesser than Genus Acipenser larvae with 10 μm and 15 to 20 (Zeiske et al., 2003). Compared to other species, P. altivelis may have less chance to encounter chemical odorants.

The supporting cells served as a mechanical support on the epithelium, and the basal cells performed the role of cytological regeneration above the basal membrane (Hara, 1986; Carter et al., 2004). The cilia covering the apical part of the supporting cells on the surface of the sensory epithelium speculated that it would diffuse water on the olfactory epithelium to facilitate ventilation of olfactory stimuli (Døving, 1986). The mucous cell in the NSE had been known for helping osmosis·ion·acid-to-base regulation, gas exchange, and providing an innate immune mechanism(Shepard, 1994; Arasu et al., 2013). From the present study, the mucous cell was stained blue by AB-PAS, meaning an acid substance. The acid mucous secreted gel-like mucin may play roles in an antibacterial effect (Hasnain et al., 2013). It may imply that mucous substance secures the olfactory epithelium from pathogenic organisms in aquatic environments. In this investigation, the nostril of the amphidromous and land-locked P. altivelis possibly exhibit both types adapted to running water system. The number of olfactory lamellae is more than that of other anadromous fish, which infers higher olfactory sensitivity than those species. Shorter and fewer cilia on ORN induce less opportunity to meet chemical stimuli on the other hand, the number of olfactory lamellae and secondary folds perhaps supplements those features. Regrettably, no differences were identified between the olfactory organ of amphidromous and land-locked ayu but it could be utilized as fundamental data of other amphidromous fish, and it is supposed to be used as taxonomic resources.