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ORIGINAL ARTICLE
Year : 2014  |  Volume : 2  |  Issue : 3  |  Page : 182-195

Light- and electron-microscopic studies of olfactory organ of Red-tail shark, Epalzeorhynchos bicolor (Teleostei: Cyprinidae)


Department of Anatomy and Histology, Faculty of Vet. Medicine, Assuit University, 71526, Egypt

Date of Web Publication6-Feb-2018

Correspondence Address:
Doaa M Mokhtar
Department of Anatomy and Histology, Faculty of Vet. Medicine, Assuit University, 71526
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.1016/j.jmau.2014.05.003

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  Abstract 


Olfaction plays a major role in various life activities of fish. The present study describes for the first time the gross morphology, histology, histochemistry, surface architecture and ultrastructure of the olfactory epithelium of Red-tail shark. It possessed a pair olfactory rosette, connected to the external by two nasal openings. The olfactory rosette was oval in shape, consisted of 45,48 lamellae. The lateral surface of olfactory lamella is covered with sensory epithelium, whereas the non-sensory area restricted mainly at the margin of the lamellae. Sensory epithelium contained ciliated, microvillous olfactory receptor cells (ORCs), crypt and rodlet cells. In addition to the presence of basal and microvillous supporting cells. The non-sensory epithelial cells were consisted of rodlet cells and stratified epithelium. The later was divided into ciliated and non-ciliated cells. Neuromasts were found in the apical third of the lamellae. Histochemistry of the olfactory epithelium revealed presence of PAS-positive granules in cytoplasm of rodlet cells were, while the ruptured ones stained strongly with PAS, AB and Sudan black B. The apical portions of supporting cells were stained with PAS and AB. Grimelus Silver method showed positive reaction of ciliated, microvillous ORCs, crypt and rodlet cells. Red tail shark seemed to have an acute sense of smell and depend mainly on the olfactory organ in detection of food and this study is important for future studies on behavior of this species. The functional significance of all olfactory epithelial cells was discussed with special references to stages and role of rodlet cells.

Keywords: Olfactory epithelium, Epalzeorhynchos bicolor, Histochemistry, Histoarchitecture, Ultrastructure


How to cite this article:
Mokhtar DM, Abd-Elhafeez HH. Light- and electron-microscopic studies of olfactory organ of Red-tail shark, Epalzeorhynchos bicolor (Teleostei: Cyprinidae). J Microsc Ultrastruct 2014;2:182-95

How to cite this URL:
Mokhtar DM, Abd-Elhafeez HH. Light- and electron-microscopic studies of olfactory organ of Red-tail shark, Epalzeorhynchos bicolor (Teleostei: Cyprinidae). J Microsc Ultrastruct [serial online] 2014 [cited 2020 Apr 4];2:182-95. Available from: http://www.jmau.org/text.asp?2014/2/3/182/224882




  1. Introduction Top


Fish are living in aquatic environment that usually devoid of light, but usually have highly developed chemosensory system. Olfaction in fish is of special interest because the olfactory epithelium act as chemical receptors for not only sense of smell, but they are also mediating other vital processes such as orientation, feeding, reproduction as well as conveying information about the surrounding environment [1],[2]. In fish, the water with dissolved chemicals pass from nostrils to the olfactory organ, consequently the olfactory receptor neurons are directly exposed to water contaminants. Therefore, fish olfactory mucosa due to its external nature is a target for environmental studies as the fish depend on it in many life activities. The highly developed sense of all vertebrates is olfaction in fishes [3].

In fish, the olfactory organ possessed a considerable variation that reflecting different developmental strategies and ecological habits. Many reports were made on histology [4],[5], SEM [6],[7],[8],[9],[10],[11],[12] TEM [13],[14] of the olfactory organs of many teleostean fish. These previous reports show variation in shape, size and lamellar arrangement as well as different arrangement of sensory and non-sensory areas. The olfactory sensory epithelium possessed either olfactory receptor cells (ORC) bearing microvilli or cilia that are common in teleosts and occur in different proportions according to species [15],[16]. Recently, special subtypes of neurons namely crypt and rod cells were recorded in some fish species [10],[17]. While the non-sensory epithelium of most fish species is consisted of stratified epithelium and mucus cells.

Red tail shark (Epalzeorhynchos bicolor) is one of fresh water fish, belongs to family Cyprinidae that originates from the streams and waterways of Thailand. It is a bottom feeder scavenger fish with an under-turned mouth that fed on decaying organic matter and algae [18]. Many studies were made on the olfactory organs of Cyprinidae [4],[19],[20]. However, there are no details about the basic structure of the olfactory rosette of Red-tail shark and distribution of their epithelium.

The present study aimed to describe the structural organization of the olfactory epithelium of Red-tail shark (E. bicolor); a popular freshwater tropical aquarium fish in Egypt by histological, histochemical analysis, transmission and scanning electron microscope focusing mainly on the morphology of the olfactory cells and arrangement of sensory and non-sensory epithelium.


  2. Materials and methods Top


Healthy mature fish of Red-tail shark were commercially purchased from ornamental shop in Assuit city, Egypt and brought to laboratory. Fish were measured and deeply anaesthetized with benzocaine (4 mg/L). Fish were ranging from 7 to 10 cm in standard body length. The whole fish were immersed in mixture of 20 mL of 2.5% glutaraldehyde and 80 mL sodium phosphate buffer. Olfactory rosettes were carefully dissected from 20 Red-tail shark at the floor of nasal cavity under a stereomicroscope.

2.1. Histological analysis

Specimens were immediately fixed in Bouin’s fluid for 20 h. The fixed materials were dehydrated in an ascending series of ethanol, cleared in methyl benzoate and embedded in paraffin wax. The embedding time was no more than 8 h. Serial longitudinal and transverse sections were obtained at 4–7 μm. For obtaining good view of raphe and olfactory lamellae on both sides of raphe, the rosette should be embed in the direction observed in [Figure 1]F.
Figure 1: General appearance of olfactory rosette. (A) Red-tail shark with black body and red colored tail showing nostrils (arrowhead) on the dorsolateral side. (B) General view of the head under stereomicroscope showing anterior nostrils (star), posterior one (arrowhead) and nasal flap (F). (Magn. 1.6 × 10). (C) The olfactory rosette (OR, arrowhead) under stereomicroscope was oval and found in the dorsolateral side of the head anterior to the eye. (Magn. 2.5 × 10). (D) SEM showing the olfactory rosette with 45–48 olfactory lamellae (OL, arrow head) radiating from central median raphe (R). Notice the connection between the capsule and lamellae (connection, arrowhead).

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2.2. Histochemical analysis

The fixed specimens in Bouin’s fluid were examined for neutral and acidic mucous by Periodic Acid-Schiff (PAS) [21] and Combined PAS-Alcian Blue stain (pH 2.5) [22]. Sudan Black B [23] was used for detection of lipid. Grimelus Silver impregnation method was used to identify the olfactory receptor cells [24].

2.3. For semithin sections and TEM preparations

Small specimens of olfactory rosettes were fixed in a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.3 for 4 h at 4 °C. They were washed in the same buffer used and then post-fixed in 1% osmic acid in 0.1 M Na-cacodylate buffer for further 2 h at room temperature. The samples were then dehydrated in ethanol and embedded in Araldite-Epon mixture. Semithin sections (1 μm in thickness) were cut and stained with Toluidine blue. Ultrathin sections, obtained by a Reichert ultra-microtome, were stained with uranyl acetate and lead citrate and examined with a Philips EM 400 electron microscope.

2.4. SEM preparation

The head of fish after opening the outer covering of the skin and exposed the two olfactory rosettes washed by 0.1 M Na-cacodylate buffer. Then they were fixed in a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.3 for 4 h at 4 °C.Thereafter, they were washed in the same buffer used and post-fixed in 1% osmic acid in 0.1 M Na-cacodylate buffer for further 2 h at room temperature. The samples were then dehydrated by aceton followed by isoamyl acetate and then subjected to critical point drying method with a polaron apparatus. Finally, they were coated with gold and observed with JEOL scanning electron microscope (JSM – 5400 LV) at KV 10.


  3. Results Top


3.1. Morphology of the olfactory organ

Red-tail shark had gray to black body color with red colored tail and shark-like dorsal fin. The nostrils located dorsolaterally in the snout, anterior to the eye [Figure 1]A. It possessed a pair olfactory organ connected to the external environment by two nasal incurrent and excurrent opening separated by skin flap [Figure 1]B. The olfactory organ consisted of right and left olfactory rosettes, located in olfactory chambers just under the flap of skin [Figure 1]C. The olfactory rosette was oval in shape, measuring 0.8–1.0 mm in diameter and comprised of 45–48 olfactory lamellae that radiated from spindle-shaped central median raphe, the outer margin of lamellae was attached to the wall of the olfactory chamber by long connection [Figure 1]D.

3.2. Histology of olfactory epithelium

The olfactory rosette was consisted of several olfactory lamellae radiating from a median raphe. The epithelium of the connection between the wall of the olfactory chamber and olfactory lamellae was consisted of stratified epithelial cells with goblet cells [Figure 2]A and [Figure 2]B. The olfactory lamellae were of unequal thickness; they were wide at the center and tapered at the end. Each lamella was consisted of two layers of epithelium separated by connective tissue core. The central connective tissue core was separated from olfactory epithelium by basement membrane and contained connective tissue fibers, fibroblasts, nerve fibers and blood vessels [Figure 2]C.
Figure 2: Semithin section of the olfactory epithelium stained with Toluidine blue. (A) The connection between the median raphe and wall of olfactory chamber consisted of stratified epithelial cells (SEC) and goblet cells (arrowhead). (B) The olfactory rosette consisted of many olfactory lamellae (OL) radiating from median raphe (R). Notice the distribution of the non-sensory epithelium (arrowhead) at the end of median raphe. (C) The lamella consisted of 2 layers of epithelium above the basement membrane (BM) separated by central core (CC). The sensory epithelium comprising the major part includes microvillous receptor cells (MRC), ciliated receptor cells (CRC), crypt cells, rodlet cells (RC, arrowheads). (D) The median raphe (R) consisted of stratified epithelial cells (2 stars), ciliated cells (arrowhead), rodlet cells (RC). (E) The apical third of olfactory lamellae contain taste bud (TB, 2 arrowheads) and ciliated receptor cells (CRC, arrowhead) and crypt cells (arrowhead). Notice presence of supporting cells (stars) and basal cells (BC).

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The olfactory epithelium that covered the lamella was mixture of sensory and non-sensory areas. The non-sensory epithelium was observed at the basal part of lamellae at the end of median raphe and consisted of stratified epithelium with prominent centrally located nuclei [Figure 2]B, while the sensory epithelium was observed on both side of lamallae. The sensory epithelium was lined with ciliated, microvillous olfactory receptor cells (ORCs), rodlet and crypt cells. The supporting cells (SCs) were located between the receptor cells [Figure 2]C.

ORCs were observed at different level of epithelium with a round cell body. Ciliated ORCs were provided by many cilia that rose above the epithelial surface and their nucleus were rounded in shape and situated deep in the epithelium. While, microvillous ORCs exhibited broad apical end with many microvillous and their nuclei were lactated more superficially in the epithelium than the ciliated ORCs [Figure 2]C. The olfactory epithelium had the tip contained many ciliated receptor cells than the middle region [Figure 2]E. Olfactory crypt cells were oval to egg-shaped neurons with oval dark nucleus and light staining cytoplasm. They located in the upper third of apical epithelium and surrounded by scarcely stained region [Figure 2]C and [Figure 2]E.

The interesting feature in this study was the identification of several stages of rodlet cells that frequently observed along the length of the olfactory lamellae in both sensory and non-sensory olfactory epithelium [Figure 2]C. Immature rodlet cells were found near the basement membrane of lamellae; the developing rodlet in the middle layer and mature rodlet were located at the surface. Immature rodlet cells were observed immediately next to the basement membrane of olfactory epithelium. They were large polyhedral in shape with eccentric vesicular nucleus [Figure 2]C. Mature rodlet cells consisted of pear-shaped cell with thick deeply stained capsule and oval basally vesicular nucleus. Their cytoplasm was filled with characteristic club shape inclusions, called rodlets. Rodlet cells showing secretory activity and some cells were observed releasing their content to the interlamellar space. Ruptured rodlet cells were concentrated in the median raphe, connection between the raphe and lamellae, the basal and the tip of lamallae. The final stage of these cells with deeply stained flattened nucleus suggested releasing their contents by holocrine mode of secretion [Figure 2]C and [Figure 2]D.

The supporting cells (SC) were elliptical to columnar in shape with prominent centrally situated oval basophilic nucleus [Figure 2]E. The basal cells were small rounded or pear shaped with a prominent round large central nucleus, situated at the basal part of the epithelium [Figure 2]E. The neuromast or taste bud was demonstrated in the cranial part of the lamella. It composed of supporting cells surrounding a central cluster of sensory hair cells [Figure 2]E.

3.3. Histochemistry of olfactory epithelium

The epithelium of olfactory lamellae was separated from the central core by PAS-positive basement membrane and AB-positive lamina reticularis of basal lamina [Figure 3]A and [Figure 3]F. The olfactory epithelium was covered by mucous layer that gave positive reaction with PAS and AB [Figure 3]B and [Figure 3]D. The cytoplasm of rodlet cells were filled with neutral mucopolysaccharides granules that stained strongly with PAS [Figure 3]A and [Figure 3]D. Moreover, the ruptured rodlet cells at the surface contained neutral and acidic mucopolysaccharides, which stained strongly with PAS and AB [Figure 3]D and [Figure 3]F. The apical portions of supporting cells contained both neutral and acidic mucopolysaccharides that stained with PAS and AB [Figure 3]D and [Figure 3]F.
Figure 3: Histochemical analysis of olfactory epithelial cells. (A and B) PAS-positive mucus layer (arrowhead) and granules of rodlet cells (stars). Notice the presence of basal cells (BC, arrowheads) on the PAS positive basement membrane (BM, arrowhead). In the basal part of the olfactory epithelium, large number of wandering lymphoid cells (L) was observed. (C) Sudan black B stain showing positive reaction of some contents of rodlet cells. (D) The mucus layer (arrowhead) covering the olfactory lamellae stained positive with AB. Rodlet cells (**) appear pink near from the basement membrane and less stained cells at the apical surface (RC, arrowhead). (arrowheads). (E) The olfactory fold showing positive reaction of microvillous receptor cells (*), ciliated receptor cells (**), crypt cells (2 arrowheads) to silver stain. Note the presence of positive granules of Rodlet cells (RC, arrowheads). (F) Olfactory fold showing positive reaction of mucus layer (arrowhead) and different stages of rodlet cells (arrowheads) to PAS and AB. Notice AB-positive reticular lamina (LR, arrowhead).

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Sudan Black B [Figure 3]C revealed presence of lipid in the mature or final stage of rodlet cells at the surface of the olfactory epithelium. Grimelus Silver method showed positive reaction of ciliated, microvillous olfactory receptor cells and crypt cells. We also observed positive reaction of granules in rodlet cells to silver stain [Figure 3]E.

3.4. SEM of olfactory epithelium

The olfactory organ was consisted of a pair of olfactory rosette. The olfactory rosette of Red-tail shark was provided with 45–48 leaf-like olfactory lamellae radiating from a central raphe. The size and shape of the lamellae varied according to their position in olfactory rosette. The large lamellae were found near the center and the small ones were present near the olfactory capsule. Each lamella was connected to the wall of olfactory chamber by long connection [Figure 4]A, [Figure 4]B, [Figure 4]C.
Figure 4: SEM of the general overview of olfactory rosette and median raphe. (A) General view showing a pair olfactory organ that consisted of right and left olfactory rosettes, located in olfactory chambers (arrow heads). (B and C) Higher magnifications of the olfactory rosette from A showing olfactory lamellae (OL) and midline raphe (R) and connection between the raphe and the wall of olfactory chamber (stars). (D–F) The epithelium of the connection between the median raphe with olfactory lamellae was consisted of stratified epithelial cells (SEC) with long cilia (arrowheads) and rodlet cells (star). (G) General view of the surface of the median raphe (R) showing numerous ciliated cells. (H and I) Higher magnification of the boxed area in G showing that epithelium of median raphe consisted of stratified epithelium with fingerprint-like microridges (SEC), ciliated non-sensory epithelial cells (cilia) and rodlet cells (RC, arrowheads). (J–L) Different magnification showing the connection between median raphe and wall of the olfactory chamber. It contained non-sensory stratified epithelium with fingerprint-like microridges (SEC) and ciliated epithelial cells (cilia).

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The epithelium of the connection between the median raphe with olfactory lamellae was consisted of stratified epithelial cells (SEC) with long cilia and rodlet cells [Figure 4]D, [Figure 4]E, [Figure 4]F. The median raphe was covered by stratified epithelial cells with their apical surface provided by labyrinth pattern of microridges. In addition to the presence of rodlet cells and ciliated non-sensory cells [Figure 4]G, [Figure 4]H, [Figure 4]I. The connection between the lamellae and the chamber showed also stratified epithelial cells with apical labyrinth pattern of microridges [Figure 4]J, [Figure 4]K, [Figure 4]L.

The olfactory epithelium was separated into broad sensory area and narrow non-sensory areas. The lateral surface of olfactory lamella was covered with sensory epithelium, whereas the non-sensory area was restricted mainly at the margin of the lamellae [Figure 5]A, [Figure 5]B, [Figure 5]C. Sensory epithelium contained ciliated, microvillous olfactory receptor cells, crypt and rodlet cells. Ciliated olfactory receptor cells were dominant over the microvillous receptor cells [Figure 5]D, [Figure 5]E, [Figure 5]F. The receptor cells were identified according to the shape of apical dendrites process that extended above the surface into ciliated and microvillous. Ciliated receptor cells were provided by tuft of long cilia that rose above the epithelial surface. Some ciliated cells possessed a long dendretic process that extended above the surface of the olfactory epithelium forming olfactory knob. These cilia appeared as rosette-like around the olfactory knob. The microvillous cells were provided by minute microvilli [Figure 5]G, [Figure 5]H, [Figure 5]I. The receptor cells were enclosed with supporting cells, which their broad apical surfaces and provided with many short microvilli [Figure 5]H. Between these cells, rodlet cells were observed [Figure 6]D. Taste buds were demonstrated at the anterior end of lamellae [Figure 5]J, [Figure 5]K, [Figure 5]L.
Figure 5: SEM of the distribution of the sensory region. (A–C) The sensory epithelium (SE) distributed on the lateral surfaces of olfactory lamellae, while the non-sensory one (NSE, arrowheads) was distributed on the margin of the lamellae. Notice the lamellae radiating from raphe (R). (D) Sensory epithelium consisted mainly of ciliated receptor cells (CRC, arrowhead) and microvillous receptor cells (MRC, star). (E and F) Higher magnification of sensory epithelium showing ciliated receptor cells (CRC, arrowheads), microvillous receptor cells (MRC, stars) and rodlet cells (RC, arrowhead). (G) lower magnification of sensory epithelium showing ciliated receptor cells (two arrowheads), microvillous receptor cells (arrowhead). (H and I) Higher magnification of boxed area on G showing the rosette-like surface of ciliated receptor cells (two arrowheads) arranged around olfactory knob and microvillous cells (**). Note the presence of microvillous supporting cells (SC, arrowhead). (J) Lower magnification showing the anterior part of olfactory lamellae with taste buds. (K and L) Higher magnification of the boxed area in J showing taste buds (TB, arrowheads).

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The non-sensory olfactory epithelium was restricted to the margin of the lamellae [Figure 6]A, [Figure 6]B, [Figure 6]C. The non-sensory epithelial cells exhibited broad polygonal apical surface of the stratified epithelium, which divided into ciliated and non-ciliated cells [Figure 6]E and [Figure 6]F. The ciliated cells were provided with long cilia, while the non-ciliated ones were free from cilia and exhibited finger print-like microridges on their broad polygonal apical surface [Figure 6]E and [Figure 6]F. The zone of transition between the sensory and non-sensory epithelium was consisted of stratified epithelial cells with apical fingerprint-like microridges and few microvillous and ciliated receptor cells [Figure 6]G and [Figure 6]H.
Figure 6: SEM of the non-sensory region. (A–C) The non-sensory epithelium distributed on the margins of olfactory lamellae (arrowheads). (D) Higher magnification of small boxed area in C showing the sensory olfactory epithelium (arrowheads) and rodlet cells (RC, arrowheads). (E) Higher magnification of large boxed area in C showing the non-sensory epithelium with ciliated cells (arrowhead) and epithelial cells with fingerprint-like microridges (EC). (F–H) Transitional zone between non-sensory epithelium (NSEC) with fingerprint-like microridges epithelial cells (EC) and sensory epithelium with microvillous ORC (**) and ciliated ORC (arrowhead).

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3.5. TEM of olfactory epithelium

The nuclei of the olfactory receptor cells were located above the level of the neighboring nuclei of supporting cells [Figure 7]A. The receptor cells nuclei have characteristics checkerboard pattern distribution of chromatin [Figure 7]A. According to the difference in staining affinity, the cells could be identified as the following: ciliated and microvillous receptor cells were stained darker than the surrounding supporting cells [Figure 7]A.
Figure 7: TEM of sensory epithelium. (A) Sensory epithelium was consisted of ciliated receptor cells (CRC) microvillous receptor cells (MRC), rodlet cells (RC), supporting cells (SC) and basal cells (BC). (B) Ciliated receptor cells with olfactory knob (OK) and cilia, microvillous ones (MRC), rodlet cells (RC) and supporting cell (SC) with apical secretory vesicles (arrowhead). (C) Higher magnification of supporting cells (SC) with apical secretory vesicles (arrowhead) showed zonula adherents (**) junctional complexes between it and receptor cells. Notice presence of neurotubules in the ciliated receptor cells (arrowheads). (D) Ciliated receptor cells (CRC, arrowhead) at the tip of lamellae. Notice the presence of epithelial cells (EC, arrowhead) with microridges. (E) Microvillous receptor cells (MRC) showed well distinct basal bodies numerous (arrowheads) with microvilli and abundant mitochondria (M). (F) The crypt cell (arrow head) was surrounding by supporting cells (SC). Notice olfactory knob (OK) of receptor cells. (G) Higher magnification of crypt cells showing mitochondria (M) and nucleus with heterochromatin (N). It was surrounded by supporting cells (SC). Cross section of cilia was observed within the cells (arrowhead, cilia). (H) Immature stage of rodlet cells on the basal part and mature one (arrowhead) in the apical region. Notice the microvillous cells (**) and basal cells (arrow head, BC). (I) Basal cells (BC) contained rER that rest on basal lamina (BL, arrowhead) with thick lamina reticularis (LR, **). Immature stage of rodlet cells contained rER, mitochondria (M), electron lucent granules (G1) and electron dense ones (G2).

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The ciliated cells had narrow apical dendrites ending in form of distinct olfactory knob with several cilia [2],[3] arising from it [Figure 7]B. Each cilium was observed arising from basal body supported with a foot, numerous neurotubules were also demonstrated at the apical end of cells [Figure 7]C. The apical portions of supporting cells were filled with many adherent electron-lucent vesicles and their lateral surfaces exhibited zonula adherent junctional complexes between them and the receptor cells [Figure 7]B and [Figure 7]C. Many ciliated ORCs were identified at the apical surface [Figure 7]D. The microvillous receptor cells were provided with less distinct olfactory knobs, beneath these knobs, there were group of abundant mitochondria arranged longitudinally [Figure 7]E.

Crypt cells were stained lighter than the surrounding supporting cells and possessed oval or egg-shaped cell body, located in the apical portion of epithelium [Figure 7]F. They contained electron-lucent staining cytoplasm with rough endoplasmic reticulum and mitochondria in the apical portion of the cell above the nucleus. Golgi apparatus was identified in a supranuclear position and cross section of cilia was observed within the cells [Figure 7]G.

The basal cells were small polyhedral cells with large nuclei contained distinct nucleoli. The differentiated or active form of these cells was characterized by nucleus with chromatin arranged in checkerboard pattern. They contain few rough endoplasmic reticulum and mitochondria [Figure 7]H and [Figure 7]I. The basement membrane of the olfactory epithelium was consisted of thick lamina densa and thick lamina reticularis layer [Figure 7]I.

The immature form of rodlet cells were found near the basement membrane and appeared as large round or polyhedral cells filled with many electron dense and lucent granules and eccentric basally located nucleus with checkerboard pattern of chromatin distribution [Figure 7]H and [Figure 7]I. The mature rodlet cell was pear in shape with its apex directed toward the surface and the broad basal part contained the basally or laterally located oval-shaped nucleus. The apical part of these cells provided with short microvilli [Figure 7]H. Some rodlet cells released their whole contents into the interlamellar space in a holocrine-like manner [Figure 8]A.
Figure 8: TEM of non-sensory epithelium. (A) Non-sensory epithelium consisted of non-sensory cells with cilia (NSC) and stratified epithelial cells (SEC) with microridges. Note mature stage of rodlet cells (RC) releasing their contents. (B) Non-sensory epithelial cells (NEC) showing broad apical surface provided with large number of cilia (arrow head). (C) Transitional zone between sensory cells with olfactory receptor cells (ORC), supporting cells (SC), rodlet cells (RC) and non-sensory epithelial cells (EC). (D) Developing stages of rodlet cells (RC). Notice the presence of epithelial cells (EC, arrowheads) with microridges. (E) Developing rodlet cells was surrounded by a thick cuticle and contained rodlet granules (RG), translucent vesicles (stars) and rER. (F) Epithelium of connection part between lamellae and wall of olfactory chamber was consisted of rodlet cells (RC), microvillous cells (Mv). Notice presence of macrophage (arrowhead). (G) Higher magnification of macrophage showed mitochondria (M), rER, apical pseudopodia and vesicles (**). (H) connection part between lamellae and wall of olfactory chamber showed microvillous cells (Mv) and some exhausted rodlet cells (arrow head). (I) Some rodlet cells in median raphe release their contents (arrowheads).

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The ciliated non-sensory cells were showed broad apical surface provided with large number of cilia [Figure 8]B. The cilia of non-sensory cells appeared longer than cilia of sensory cells. Mitochondria were packed under the apical end of the cells [Figure 8]B. Moreover, the ciliated non-sensory cells can distinguish from the sensory ones by absence of olfactory knob. Transitional zone between sensory and non-sensory epithelium consisted of olfactory receptor cells, rodlet cells and epithelial cells with microridges [Figure 8]C. Epidermal cells or non-sensory epithelial cells with irregular apical end or serrated appearance were seen in non-sensory part [Figure 8]D. The rodlet cell was surrounded by a thick cuticle and within their cytoplasm, rER, translucent vesicles and many club shaped rodlet inclusion were observed. Each inclusion consisted of a high dense core surrounded by a less dense material. The inclusions organized as their wide part directed toward the basal nucleus and the narrow part directed toward the apex of the cells [Figure 8]E.

Epithelium of connection part between lamellae and the wall of the olfactory chamber was consisted of rodlet cells, microvillous cells and macrophage [Figure 8]F. Macrophage was characterized by kidney-shaped nucleus and its cytoplasm contained mitochondria and rER with apical pseudopodia and vesicles [Figure 8]G. Empty and exhausted rodlet cells were also observed in the median raphe and connection part between lamellae and the wall of the olfactory chamber as they released their contents into the lumen [Figure 8]H and [Figure 8]I.


  4. Discussion Top


Olfaction plays a major role in fish as the olfactory organ directly interact with the environment and involving in various life activities include feeding, reproduction and social interaction. In contrast to many other teleostean, Red-tail shark is characterized by presence of two nasal (incurrent and excurrent) openings, which allow and regulate the passage of ventilator water flow through it and prevent the large particles from entrance to the nasal cavity. The olfactory rosette of Red-tail shark was oval in shape, therefore this species can be classified into eye-nose fish that indicated that the fish possess similarly optic and olfactory faculties according to [25]. In many teleostean such as, catfish, minnows, carps, pikes and salmons, the olfactory organ is multi-lamellar structure with variable configuration [26]. These findings agreed with our observations to the number of olfactory lamellae in Red-tail shark that ranged from 45 to 48. The multilamellar arrangement may increase the surface needed for sensory activity [27]. The fish also classified into the number of lamellae into microsomatic with few lamellae and macrosomatic with many lamellae. Red-tail fish is macrosomatic fish which mean that they rely mainly on olfaction more than vision.

The olfactory lamellae of Red-tail shark had unequal thickness as the tip of olfactory fold was thinner and contained many receptor cells than the middle portion, this may be due to that anterior epithelial cells comes in contact with the incoming water flow and consequently provided by high density of receptor cells.

The sensory epithelium exhibits various distribution patterns according to fish species such as continuous, separated regularly by non-sensory epithelium, interspaced irregularly and scattered in islets [1]. In Red-tail shark, the lateral surface of olfactory lamella is covered with sensory epithelium, whereas the non-sensory area restricted mainly at the margin of the lamellae. The presence of broad sensory area is thought to increase the capability of olfactory organ to interact with water soluble compounds during olfaction.

Buck and Axel [28] clarified that the initiating olfactory processing started at the apical tip of the olfactory receptor neurons. Probably, the olfactory knob with its cilia and microvilli of sensory receptor cells indicates different functional activity and ability for detection of different odorants present in the water. Hino et al. [29] added that the fish have the ability for orientation of water soluble compounds through the sensory olfactory epithelium during water ventilation.

Red-tail shark is a freshwater fish fed on decaying organic matter and algae, therefore, high density of ciliated ORCs in the olfactory rosette were of great importance in detection of food and were adapted to food and feeding habit of this species. The cilia are the site of transduction process and are stimulated by odor-bearing substances is an established fact and may have a role in circulation of water current in the olfactory chamber and in interlamellar space. The high distribution of ciliated cells in the olfactory lamella of Red-tail shark, especially in the tip of the lamellae confirms this idea. Ciliated ORC might be termed as generalists, because its abilities for detection of odorants including bile salts, amino acids and other odorants, while microvillous ORC might be called as specialist, which is more specific to nucleotides and amino acids [30]. Hamdani et al. [31] supposed that the microvillous olfactory receptor cell may have role in the feeding behavior of Crucian carp (Carassius carassius), while the ciliated receptor cell mediates the alarm reaction elicited by pheromone [32]. Therefore, these sensory elements participate in different behavioral pattern in teleosts.

We observed by electron microscope presence of neurotubules in dendrite of all receptor cells. Such observation was also recorded in many fish species [33],[34]. These neurotubules may have the main role in maintaining the shape of the dendritic process and acting as minute channels for transportation of various substances, while [35] supposed a supportive role to these tubules.

The olfactory crypt cell was first identified and named in teleosts by [36]. Crypt cells were detected in many fishes, including zebrafish, catfish, goldfish and salmonids [17], but they are apparently absent from other species, including two types of lungfishes [8],[13]. The function of these cells were still unknown, however crypt cells are supposed to detect of sex pheromones [37]. In addition, crypt cells act as chemoreceptors to amino acids according to [38]. Saito et al. [39] added that the crypt cells of zebrafish expressed TrkA-like proteins and might be involved in olfactory epithelium regeneration. We supposed a chemoreception and epithelium regenerative function of these cells based on their ultrastructure as they contained abundant mitochondria, rER and possessed apical cilia.

Rodlet cells are the most interesting feature in the present study. They distributed among sensory and non-sensory olfactory epithelium as well as median raphe. Rodlet cells are commonly observed in tissues and organs of fish include kidney, gills, heart and gut [40]. The accurate function of the RCs is unsolved until now. Several opinions of many authors on the nature and function of rodlet cells were recorded; Vickers [41] thought that they maybe modified goblet cells. Leino [42] supposed secretory activity to these cells, while [43],[44] supposed a regulatory role of these cells in ion transportation and osmoregulation. Bielek [45] considered these cells as migrating secretory cells. These may also considered as non-specific immune cells, involved in immunity as their number is increased in parasitic infection [46]. Reite and Evensen [47] considered them a type of eosinophilic granulocyte. While In the present study, TEM showed presence of electron dense secretory granules in immature stage and in developing stage, the rodlets were surrounded by capsule of fibrillar materials, and finally in mature stage the whole content of these cells came out. We supposed a secretory function to these cells with a holocrine mode of secretion based on the complete discharging of secretory materials in semithin sections and ultrastructural view combined with their location around blood vessels. This view is supported by the findings of [42] who mentioned that the RCs were developed from undifferentiated cells near epithelial basement membranes and then migrated toward the surface to secret their contents in a holocrine mode. He added that the process of secretion usually associated by contraction of fibrillar border inside to their plasma membrane. In the present study the histochemical analysis revealed positive reaction for immature stages of rodlet cells with PAS and Toluidine blue and positive reaction of developing and mature stage of rodlet cells to Alcian Blue and Sudan Black B stain. The variation in results of histochemical analysis is unclear. But it may indicate different secretory components related to different functions during different stage of maturation and further studies on component of these cells should be done.

The supporting cells were distributed along the lamellar surface; in sensory region they enveloped many receptor cells. Transmission electron microscope revealed presence of abundant mitochondria and many secretory vesicles in the apical surface of the supporting cells which may indicate a secretory function of these cells. The abundance of mitochondria may have role in production of energy involved in a secretory function for these cells. According to histochemical analysis, the lamellae were covered by PAS-and AB-positive mucus coat that may be produced from the apical secretory vesicles of these cells, which indicated presence of neutral and acid mucopolysaccharides that may form a thick lubricant coat on the neighboring olfactory cells. The supporting cells took the mosaic appearance between the sensory cells and zonula adherents junctional complexes were found between them and receptor cells. Thornhill [48] reported that the zonula adherents acts as a sieve to prevent passage of substances through the interlamellar spaces.

The basal cells were oval or pear-shaped cells satiated above the basal lamina. These cells may act as stem cells for regeneration of the olfactory epithelium, which characterized by relatively short life span according to [26],[49] and may be replaced throughout the life by these progenitor basal cells. This view is supported by presence of rough endoplasmic reticulum in the cytoplasm of these cells.

Neuromast or taste bud is mechanosensory organ that involved in detection of predators and capture of prey [50]. Webb [51] classified the neuromasts in fish into two types; presumptive which present in canals and superficial neuromasts that present on epithelium of the tail, trunk and head region. SEM and semithin section revealed presence of few superficial neuromasts on the apical third of olfactory lamellae that consisted of sensory and supporting cells with prominent taste pore. Their position in the apical region of the lamellae allows it to react quickly with the incoming signals from the outside. Supporting cells are involved in production of a gelatinous materials “cupula” that help the olfactory organ to communicate with the external environment [52]. While sensory cells help in orientation of chemical stimuli and consequently in detection of palatable food items [53].

The non-sensory epithelium was consisted of ciliated stratified epithelial cells with long cilia and non-ciliated cells with fingerprint-like microridges on the apical surface. These long cilia may have mucus-propelling action. The ciliated non-sensory cells participate in ventilation of olfactory chamber by beating action of cilia [1]. The microridges may increase the surface of non-sensory epithelium and help in holding the mucus along the olfactory epithelium and give protection to the receptor sensory cells from the mechanical abrasion. In addition, the cells of non-sensory regions may involve in mechanical support of receptor cells.

The epithelium of median raphe contained stratified epithelium with labyrinth pattern microridges. The arrangement of microridges may protect the sensory epithelium from mechanical injuries and help in holding the mucus over the epithelium. Few mucus cells were present in the connection between the wall of olfactory chamber and median raphe. These cells secrete mucous, which protect the olfactory epithelium from external injuries and allows smooth flow of water through the olfactory chamber. As Red-tail shark is scavenger fish, the mucin help in binding of microscopic debris that enter with incoming water as well as help in decrease friction of water in the nasal cavity. Moreover, the mucus over the olfactory lamellae forms a suitable medium for diffusion of odorants. Banerjee [54] added that the mucous layer may trap the ions to delay penetration of toxicants, especially the heavy metals into underlying tissues.

Red-tail shark is an aquarium fish, although the detailed description on the olfactory epithelium of other aquarium fish is rare except for guupy [5] which consisted of ciliated, microvillous ORCs and crypt cells. Also the structural organization of the olfactory epithelium of Red-tail shark was not yet studied that appeared to be differed from closely related cyprinides species [19],[20].

Finally, Red tail shark is a fresh water teleost subsists on decaying organic matter, algae, etc. and seemed from multilamellar arrangement and dense population of cells that it have an acute sense of smell and depends mainly on the olfactory sensory cells in detection of food and may be other vital activities. Therefore, presence of various receptor cells and neuromasts in the olfactory rosettes adapted accordingly in relation to food and feeding habit of the fish. Further researches and experimental studies on the olfactory organ of Red-tail shark are recommended.

Conflict of interest

None declared.

Acknowledgments

The authors thank Mr. Ahmed Ibrahim, the technician in electron microscope unit for providing the facilities for achieving this study.



 
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