STRUCTURAL DIVERSITY OF OLFACTORY ORGANS IN OSTEOGLOSSIFORMES Sudhanshu Shekhar
Research Scholar, Department of Zoology, Y B N University Namkum Ranchi, Jharkhand Abstract- Osteoglossomorpha, an ancient Teleostei group, exhibits many ancestral structural features. Herein, we describe the diversity in morphology of olfactory organs of air-breathing Pantodon buchholzi, Arapaima gigas, and Gymnarchus niloticus in terms of adaptations to short ventures out of water and compare the results with the water- breathing Osteoglossum bicirrhosum. We demonstrate the presence of olfactory rosettes within the olfactory chamber in all studied species and additionally a peculiar cudgel- shaped structure in P. buchholzi. The typical olfactory rosette with centrally located elongate median raphe and two rows of olfactory lamellae was found only in P. buchholzi. In A. gigas, the olfactory lamellae arranged in semicircular manner were merged to small median raphe. Osteoglossum bicirrhosum and G. niloticus lacked median raphe, while the olfactory lamellae were ordered nearly parallel or circular, respectively. Olfactory epithelium lining the olfactory lamellae was unfolded only in P. buchholzi and G. niloticus. In contrast, in O. bicirrhosum and A. gigas convexities formed by nonsensory cells and concavities made by the sensory component were observed. Among the olfactory sensory neurons, ciliated and microvillus neurons were found in all studied species. Crypt-like cells were noted only in P. buchholzi, whereas rod-like cells were unique for A. gigas. The substantial morphological variation of the olfactory organ structure and ultrastructure in the studied osteoglossiforms may be explained by the early division of Osteoglossomorpha within Teleostei and long-term, independent evolution within the families.
Keywords:- Olfactory rosette; olfactory sensory neurons; airbreathing fishes; Teleostei;
evolution; olfactory organ structure; Osteoglossiformes.
1. INTRODUCTION
Osteoglossomorpha is one of the most ancient lineages among Teleostei (Betancur et al., 2017), and its representatives show numerous ancestral structural features. The species are highly specialized and are varied in terms of numerous aspects of morphology, body size (body length ranging from 4 cm in Petrocephalus catostoma to about 300 cm in Arapaima gigas) (Froese & Pauly, 2019), and breeding (e.g., internal vs. external fertilization, occurrence of four types of sperm) (Jamieson, 2009; Mattei, Marchand & Quilichini, 2019).
Some of them (i.e., Pantodon buchholzi, Arapaima gigas, Heterotis niloticus, a few notopterids, and Gymnarchus niloticus) possess gas bladders adapted to aerial respiration (Graham, 1997). Osteoglossomorphs live in various water zones. They are either pelagic/
benthopelagic (i.e., P. buchholzi, O. bicirrhosum, and A. gigas) or demersal (i.e., G.
niloticus) (Froese & Pauly, 2019).
Within the superorder Osteoglossomorpha, there are two orders:-
1. Hiodontiformes, with only two living species classified in the family Hiodontidae and 2. Osteoglossiformes, in which five families are distinguished (i.e., Pantodontidae, Osteoglossidae, Notopteridae, Gymnarchidae, and Mormyridae) (Betancur et al., 2017).
Previously, six families were recognized; however, the phylogenetic analysis of Betancur et al. (2017) suggested the fusion of Osteoglossidae and Arapaimidae into one family. Despite the relatively close phylogenetic relationship of O. bicirrhosum and A. gigas, they are different in terms of breathing (water or air, respectively), which should imply morphological and developmental differences. These dissimilarities were found, for example, in the development of gills (Brauner et al., 2004) or eyes (Saidel and Braford, 1985).
One of the most important sense organs among fish is the olfactory organ, which allows recognition and identification of chemical substances (odorants) responsible for finding food and a partner for breeding or for receiving signals about danger, social interactions, and migration (Sorensen & Caprio, 1998; Bone & Moore, 2008). In species inhabiting water environments, the olfactory organ is typically organized as an olfactory rosette formed by olfactory lamellae and median raphe (e.g., Jakubowski & Kunysz, 1979;
Hansen & Zielinski, 2005).
The olfactory rosette can differ between species in terms of number and arrangement of olfactory lamellae, that is, it may consist of one lamella arranged lengthwise or crosswise, or many lamellae ordered parallelly or radially. Additionally, lamellae may (or may not) surround short or long median raphe located centrally or on one side of the olfactory chamber (Yamamoto, 1982). Size and often the number of lamellae increase during fish development up to maturity. Some fish do not possess olfactory rosettes and the olfactory epithelium lines walls of the olfactory chamber, for example, in syngnathids (Dymek et al., 2020) and mudskippers (Kuciel, Zuwala & Jakubowski, 2011; Kuciel, Zuwała
& Satapoomin, 2013).
They are active land dwellers and use pectoral fins and the tail to leave the water, and to move on mud or sand surfaces; therefore, they spend considerable time exposed to air (Graham, 1997). The olfactory organ of mudskippers is unusual among fish, because their nasal cavity consists of an elongated canal that widens into a chamber-like sac. There is no olfactory rosette, and the olfactory epithelium is morphologically variable depending on the species (Kuciel et al., 2014,2011,2017,2013). The olfactory epithelium, present in every fish species studied thus far, is divided into sensory and nonsensory compartments.
These two areas can be arranged differently:
1. Continuously—the sensory part is located concentrically and the nonsensory one is located distally within the olfactory lamella, like in Oncorhynchus masou (Pfeifer, 1963);
2. Zonally— the sensory and nonsensory areas form bands, as in Silurus glanis (Jakubowski & Kunysz, 1979); or
3. Irregularly— sensory and nonsensory compartments are interlaced, as in Gasterosteiformes (Yamamoto & Ueda, 1978; Honkanen & Ekstrom, 1991;
Honkanen & Ekstrӧm, 1992; Wilson & Orr, 2011).
The non-sensory compartment is primarily formed by non-sensory ciliated cells.
Cilia exhibit positive immunoreactions for ASIC2, rendering this method useful for detection of these cells (Vi~na et al., 2015). Within the olfactory sensory epithelium, three types of cells can be distinguished: olfactory sensory neurons (OSNs), supporting cells, and basal cells. Cylindrical-shaped supporting cells are placed between the OSNs to keep them in the right position (Hansen & Zielinski, 2005).
Basal cells are located near the basal lamina and can develop into all elements of the sensory and non-sensory epithelium as well as regenerate the sensory neurons (Hansen et al., 1999; Iqbal & Byrd-Jacobs, 2010). OSNs exhibit differing morphology, various receptor classes, and target different glomeruli in the olfactory bulb. They are also defined by specific molecular markers. Therefore they can be distinguished as ciliated, microvillus, and crypt neurons (Hansen et al., 2003) and two additional types discovered in Danio rerio:
Kappe neurons (Ahuja et al., 2014) and pear-shaped neurons (Wakisaka et al., 2017).
The ciliated OSN is an elongated bipolar neuron with a knob, which projects highly above the epithelial level. It expresses G protein-coupled odorant receptors of the OR family and also receptors of the TAAR family, and is marked by an OMP promoter (Mombaerts et al., 1996; Hansen, Anderson & Finger, 2004; Sato, Miyasaka & Yoshihara, 2005; Wagner et al., 2006; Johnson et al., 2012). This type of OSN targets most of the glomeruli in the dorsal and medial parts of the olfactory bulb (Sato et al., 2005).
In the apical part of the neuron, numerous cilia, typically with an axonemal structure, extend into the lumen of the olfactory chamber. At the base of the cilium, there is a basal body that consists of two perpendicularly arranged centrioles (Hansen & Zielinski, 2005). The ciliated OSN possesses numerous mitochondria and a spherical nucleus located basally. Rod-like (giant) cells are distinguished as a subpopulation of the ciliated OSNs;
however, some authors describe them as a further type of OSN (Bannister, 1965; Ichikawa
& Ueda, 1977; Yamamoto & Ueda, 1978; Rhein et al., 1981; Muller & Marc, 1984; Datta &
Bandopadhyay, 1997; Ghosh, 2018; Webb et al., 2019).
This type of neurons expresses genes of the V1R and V2R receptor classes and is marked by TRPC2 or Rag1 (Mombaerts et al., 1996; Hansen et al., 2004; Feng et al., 2005;
Sato et al., 2005; Wagner et al., 2006; Johnson et al., 2012). Target glomeruli of microvillus OSNs are in the middle and ventral parts of the olfactory bulb (Sato et al., 2005). The crypt OSN is an oval cell with cilia and microvilli located within the invagination (crypt). These
neurons are situated in the apical part of the olfactory epithelium and are enclosed by supporting cells of glial characteristics (Hansen & Finger, 2000; Schmachtenberg, 2006;
Bazaes & Schmachtenberg, 2012).
2. MATERIALS AND METHODS 2.1 Specimens
The research material included 8 specimens of Pantodon buchholzi (Pantodontidae) and 4 specimens of each of the following species: Osteoglossum bicirrhosum, Arapaima gigas (Osteoglossidae), and Gymnarchus niloticus (Gymnarchidae). All specimens were obtained from a local aquarist shop. The total body lengths (TLs) of the studied specimens are summarized in of the individuals was carried out in an aqueous tricaine solution (0.1% MS 222; Sigma, St. Louis, MO, USA).
2.2 Histology
Bouin fixative was used to fix decapitated heads for the histological study using a light microscope. Next, tissue dehydration with an increasing gradient of ethanol concentrations and overexposition in toluene was carried out. The materials were embedded in Paraplast Regular at 60°C (Sigma). 5-µmthick sections were prepared and stained with Mallory`s trichrome. Then they were enclosed in a Thermo Scientific Shandon Consul Mount. The slides were observed under a Nikon Eclipse E600 light microscope and photographed using a Nikon COOLPIX P6000 camera.
2.3 Electron microscopy
After dissection of the olfactory rosettes, tissues for the electron microscopes were fixed in Karnovsky fixative (pH 7.2) prepared with 0.1 M cacodyl buffer (pH 7.2). Next, tissues for scanning electron microscopy (SEM) were dehydrated with a rising ethanol concentration gradient and placed in 100% acetone. Further, they were dried at the CO2 critical point and sputtered with gold. The material was observed on a HITACHI S-4700 scanning electron microscope in the Institute of Geological Sciences at Jagiellonian University in Cracow.
The fixed material was put in a 1% osmium tetroxide solution in 0.2 M cacodylate buffer (pH 7.2) for transmission electron microscopy (TEM). Before embedding the tissues in Epon 812 resin (Sigma), they were dehydrated in a series of graded ethanol solutions. After cutting the embedded material into ultra-thin sections and contrast staining with uranyl acetate and lead citrate, the strips were viewed in a JEOL-100SX transmission electron microscope (Jeol, Tokyo, Japan) in the Department of Cell Biology and Imaging at Jagiellonian University in Cracow. All measurements were made using ImageJ software.
3. RESULTS
The olfactory organs are paired structures in all of the examined species. Round anterior and posterior nostrils lead to the olfactory chamber in which the olfactory rosette is located.
Additional nasal sacs are absent in the analyzed osteoglossiforms.
3.1 The Olfactory Organ of Pantodon Buchholzi Consists of a Typical Olfactory Rosette and a Peculiar Cudgel-Shaped Structure
The anterior nostril takes on a tubular form, whereas the posterior nostril does not protrude above the surface of the skin. Within the high (~950 µm) oval olfactory chamber (999 52 µm length and 629 57 µm in width), there is an olfactory rosette and a cudgel- shaped structure. The olfactory rosette consists of olfactory lamellae (496 41 µm in height), which are located close together (average distance between two lamellae measured in half of the length of the lamella is 43 16 µm) and abut to the centrally placed, elongated median raphe (average 718 41 µm in length) and fused with the walls of the olfactory chamber.
Olfactory lamellae are arranged perpendicularly to the direction of water flow. The lengths of the lamellae in the rosette differ. Based on measurements of length of the top of lamellae, the shortest lamellae (249 32 µm in length) are located in the anterior part, whereas the longest (350 39 µm in length) are located in the central and posterior parts of the olfactory chamber.
The core of each lamella consisting of connective tissue is lined with an unfolded epithelium divided into sensory and non-sensory compartments arranged zonally; the
sensory epithelium lines part of the lamella near the median raphe and covers 64% of the whole lamellar area, whereas the non-sensory ciliated epithelium is located distally and covers the
Figure 1 Structure and ultrastructure of the olfactory organ of Pantodon buchholzi.
(A) Olfactory lamellae (ol) located close together and merged to the central median raphe (mr) are visible at the bottom of the olfactory chamber. A cudgel-shaped structure (cs) is visible on the posterior wall of the olfactory chamber. (B) Histological
structure and arrangement of olfactory lamellae (ol) and median raphe (mr). (C) Histology of cudgelshaped structure. Ciliated epithelial cells (cl) cover connective tissue (ct). (D) Zonal arrangement of sensory (sc) and nonsensory (nc) components lining the olfactory lamellae. (E, F) Histological structure of olfactory lamellae and
median raphe.
remaining area. The sensory epithelium surface area in the single olfactory organ in P.
buchholzi is on average about 10.05 mm2. Within the sensory compartment, three types of olfactory sensory neurons are visible: ciliated microvillus and crypt-like OSNs. Ciliated OSNs dominate within the sensory epithelium, but microvillus OSNs are also relatively common. Crypt-like OSNs exhibit few cilia that project into the crypt. We cannot precisely classify them as crypt or Kappe neurons due to their morphological similarities.
Basal cells found within the lowest part of the sensory epithelium are in contact with the basal lamina. The non-sensory epithelium is composed of ciliated epithelial and mucus cells. Additionally, a cudgel-shaped structure is visible on the posterior wall of the olfactory chamber. The average diameter of the spherical part of the structure is 251 50 µm, while the length from the wall to the top of the structure is 254 50 µm. The cudgel-shaped structure consists of connective tissue with dominant collagen fibers and ciliated epithelial cells.
3.2 The Olfactory Rosette of Osteoglossum Bicirrhosum is formed by one row of Lamellae with Process-Like Structures
Anterior and posterior nostrils do not protrude above the surface of the skin. Within the very tall (height: ~1000 µm) oval olfactory chamber (length: 2170 47 µm), O. bicirrhosum possesses a large olfactory rosette composed of one row of high olfactory lamellae (799 31 µm in height) that are arranged perpendicularly to the direction of water flow. There is no median raphe. The dorsal portion of each lamella in the central part forms a process-like structure. Both sides of the lamellae are joined to the walls of the olfactory chamber.
The average distance between lamellae measured in the proximal part of the lamellae is similar (200 12 µm). The lamellae are shorter in the anterior and posterior part of the olfactory rosette (462 16 µm), but longer in the central part (862 16 µm). The epithelium lining the olfactory lamella and the process like structure is highly folded and it forms palmate-shaped concavities and convexities that line the remaining area.
The convexities contain non-sensory ciliated epithelial cells while in the concavities, OSNs separated by supporting cells are visible. Sensory epithelium covers 22% of the lamellar surface, and in one olfactory organ it occupies on average 0.42 mm2. Ciliated OSNs constitute the largest fraction of OSNs while microvillus OSNs are also great in number. Basal cells are likewise visible in the olfactory epithelium. Numerous mucus cells are in the direct vicinities of the OSNs.
3.3 In Arapaima Gigas Olfactory Lamellae with Processes are Arranged in a Semicircle and Merge with a Small Median Raphe
The anterior nostril takes a tubular form, whereas the posterior nostril does not protrude above the surface of the skin. The round olfactory chamber of A. gigas (diameter: 1957 57 µm, height: ~750 µm) contains an olfactory rosette consisting of olfactory lamellae (height:
550 32 µm) arranged in a semicircle with a process in the distal part directed centrifugally merged to the short, located centrally on the proximal wall median raphe and to the distal wall of the olfactory chamber.
The longest lamellae (length: 1580 34 µm) are present in the central part of the olfactory rosette, whereas the shortest (length: 830 27 µm) are located peripherally. The average distance between two lamellae measured in the proximal part of the lamella is 107 35 µm. Each lamella is covered by parallelly arranged, long but narrow, bands of concavities (length: 500 21 µm; width: 19 4 µm) and convexities lining the remaining area.
On cross sections, the distal parts of the lamellae are lined with non-sensory ciliated epithelial cells only, whereas proximal parts of the lamellae exhibit a deeply folded epithelium forming convexities comprising the non-sensory epithelium and concavities containing OSNs. In total, 9% of the lamellar surface is lined with sensory epithelium, which in one olfactory organ covers on average 0.74 mm2. Ciliated OSNs and rod-like OSNs are more numerous than microvillus OSNs within the sensory compartment. Basal cells in contact with basal lamina are also visible within the lowermost part of the sensory epithelium.
4. DISCUSSION
Teleosts show great variation in many aspects of body structure. This variation is correlated with adaptation to different environmental conditions, various inhabited ecological niches, and is a consequence of multiple evolutionary pathways. Osteoglossomorpha is an ancient Teleostei group that is diverse not only in terms of general morphology (Froese & Pauly, 2019) but also in terms of the structure of their olfactory organ. Nostrils show different shapes and positions in fish, depending on factors such as swimming speed and inhabited depth.
In O. bicirrhosum, both nostrils are at the level of the skin. A simple opening was also observed in some hatchet fish (Derscheid, 1924). In contrast, in P. buchholzi, A. gigas, and G. niloticus, anterior nostrils are tubular in shape (nasal ridge), whereas posterior nostrils do not protrude above the surface of the skin. Nostrils situated at the end of a tubule were also observed in Spinachia spinachia (Theisen, 1982) or Arothron (Tetraodon) nigropunctatus (Wiedersheim, 1887).
The tubular form of nostrils probably enables more effective suction of water into the olfactory chamber (Døving, 1986). One of the most extensively studied fish species in terms of olfaction is Danio rerio (Hansen & Zeiske, 1998; Sato et al., 2005; Jesuthasan &
Mathuru, 2008; Oka et al., 2012; Ahuja et al., 2013, 2014; Vi~na et al., 2015). It possesses an oval olfactory rosette typical of teleosts, consisting of a centrally located elongate median raphe from which olfactory lamellae project (Hansen & Zeiske, 1998).
This type of olfactory rosette is found in many fish species, including Cyprinus carpio, Oncorhynchus mykiss, and Dissostichus mawsoni (Ishikawa, Masahito &
Takayama, 1978; Hansen & Zeiske, 1998; Hansen et al., 1999; Ferrando et al., 2019b), as well as in air-breathing A. anguila (Atta, 2013) and H. fossilis (Bandyopadhyay & Datta,
1998). Among studied osteoglossiforms, only P. buchholzi possesses similar type of olfactory rosette with one edge of the olfactory lamellae adjoining to the median raphe and with the opposite to the wall of the olfactory chamber.
Other analyzed species exhibit different types of olfactory rosettes. In O. bicirrhosum there is one row of nearly parallel olfactory lamellae without a median raphe. The olfactory rosette of A. gigas consists of semi circularly arranged olfactory lamellae joined to the short median raphe and to the distal wall of the olfactory chamber, which may be classified as one type of arrow-like olfactory rosette characteristic for some species of Cyprinidae and Salmonidae (Kleerekoper, 1969; Yamamoto, 1982; Zeiske, Theisen & Breucker, 1992; Chen
& Arratia, 1994).
On the bottom of the wide and low olfactory chamber of G. niloticus, there are a few concentrically arranged short olfactory lamellae without a median raphe, a similar arrangement was observed in Esox lucius (Holl, 1965). Olfactory lamellae in P. buchholzi are located close together and perpendicular to the direction of water flow. The olfactory lamellae are not very tall; therefore, the area between the olfactory rosette and nasal bridge that separates both nostrils is large.
This organization probably makes it possible to retain a portion of water in the space between the lamellae on account of surface tension. Additionally, an unusual cudgel shaped structure is present on the posterior wall of the olfactory chamber. This kind of structure has not been described in fish yet. During aerial locomotion, that is, jumping out of water and moving through air, the water that flows through the anterior nostril probably passes toward the back of the olfactory chamber where it can be retained by the cudgel shaped structure.
REFERENCES
1. Ahuja, G., Ivandic, I., Salt€urk, M., Oka, Y., Nadler, W. & Korsching, S.I. (2013). Zebrafish crypt neurons project to a single, identified mediodorsal glomerulus. Sci. Rep. 3, 2063.
2. Ahuja, G., Nia, S.B., Zapilko, V., Shiriagin, V., Kowatschew, D., Oka, Y. & Korsching, S.I. (2014). Kappe neurons, a novel population of olfactory sensory neurons. Sci. Rep. 4, 1–8.
3. Atta, K.I. (2013). Morphological, anatomical and histological studies on the olfactory organs and eyes of teleost fish: Anguilla anguilla in relation to its feeding habits. J. Basic Appl. Zool. 66, 101–108.
4. Bandyopadhyay, S.K. & Datta, N.C. (1998). Surface ultrastructure of the olfactory rosette of an air- breathing catfish, Heteropneustes fossilis (Bloch). J. Biosci. 23, 617– 622.
5. Bannister, L.H. (1965). The fine structure of the olfactory surface of teleostean fishes. J. Cell Sci. 3, 333–
342.
6. Bazaes, A. & Schmachtenberg, O. (2012). Odorant tuning of olfactory crypt cells from juvenile and adult rainbow trout. J. Exp. Biol. 215, 1740–1748.
7. Betancur, R., Wiley, E.O., Arratia, G., Acero, A., Bailly, N., Miya, M., Lecointre, G. & Ortı, G. (2017).
Phylogenetic classification of bony fishes. BMC Evol. Biol. 17, 1–40.
8. Bonde, N.O. (1996). Osteoglossids (Teleostei: Osteoglossomorpha) of the Mesozoic. Comments on their interrelationships. In Mesozoic Fishes - Systematics and Paleoecology: 273–284.
9. Arratia, G. & Viohl, G. (Eds.). Munich: Verlag Dr. Friedrich Pfeil. Bone, Q. & Moore, R. (2008). Biology of fishes. New York: Taylor & Francis.
10. Brauner, C.J., Matey, V., Wilson, J.M., Bernier, N.J. & Val, A.L. (2004). Transition in organ function during the evolution of air-breathing; insights from Arapaima gigas, an obligate airbreathing teleost from the Amazon. J. Exp. Biol. 207, 1433– 1438.
11. Catania, S., Germana, A., Laura, R., Gonzalez-Martinez, T., Ciriaco, E. & Vega, J.A. (2003). The crypt neurons in the olfactory epithelium of the adult zebrafish express TrkA-like immunoreactivity. Neurosci.
Lett. 350, 5–8.
12. Chen, X. & Arratia, G. (1994). Olfactory organ of Acipenseriformes and comparison with other actinopterygians: patterns of diversity. J. Morphol. 222, 241–267.
13. Das, J.R., Saha, N. & Ratha, B.K. (1991). Tissue distribution and subcellular localization of glutamate dehydrogenase in a freshwater air-breathing teleost, Heteropneustes fossilis. Biochem. Syst. Ecol. 19, 207–212.
14. Datta, N.C. & Bandopadhyay, S. (1997). Ultrastructure of cell types of the olfactory epithelium in a catfish, Heteropneustesfossilis (Bloch). J. Biosci. 22, 233–245.
15. Derscheid, J.M. (1924). Structure de l’organe olfactif chez les poissons. Premiere partie. Osteichthyes, Teleostei. Malacopterygii. Ann. Soc. Roy. Zool. Belg. 54, 79–162.
16. Døving, K.B. (1986). Functional properties of the fish olfactory system. In Progress in Sensory Physiology:
39–104. Ottoson, D. (Ed.). Berlin: Springer, Berlin Heidelberg.
17. Fange, R. (1983). Gas exchange in fish swimbladder. Rev. Physiol. Biochem. Pharmacol. 97, 111–158.
18. Feng, B., Bulchand, S., Yaksi, E., Friedrich, R.W. & Jesuthasan, S. (2005). The recombination activation gene 1 (Rag1) is expressed in a subset of zebrafish olfactory neurons but is not essential for axon targeting or amino acid detection. BMC Neurosci. 6, 46.
19. Ferrando, S., Amaroli, A., Gallus, L., Aicardi, S., Blasi, D.Di, Christiansen, J.S., Vacchi, M., Ghigliotti, L.
& Meredith, T.L. (2019a). Secondary Folds Contribute Significantly to the Total Surface Area in the Olfactory Organ of Chondrichthyes. Front. Physiol. 10, 1–14.
20. Ferrando, S., Amaroli, A., Gallus, L., Di Blasi, D., Carlig, E., Rottigni, M., Vacchi, M., Parker, S.J. &
Ghigliotti, L. (2019b). Olfaction in the Antarctic toothfish Dissostichus mawsoni: clues from the morphology and histology of the olfactory rosette and bulb. Polar Biol. 42, 1081–1091.
