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Italian Journal of Zoology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tizo20 Surface ultrastructure of the olfactory epithelium of loach fish, Triplophysa dalaica (Kessler, 1876) (Cypriniformes: Balitoridae: Nemacheilinae) B. Waryani a b c , R. Dai a , Y. Zhao b , C. Zhang b & A. R. Abbasi c a School of Life Sciences, Beijing Institute of Technology b Key Laboratory of Zoological Systematic and Evolution , Chinese Academy of Sciences c Department of Fresh Water Biology and Fisheries , University of Sindh Jamshoro , Pakistan Published online: 20 Mar 2013.

To cite this article: B. Waryani , R. Dai , Y. Zhao , C. Zhang & A. R. Abbasi (2013) Surface ultrastructure of the olfactory epithelium of loach fish, Triplophysa dalaica (Kessler, 1876) (Cypriniformes: Balitoridae: Nemacheilinae), Italian Journal of Zoology, 80:2, 195-203, DOI: 10.1080/11250003.2013.771711 To link to this article: http://dx.doi.org/10.1080/11250003.2013.771711

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Surface ultrastructure of the olfactory epithelium of loach fish, Triplophysa dalaica (Kessler, 1876) (Cypriniformes: Balitoridae: Nemacheilinae)

B. WARYANI1,2,3,R.DAI1,Y.ZHAO2,C.ZHANG2*, & A. R. ABBASI3

1School of Life Sciences, Beijing Institute of Technology, 2Key Laboratory of Zoological Systematic and Evolution, Chinese Academy of Sciences, and 3Department of Fresh Water Biology and Fisheries, University of Sindh Jamshoro, Pakistan

(Received 11 January 2012; accepted 19 January 2013)

Abstract The olfactory organs of loach Triplophysa dalaica were examined under a scanning electron microscope, sampled from the Dali-Nor Lake, Inner Mongolia, China. The olfactory organs are oval in shape, lying in the olfactory chamber on the dorsal side of the head. Ten pairs of lamellae in the rosette are present on each side of the middle raphe. Lamellae are covered with cilia at some margins. The surface of the olfactory lamellae is divided into three epithelial regions: sensory, ciliate non-sensory and non-ciliated non-sensory epithelium. Sensory epithelium consists of receptors and supporting cells, ciliated non-sensory epithelium is covered with dense cilia and non-receptor epithelium is covered with ciliated stratified epithelial cells and mucus cells. Cells other than epithelial cells present on the surface of the rosette are related to the significant olfactory function in the fish concerned.

Keywords: Loach, olfactory epithelium, electron micrograph, scanning electron microscope, histology

Introduction organs were focused on macro- or micro-observation of their structure for comparative anatomies within In most fishes, olfactory organs are present on the a taxonomic context (Pfeiffer 1964; Su & Li 1982). anterior dorsal side of the head. Olfactory organs Goniakowska-Witalinska et al. (1992, 1995) stud- are composed of olfactory rosettes, bulbs, lobes, ied the neuroendocrine cells in gills of bowfin and nerves and receptors of smell and touch. Externally in the lung of tiger salamander. The studies show they are connected with nasal openings and function the relatively complex pattern of the neuroendocrine in prey location, migration, propagation, shoaling cells. They found that the neuroendocrine cells in and species recognition (Song 1987). Olfaction is Downloaded by [Institute of Zoology] at 23:59 13 December 2013 the gill epithelium of Amia and lung of Ambystoma achieved mainly by olfactory cells on the surface of tigrinum are of unusual type both ultrastructurally lamellae (Ichikawa & Ueda 1977; Hansen & Zeiske and immunocytochemically. With advanced tech- 1998). Water contains dissolved chemicals, which niques for observation at the cellular level, the flow into the olfactory sac through the nostril infil- emphases have been centered on the study of mor- trate by cell reactions of the olfactory cells (Fishelson phology, structure and physiology of the olfactory 1995). In different groups of fishes there are varia- organ and its ecological adoptability (Banerjee 1993; tions in the ratio of the length of the olfactory sac to Giorgi et al. 1994; Wang et al. 1994; Fishelson eye diameter, the number of lamellae on the olfactory 1995; Liu et al. 2005; Ma et al. 2005). A number organ and the arrangement of olfactory cells of of studies have been carried out on the size of the the epithelium in sensory and non-sensory areas olfactory structures relating to species ecology, which (Yamamoto 1982; Meng et al. 1987; Bandyopadhyay indicates that differences in the size of olfactory & Datta 1998). Earlier studies on the olfactory structures are directly related to olfactory sensitivity

*Correspondence: Z. Chunguang, Institute of Zoology, Chinese Academy of Sciences National Zoological Museum, Key Laboratory of Zoological Systematics and Evaluation, Chinese Academy of Sciences, No.1-5, Beichen West Road, Chaoyang District, Beijing, 100101, P.R. China. Tel: +86 010-64807116. Fax: +86 010-64807116. Email: fi[email protected]

© 2013 Unione Zoologica Italiana 196 B. Waryani et al.

(Northcutt 1977, 1978; Theisen et al. 1986; Lisney Histological studies & Collin 2006; Lisney et al. 2007; Yopak et al. 2007; Olfactory tissues were immersed in aqueous Bouin’s Schluessel et al. 2008; Theiss et al. 2009). solution for a period of 20 hours for fixation. Fixed Loach, Triplophysa dalaica (Kessler, 1876) tissues were washed in distilled water, repeatedly (Cypriniformes: Balitoridae: Nemacheilinae), is a rinsed in 70% ethanol and dehydrated through a benthic fish found in slow running or standing water, graded series of ethanol followed by acetone and that feeds upon aquatic invertebrates (Kessler 1876; cleared in benzene. The tissues were embedded in Zhu 1989). Zhang et al. (1994) conducted studies paraffin wax at 56–58◦C under thermostat vacuum on the surface ultrastructure of gills, olfactory paraffin-embedding bath for a period of 1.5 hours. organs and barbells of Triplophysa (Hedinichthys) Sections were obtained using a rotary microtome. yarkandensis yarkandensis. Loach, T. dalaica,isa The sectioned tissues were stretched on Mayer’s freshwater fish species but it can tolerate living albuminised glass slide and stained with eosinhaema- in saline waters as well. No significant work has toxylin stain. Stained slides were examined and pho- appeared in the literature relating to the olfactory tographed under a Zeiss W-Pl 10 ×/23 compound organ of Loach living in saline waters. microscope. Loach, T. dalaica, living in saline water of Dalai- Nor Lake in Inner Mongolia, China was selected for the present study. The present work explores the Results changes on the surface of the olfactory organ. This Figure 1a–c shows the structure of the complete paper describes the detailed morphology and struc- fish Triplophysa dalaica; the head region shows the ture of the olfactory organ of T. dalaica using optical anterior and posterior olfactory openings and dis- and scanning electron microscopy and is the first sected head from the dorsal side, which shows the research report on the microstructure of the olfactory complete olfactory organ lying in the chambers. organ of this species. Paired olfactory rosettes are situated on the anterior dorsal side of the head in olfactory pits or olfactory Materials and methods chambers (Figure 1b). Externally, each pit has two Fish samples were collected in July 2010 from Dalai- separate openings, anterior and posterior, present Nor Lake (43.97◦N, 117.47◦E), the second largest on both sides of the head. Anterior and posterior lake in Inner Mongolia, China. The lake is located nostrils are close to each other. Both nostrils are north of Beijing at a distance of 200 km. Fish sam- oval in shape; the posterior nostril is wider than the ples were preserved in 10% formalin and brought to anterior nostril. A prominent flap of skin called the the Zoological Museum of the Institute of Zoology, nasal flap is present between the anterior and poste- Chinese Academy of Sciences, Beijing, China. rior openings (Figure 1b). The tips of the olfactory Standard body length, eye diameter, distance from lamellae are clearly visible within the sac (Figure 1b). front margin of the anterior and back margin of the Olfactory rosettes are oval, cup-shaped and are posterior nostrils of each fish were measured. The present in the depressions of the head (Figure 1c). heads of the fishes were dissected from the dorsal side Each rosette is connected with the olfactory bulbs and olfactory bulbs are of an oval shape (Figure 1c).

Downloaded by [Institute of Zoology] at 23:59 13 December 2013 and examined under the Zeiss stereoscope binocular microscope to study details of olfactory rosettes and Table I shows the average length of fish (85.1 mm), their relationship with the fish brain. The olfactory length of the head (22.4 mm), eye diameter organs were exposed and measured and were dis- (3.0 mm), distance between the snout of the fish sected out immediately and fixed in 70% alcohol. and the anterior nostril (5.5 mm), distance between Later, olfactory lamellae of the rosette were removed posterior nostril and eye length (1.6 mm), width of one by one from each rosette under the binoc- the olfactory tract (1.8 mm) and diameter of poste- ular scope. The olfactory organ was divided into rior nostril (1.4 mm). The standard deviation is also three portions, and from each portion two lamellae indicated at the bottom of each column of the table, were fixed in 70% alcohol for the scanning electron against the mean. microscope (SEM). The rosette contains a central elongated raphe; the For SEM, the dehydrated samples were dried lamellae originate from the raphe, which are 10 in number on each side (Figure 2b). The size and with liquid carbon dioxide (CO2) using the criti- cal point dryer CPD 030 and sputter coater SCD shape of the lamellae vary according to their posi- 005. The samples were observed with the help tion on the rosette. The length of posterior lamellae of Environmental Scanning Electron Microscope (near the water outlet) is greater; it is a tongue- QUANTA 200. like structure and well developed compared to the Surface ultrastructure of the olfactory epithelium of Triplophysa dalaica 197

Figure 1. Triplophysa dalaica: a, full-size picture; b, dorsal view of the head showing the anterior nostril, posterior nostril, and the nasal flap; c, antero-dorsal view of the head showing the olfactory organs, cerebral hemisphere, olfactory tract, and the olfactory bulb. ANO, anterior nostril; CH, cerebral hemisphere; NF, nasal flap; OB, olfactory bulb; PNO, posterior nostril; OR, olfactory organs; OT, olfactory tract.

Table I. Length of olfactory sac and width to eye diameter ratios for Triplophysa dalaica (mean ± Se; n = 10).

No Standard Head Eye Distance B/TA. Distance B/TP. Olfactory P. No s t r i l Length Length diameter Nostril & Snout Nostril & Eye L/W diameter

1 40.0 16.9 2.4 4.0 1.1 2.0/1.4 1.2 2 49.1 21.0 2.5 6.0 1.5 2.2/1.3 1.0 3 92.0 22.5 3.2 5.9 1.3 2.7/1.4 2.0 4 88.0 21.5 3.0 4.6 1.7 2.5/1.4 1.4 5 91.0 22.7 3.4 5.6 1.4 2.5/1.4 1.8 6 97.8 24.0 3.1 6.0 1.6 2.9/1.3 1.2 7 98.5 24.3 3.0 5.2 1.8 2.5/1.4 1.5 8 92.6 21.6 2.8 4.4 1.7 2.5/1.5 1.3 9 100.8 25.9 3.6 7.4 1.8 2.6/1.5 1.6 10 101.1 23.4 3.1 6.0 1.8 2.6/1.3 1.2 Mean ± SD 85.1 ± 21.9 22.4 ± 2.4 3.0 ± 0.4 5.5 ± 1.0 1.6 ± 0.2 1.8 ± 0.2 1.4 ± 0.3

B/T, between; A., anterior; P., posterior; L, length; W, width; SD, standard deviation. Downloaded by [Institute of Zoology] at 23:59 13 December 2013 anterior lamellae (near the water inlet). The anterior as well as the actual surface configuration of this lamellae are smaller at the distal part but gradually region of the lamellae. Mucus cells form the major increase in size towards the middle region of the part of the indifferent olfactory epithelium, and their rosette (Figure 2a). For structural observations of openings are clearly evident (Figures 3a, b, e). the lamellae the olfactory organ is divided into three In Figure 4d, supporting cells (SC) are visible regions: anterior, middle and posterior. Two lamellae in the olfactory epithelium. Among the receptor were examined from each region. On the basis of sur- cells the microvillus (MV) (Figures 2c, 4d) and a face characteristics and the distribution pattern of the separate portion of the lamellae (Figures 2f, 4d) are cell types, the surface of the lamellae is distinguished prominent. into sensory, ciliated non-sensory and non-ciliated Ciliated non-sensory cells are visible on all sur- non-sensory epithelium. faces of the lamellae except in Figures 2e, 3c The posterior portion of the lamellae covers the and 4a. Ciliated non-sensory cells were abun- innermost proximal oval region of the lamellae, dant in the middle portion of the lamellae and which is the portion of ciliated non-sensory cells are extended to mask other types of cells present (CN) (Figure 1b). Figure (3b and e) show the region on the surface of the organ. In addition to with profuse cilia, which covers the other cell types ciliated receptor cells and ciliated non-sensory cells, 198 B. Waryani et al.

Figure 2. Triplophysa dalaica: Scanning Electron Microscope (SEM) of different parts of the olfactory rosette. a, whole olfactory organ an adult fish, lamellae and raphae; b, raphae and parallel arranged lamellae; c, lamellae showing ciliated non-sensory cells and non-ciliated non-sensory cells; d, lamellae showing ciliated non-sensory cells on the middle region of the lamellae; e, on the posterior part of the lamellae large receptor cells of olfactory knobs of ciliated are present around the larger pits, mucus droplets are visible and microvillous shown in this area; f, middle part of lamella showing ciliated non-sensory cells, microvillus of supporting cells scattered and rod cells. CN, ciliated non-sensory cells; CR, ciliated receptor cells; L, lamellae; M, mucus drop; MV, microvillous; NN, non-ciliated non-sensory cells; P, pits; Downloaded by [Institute of Zoology] at 23:59 13 December 2013 R, raphae. Scale bars: a, 1.0 mm. b, 100.0 µm. c, 20.0 µm. d, 10.0 µm. e, 10.0 µm. f, 5.0 µm.

occasionally a few rod-shaped cells were present in non-receptor cells indicating the regional dominance the ciliated sensory area (Figure 4d). Small pits of a particular cell type. Supporting cells lie on the were present all over the surface of the lamellae, surface of the lamellae (Figures 2f and 3f). A number which were considered the openings of the mucus of mucus cells were present in the middle region cells. of the lamellae (Figure 3c). Rod-shaped cells, in The middle portion of the lamellae displays a con- addition to ciliated receptor cells and ciliated non- siderable number of ciliated non-sensory cells, which sensory cells, appeared in the ciliated sensory area are arranged in the form of ciliated tufts (Figures 2d (Figures 3d and f). A single cilium is formed by and f, 3d and f). The ciliated receptor cells (CR) are elongation of the tubercle at the top of ciliated cells. found scattered in the mass of ciliated non-receptor In the same part of the lamellae some of the pits of cells, which shows that 4–6 ciliated processes arise mucous balls are clearly visible (Figure 4a). from the cell bodies of these receptor cells (Figures 3 According to SEM studies, the non-receptor c and d). In the same part of the lamellae numerous epithelium is made up of patches of ciliated non- microvilli (MV) are scattered in between the ciliated sensory cells among which the glandular duct cells Surface ultrastructure of the olfactory epithelium of Triplophysa dalaica 199

Figure 3. Triplophysa dalaica: a, SEM of anterior region of the lamellae a clump of ciliated non-sensory cells showing and the pits are spread in this region; b, ciliated non-sensory region of the anterior part of the lamellae showing mucus droplets and pits in this region; c, middle part of the lamellae showing ciliated receptor olfactory knob cells are abundant, pits (P) are visible and arrow showing the mucus droplets; d, middle part of the lamellae showing large receptor cells of olfactory knobs of ciliated non-sensory cells present in this part, rod cells are clearly visible and arrow shows the mucus droplets; e, anterior part of the lamellae showing ciliated receptor cells, pits are visible and large mucus drop is present; f, middle part of the lamellae showing ciliated non-sensory cells surroundings, microvilli among the non-sensory Downloaded by [Institute of Zoology] at 23:59 13 December 2013 area and rod cells are clearly visible, pits also present and arrow shows the mucus droplets. CN, ciliated non-sensory cells; CR, ciliated receptor cells; M, mucus drop; MV, microvillous; P, pits; RD, rod cells. Scale bars: a, 20.0 µm. b, 20.0 µm. c, 10.0 µm. d, 10.0 µm. e, 10.0 µm. f, 10.0 µm.

are present which are covered with ciliated non- (B) dense cells in the ciliated non-sensory area in sensory cells. Ciliated receptor cells show irregular which single cells cannot be seen (Figure 4b). The knob-like structures on the surface of the olfactory pits are clearly visible in all the portions of the epithelium (Figure 2e). In the ciliated sensory area, lamellae. numerous ciliated receptor cells are distributed with In histological micrographs the olfactory ciliated non-sensory cells (Figure 4c). On the sur- epithelium (OE) of Triplophysa dalaica encloses face of the olfactory epithelium the non-sensory area a central lumen called the central core (CC), which shows roughly ciliated non-sensory cells (Figure 4b). contains blood vessels (BV), connective tissues and Ciliated non-sensory cells show two patterns of dis- nerve fibers. The olfactory epithelium is separated by tributions on the lamellae: (A) loose or even sin- the basement membrane (BM) (Figures 5a–f). Crypt gle cells in the ciliated sensory area, intercalated cells (CRC) on the surface of the olfactory rosette are with ciliated receptor cells (Figures 2e and 4c); few in number (Figures 5a–c, e). Olfactory lamellae 200 B. Waryani et al.

Figure 4. Triplophysa dalaica: a, SEM of anterior part of the lamellae is covered with the pits and mucus droplets; b, posterior part of the lamellae showing the micro-ridge and the ciliated non-sensory cells in this region; c, posterior part of the lamellae showing the ciliated non-sensory cells and the pits; d, anterior part of the lamellae showing the ciliated non-sensory cells, surroundings microvillus among the non-sensory area, large pits are visible and the rod cells are clearly present in this region. CN, ciliated non-sensory cells; M, mucus drop; MV, microvillous; P, pits; RD, rod cells. Scale bars: a, 20.0 µm. b, 20.0 µm. c, 20.0 µm. d, 10.0 µm.

are connected with raphae (Fig 5b). Receptor cells cells are sensory in function. They form a part of the and microvillus cells (MC) are present on the olfactory transduction mechanism, which is stimu- surface of the olfactory epithelium (Figures 5b–d). lated by odour-bearing substances and detection of Scattered sensory receptor cells are distributed on food (Zeiske et al. 2003). From histological obser- the non-sensory surface area of the olfactory lamellae vations a small number of crypt cells were found (Figures 5d–f). in the lamellae. Crypt cells were also found in cat- fish, sword tail, and needle fish (Hansen et al. 1997). Hansen & Zeiske (1998) reported that the crypt cell Discussion is a receptor neuron in the peripheral olfactory organ

Downloaded by [Institute of Zoology] at 23:59 13 December 2013 A wide range of diversity in surface area and arrange- of the zebra fish, Danio rerio. ment of the olfactory epithelium of teleosts has been In Schizothorax plagiostomus, olfactory epithelium discussed in a number of studies (Kleerekoper 1969; is characterized by ciliated non-receptor cells, ciliated Yamamoto 1982; Hansen & Zielinski 2005). Zielinski receptor cells and microvillus cells. In S. richard- and Hara (2001) also correlated the variations in the sonii, mucus cells and patches of sensory epithe- morphology of the teleost olfactory organ according lium were predominant (microvillus and rod cells). to its ecological and phylogenic parameters. In the In S. plagiostomus and Schizothoraichthys progastus present study of T. dalaica the surface of the olfactory ciliated receptor cells and patches of microvillus cells lamellae consists of ciliated non-sensory cells, non- occurred in the mass of indifferent epithelium, and ciliated non-sensory cells, receptor cells, microvillus, the ciliated non-receptor cells were found in many and crypt cells. The olfactory epithelium of channel clusters forming ciliary tufts (Yamamoto 1982). catfish contains three intermingled types of olfactory In the olfactory epithelium of gadoid fishes simi- receptor neurons: ciliated, microvillus, and crypt, lar ciliary aggregations appeared but with a different which are responsible for the detection of bile salt configuration (Lowe 1974). Ciliated receptor cells and amino acid odorants (Hansen et al. 2003). The and olfacto-sensory functions were also reported by present study reveals that the receptor cells domi- Lowe and Macleod (1974), Zielinski & Hara (1988), nate the microvillus and crypt cells. The receptor and Hara & Zielinski (1989). Surface ultrastructure of the olfactory epithelium of Triplophysa dalaica 201

Figure 5. Triplophysa dalaica: a, section of the lamellae showing olfactory epithelium, central core, which connects with the blood vessels and crypt cells are present on the surface of olfactory lamellae; b, olfactory lamellae are connected with raphae, separated by central core olfactory epithelium showing crypt cell and scattered microvillus cells; c, sensory olfactory epithelium showing microvillus cells, crypt cell and blood vessels in central core; d, section of non-sensory olfactory epithelium showing microvillus cells, receptor cells, and blood vessels; e, olfactory lamellae showing presence of the basement membrane in between central core connected with blood vessels, olfactory epithelium, crypt cell is present and scattered receptor cells on the surface of the epithelium; f, olfactory lamellae showing olfactory epithelium, central core connected with blood vessels, non-sensory epithelium showing receptor cells. BM, basement membrane; BV, blood vessels; CC, central core; CRC, crypt cells; MC, microvillus cells; MV, microvillous; OE, olfactory epithelium; R, raphae.

In this study it was observed that rod-shaped cells area, numerous ciliated receptor cells are seen inter- were present in the ciliated sensory area. Small pits calating with loosely distributed ciliated non-sensory present all over the surface of the lamellae were con- cells (Figure 4c).

Downloaded by [Institute of Zoology] at 23:59 13 December 2013 sidered to be openings of the mucus cells. Yamamoto Yamamoto (1982) suggested an ecomorphologi- (1982) stated that the appearance of rod cells is cal system for classifying fish olfactory organs with a sign of aging of ciliated receptor cells and sug- regard to shape, number of lamellae, and the dis- gested that the ciliated receptor cells with a tubercle tribution pattern of the sensory epithelium. The topped with a single cilium is probably a transitional general shape of the rosette and arrangement of the type from non-ciliated to rod cells. The single cil- lamellae are identical in Triplophysa dalaica, which ium may be formed by elongation of the tubercle resembles Yamamoto’s category Type G: olfactory at the top. In the same part of the lamellae some rosettes with fishbone-shaped lamellae. Following of the pits and mucous balls are clearly visible. this classification for the distribution pattern of the According to SEM studies the non-receptor epithe- ciliated epithelium of olfactory lamellae, the arrange- lium is made up of patches of ciliated non-sensory ment in Triplophysa dalaica is Type I, according to cells among which the glandular duct cells typi- the findings of Zeiske et al. (1992). This classifica- cally showed the whole part of the lamellae covered tion shows that in older olfactory organs, the larger with ciliated non-sensory cells. The surface of the the rosette, the more lamellae. However, the number olfactory cilia was not smooth, but showed irregular of lamellae remains constant after the fish reaches knob-like profiles (Figure 2e). In the ciliated sensory a certain stage in growth (Yamamoto 1982). In the 202 B. Waryani et al.

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