Histochemical and immunohistochemical study was carried out on nitrinergic innervation and neuroendocrine
system in the gill epithelium of the abyssal fish Coelorhynchus coelorhynchus. The results showed that nNOS-positive nerve
fibers, originating from the branchial arch were present in the subepithelial tissue of branchial primary filament. nNOS-positive
neuroendocrine cells were also present in the primary filaments and secondary lamellae. Numerous mucous cells in the gill
epithelium were AB/PAS-positive, while sialic acid was absent as confirmed by neuraminidase reaction and WGA lectin
histochemistry. The mucus compounds in abyssal teleost fish are different from those found in pelagic species, being related
to their living conditions. In abyssal species, greater numbers of chloride and neuroendocrine cells are involved in the movement
of water and electrolytes. Neuroendocrine cells possess oxygen receptors which mediate the cardiovascular and ventilatory
response to oxygen deficiency, as reported in teleost species. Besides, NO contributes through nervous stimulation to the
regulation of vascular tone and blood circulation in the gill

Albanese, Maria Pia

Calabrò, Concetta

Lauriano, Eugenia Rita

Licata, Aurelio

Martella, Silvestro

English

Via Medica Journals

FOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 43, No. 1, 2005pp. 51-56Morphological, histochemical and immunohistochemicalstudy of the gill epithelium in the abyssal teleost fishCoelorhynchus coelorhynchusConcetta Calabrò, Maria Pia Albanese, Eugenia Rita Lauriano, Silvestro Martellaand Aurelio LicataDepartment of Animal Biology and Marine Ecology, Faculty of Science, University of Messina, ItalyAbstract: Histochemical and immunohistochemical study was carried out on nitrinergic innervation and neuroendocrinesystem in the gill epithelium of the abyssal fish Coelorhynchus coelorhynchus. The results showed that nNOS-positive nervefibers, originating from the branchial arch were present in the subepithelial tissue of branchial primary filament. nNOS-positiveneuroendocrine cells were also present in the primary filaments and secondary lamellae. Numerous mucous cells in the gillepithelium were AB/PAS-positive, while sialic acid was absent as confirmed by neuraminidase reaction and WGA lectinhistochemistry. The mucus compounds in abyssal teleost fish are different from those found in pelagic species, being relatedto their living conditions. In abyssal species, greater numbers of chloride and neuroendocrine cells are involved in the movementof water and electrolytes. Neuroendocrine cells possess oxygen receptors which mediate the cardiovascular and ventilatoryresponse to oxygen deficiency, as reported in teleost species. Besides, NO contributes through nervous stimulation to theregulation of vascular tone and blood circulation in the gill. Key words: Gill - nNOS - Glycoconjugates - Lectins - Abyssal fish IntroductionThe mucus of fish gill epithelia is involved in manybiological activities, such as swimming and defenceagainst pathogenic microorganisms, parasites and pol-lutants [8]. The mucus contains sialic acid responsiblefor reduction of the pH [10], glycoconjugates [6, 9, 14]and many other compounds. The demonstration ofnNOS in the invertebrate epithelial cells [12, 13] and inthe gill epithelia of the catfish [34, 35], has suggested aregulatory role of NO in the secretory function. NOcontrols smooth muscle relaxation [18], the water-saltbalance [15] and is involved in a diffuse chemoreceptorsystem sensitive to hypoxia, as shown in marine andfreshwater teleost fishes [1-3, 7, 11, 25-31]. In the fishgills, the subepithelial nNOS- and VIP-positive nervefibers act as non-adrenergic non-cholinergic (NANC)transmitters in the neuronal inhibitory regulation of gillrespiration [16]. They also play a similar role in inver-tebrates with cutaneous respiration [12]. In the gill epi-thelia nNOS-positive neuroendocrine cells are involved,by paracrine mechanism, also in the modulation of epi-thelial integrity, secretion of mucus [21] and water andsalt balance [5, 30-35]. An increase in the number ofneuroendocrine cells has been shown in recent studieson fish gill in the environment with oxygen deficiency[17]. The aim of the present study was to investigate theglycoconjugate distribution pattern and nitrinergic in-nervation of the gill epithelium in the abyssal fishCoelorhynchus coelorhynchus in order to clear the in-volvement of NO in the neuronal regulation of thebranchial epithelia function in environments withoxygen deficiency. Materials and methods Specimens of Coelorhynchus coelorhynchus, caught in the Straits ofMessina (Italy), were transferred to our laboratory where dissectionof gills was carried out. The dissected tissue samples were fixed inBouin or Carnoy fluids. For histochemical and immunohistochemi-cal study, tissue samples were fixed in 4% paraformaldehyde buf-fered with 0.01 M sodium phosphate (PBS), pH 7.4, at 4˚C for 2 hand embedded in paraplast.Serial sections (5 µm thick) were stained with Mallory mod.Ignesti (Carazzi hemalum, 1% acid fuchsin, Mallory’s solution) andCorrespondence: A. Licata, Dept. Animal Biology and MarineEcology, University of Messina, Contrada Sperone 31, 98166 S. Agata Messina, Italy; e-mail: alicata@unime.it52 C. Calabro et al.Galgano I (Mayer’s hemalum, 0.1% acid fuchsin, 1% phosphomo-lybdic acid, Mallory’s solution) methods. Neuraminidase enzymatic digestion was carried out by Vibriocholerae neuraminidase (500U/ml) diluted in acetate buffer, pH 5.5,for 16-24 h at 39-41˚C. The conventional histochemical methodsused in this study are reported in Table 1.The following biotinylated lectins (SIGMA, St. Louis MO, USA)were tested: ConA, WPA, WGA, SBA, PHA-L. The lectins and theirsugar specifities are shown in Table 2. Sections were incubated withthe corresponding inhibitory sugar in control experiments to test thespecificity of lectin binding. To reveal nNOS, tissue sections were incubated overnight at 4˚Cin a humid chamber with the polyclonal anti-human brain nNOSantibodies, diluted 1:200 (rabbit anti-nNOS type I antibodies, code606-259-1550; Biomol, Hamburg, Germany). Afterwards, sectionswere washed in PBS and incubated for 1 h with a goat anti-rabbitIgG-peroxidase conjugate (1:100; Sigma Munich, Germany). Perox-idase activity was visualized by incubation of the sections for 5-12min at room temperature in a solution of 0.015% 3,3’-diaminoben-zidine in 0.01 M Tris buffer, pH 7.6, containing 0.005% H2O2.Negative controls included omission of the primary antibody or itssubstitution with non-immune rabbit serum.ResultsThe gills of Coelorhynchus coelorhynchus are formedby four holobranchs, each consisting of two hemi-branchs with primary filaments and secondary lamellae.Fig. 1. PAS-positive mucous cells in primary filament of the abyssal fish C. coelorhynchus. × 1400. Fig. 2. AB/PAS pH 1-stained mucouscells in the primary filament of the branchial epithelia. × 1400. Fig. 3. Some AB-stained cells (arrows), after neuraminidase digestion, showingabsence of sialic acid in the gill epithelia. × 900. Figs. 4, 5. Mucous cells in the epithelium of secondary lamellae positive to PHA-L (Fig.4) and SBA (Fig. 5) lectins. Fig. 4: × 900; Fig. 5: × 1400. Figs. 6, 7. No labelling for ConA (Fig. 6) and WGA (Fig. 7) in the gill epithelia.× 750. Fig. 8. Some nNOS-positive neuroendocrine cells (arrows) in the gill epithelium. × 750. Fig. 9. In the epithelium of primary filamentand secondary lamellae, nNOS-positive neuroendocrine cells (arrows) are seen. × 900. Fig. 10. nNOS-positive neuroendocrine cells (arrows)in the outer layer of gill epithelium. × 1400. Fig. 11. nNOS-positive neuroendocrine cells (arrows) at the base of secondary lamellae and inthe primary filaments. × 1400. Fig. 12. nNOS-positive nerve fibers (arrows) innervating the gill epithelium can be seen in the connectivetissue. × 1400.Table 1. Conventional histochemical methods for visualization andidentification of glycoconjugates (GCs) Procedures GCs revealedPAS GlycoproteinsAB pH 1 Proteoglycans with sulphate group AB pH 2.5 Proteoglycans with carboxyl groupsAB pH 2.5/PAS Proteoglycans with carboxyl groupsand glycoproteins Neuraminidase digestion Sialic acidAB (C.E.C.) Sialic acidGlycoconjugates and nNOS in the gill of C. coelorhynchus                53The gill filaments are vascularized by afferent and effer-ent arteries. The filament and lamellae epithelium con-tain pillar, chloride and mucous cells. After Mallory(mod. Ignesti) and Galgano I staining the cytoplasm ofthe pillar and chloride cells was red-orange. Chloridecells were found only in the primary filaments and ininterlamellar zones. In the secondary lamellae, numer-ous capillaries, deriving from the afferent and efferentbranchial arteries, were beneath the epithelium consist-ing of two layers of cells.Mucous cells in the primary filament and secondarylamellae were PAS- (Fig. 1) and AB at pH 1-PAS-posi-tive (Fig. 2). After C.E.C. staining at pH 5.8 and 0.2 Mof MgCl2, the alcianophylia decreased, confirming thepresence of acid proteoglycans with carboxyl groups.Sialomucins were absent after acid hydrolysis accordingto Quintarelli et al. [19] and after neuraminidase diges-tion (Fig. 3), with and without saponification, followedby staining with AB at pH 2.5. By lectin method, the mucous cells in the primaryfilaments and secondary lamellae were positive forPHA-L (Fig. 4) and SBA (Fig. 5) and negative for ConA(Fig. 6) and WPA. The cells that showed no staining withWGA indicated the absence of GlcNac and sialic acid(Fig. 7).At the base of interlamellar space and in the epithe-lium of secondary lamellae, nNOS-positive neuroendo-crine cells were found (Figs 8-10). In the subepithelialtissue of primary filaments, nNOS-positive nerve fibersoriginating from the branchial arch, were observed (Fig.11). Controls for nNOS, after omission of the primaryantibody, were negative.DiscussionOur results have shown in the gill epithelium of Coelor-hynchus coelorhynchus numerous glandular mucouscells with different chemical composition. In fact, themucous cells showed glycoproteins and acid proteogly-cans with sulphate or carboxyl groups. This is in accord-ance with results reported in Anguilla anguilla [4], inMugil cephalus and in Anoptichthys jordani [22, 23].Sialic acid absence was confirmed by neuraminidasedigestion and WGA staining. The absence of glucoseand mannose residues as well as sialic acid does notagree with previous observations regarding the chemicalcomposition of mucus in the gills of pelagic fishes [9].The mucus compounds differ in various teleost speciesaccording to their different living conditions. The differ-ent chemical composition of the mucus in the gills couldbe correlated with the abyssal life of C. coelorhynchus.The absence of sugar residues involved in oxygen recep-tion, revealed by negative WPA and WGA results, doesnot support the oxygen diffusion. In the freshwater fish, Barbus filamentosus, adaptedto sea water, the excretion of NaCl was related to anincrease in the number of chloride cells with Mg-de-pendent ATPase activity in the primary filaments [24].In C. coelorhynchus, chloride cells are also present inclusters, as an excretory gland, in the primary filament.The NaCl excretion occurs through the vascular networkin the subepithelial connective tissue of the primaryfilaments, as shown in other species [11]. With respectto pelagic fishes, osmoregulation in this abyssal speciesrequires a greater abundance of cells involved in thatactivity. This was confirmed by the presence of nNOS-positive neuroendocrine cells in the primary filaments;such cells are involved in the movement of water andelectrolytes as shown in other teleost species [30-32, 34,35]. Some cells of the diffuse neuroendocrine systemseem also to possess receptors for oxygen that mediatethe cardiovascular and ventilatory response to oxygendeficiency, as shown in the catfish Heteropneustesfossilis [16]. In conditions of low oxygen exchange infishes, the release of NO contributes, through nervousstimulation, to the regulation of vascular tone in thegills. As shown in higher vertebrates, vasoactive sub-stances like NO control the pulmonary blood circula-tion [20].In the subepidermal connective tissue, nNOS-posi-tive nerve fibers were observed in proximity to theafferent arterial ramifications and in proximity to theneuroendocrine cells. In H. fossilis [15], nNOS-positivenerve fibers were associated with ramifications of theefferent arterioles and located in proximity to neuroen-docrine cells at the base of the epithelium. The bioactivesubstances produced by cells of the diffuse neuroendo-crine system (DNES) have important paracrine activitiesin osmoregulation and chemoreception of chemicalagents. Neuroendocrine cells, in response to neuronal orTable 2. Lectins employed and their major binding specificitiesLectins Origin Carbohydrate specificity Inhibitor sugarConA Canavalia ensiformis α-D-mannose,  α-D-glucose D-MannPHA-L Phaseolus vulgaris oligosaccharides D-GalNAcSBA Glycine max N-acetyl-D-galactosamine> D-galactose D-galNAcWPA Lotus tetragonolobus α-L fucose L-fucose WGA Triticum vulgaris N-acetyl-D-glucosamine> sialic acid D-GlcNac54 C. Calabro et al.environmental stimuli, release NO by a paracrine mech-anism for local regulation of muscle tone, as alreadysuggested in the gills of H. fossilis [15].The most important result of the present study is thedemonstration of numerous neuroendocrine cells in theprimary filaments and secondary lamellae and ofchloride cells only in the primary filaments, which couldbe related to the abyssal habitat of this species. In fact,the DNES is involved in oxygen reception and thereleased NO regulates several functional activities in-cluding those of chloride cells, such as the excretion ofsalts. This is the first data about the nitrinergic innervationand the DNES in the gills of an abyssal species. Furtherstudies are necessary to clarify the role of NO andneuroendocrine cells and their involvement in theoxygen reception in abyssal fish. References [1] Ainis L, Fasulo S, Licata A, Zaccone G (1991) On the occurenceof neuroendocrine cells in the fish gill: an immunohistochemicalstudy. Atti Acc Pelor Sc Fis Mat Nat 49: 291-305  [2] Bailly Y, Dunel-Erbe S, Geffard M, Laurent P (1989) Thevascular and epithelial serotonergic innervation of the actinop-terygian gill filament with special reference to the trout, Salmogairdneri. Cell Tissue Res 258: 349-363 [3] Bailly Y, Dunel-Erb S, Laurent P (1992) The neuroepitelial cellsof the fish gill filaments: indolamine immunocytochemistry andinnervation. Anat Rec 233: 143-161  [4] Colombo G (1960) Ricerche istochimiche sulla funzione os-moregolatrice delle branchie nelle anguille europee (Anguillaanguilla L.). Arch Ocean Limnol 12: 117-130 [5] Dunel-Erb S, Bailly Y, Laurent P (1982) Neuroepithelial cellsin fish gill primary lamellae. J Appl Physiol 53: 1342-1353 [6] Fasulo S, Licata A, Contini A, Ainis L, Mauceri A, Zaccone G.(1991) Peroxidase localization of lectin binding sites on plasmamembrane of the surface epidermis in the rusty blenny, Blenniussanguinolentus (Pallas, 1811). Acta Zool 72: 217-222 [7] Fasulo S, Mauceri A, Tagliaferro G, Ricca MB, Lo Cascio P,Ainis L (1998) Immunoreactivity to calcium-binding proteins(CaBPs) in the epithelia of skin and gill of the catfish, Heterop-neustes fossilis. Ital J Zool 65: 149-153 [8] Fletcher TC (1978) Defence mechanism in fish. J HistochemCytochem 32: 681-689  [9] Harris J, Hunt S (1973) Epithelial mucins of the Atlantic salmon(Salmon salar L). Biochem Soc Trans 1: 153-155[10] Kirschner LB (1978) External change and Na+ regulation. In:Osmotic and volume regulation. Alfred Benzon Symposium 11.Academic Press, New York, pp 310-321 [11] Laurent P, Dunel-Erbe S (1980) Morphology of gill epithelia infish. Am J Physiol 238: 147-159[12] Licata A, Ainis L, Martella S, Ricca MB, Licata P, Lauriano ER,Zaccone G (2002). Immunohistochemical localization of nNOSin the nerve fibers of the earthworm Lumbricus terrestris L(Annelida, Oligochaeta). Acta Histochem 104: 289-295[13] Licata A, Ainis L, Martella S, Ricca MB, Licata P, Pergolizzi SCalabro C, Zaccone G (2003) Immunohistochemical localiza-tion of neural nitric oxide synthase and VIP in the mantleintegument of the mussel, Mytilus galloprovincialis. Acta His-tochem 105: 143-149 [14] Manfredi-Romanini MG (1992) Polysaccharides and glycon-jugates in nonmammalian vertebrates. In: Handbuch der Histo-chemie. Graumann W [Ed], Gustav Fischer Verlag, Stuttgart-Jena-New York, pp 259-315[15] Martinez A (1995) Nitric-oxide synthase in invertebrates. His-tochem J 27: 770-776[16] Mauceri A, Fasulo S, Ainis L, Licata A, Lauriano ER, MartinezA, Mayer B, Zaccone G (1999) Neuronal nitric oxide synthase(nNOS) expression in the epithelial neuroendocrine cell systemand nerve fibres in the gill of the catfish, Heteropneustes fossilis.Acta Histochem 101: 437-448[17] Mauceri A, Tigano C, Ferrito V, Barbaro B, Calderaro M, AinisL, Fasulo S (2002) Effect of natural confinement on the gill celltypes and bony elements of Lebias fasciata (Teleostei, Cypri-nodontidae): a morphological and immunohistochemical ana-lysis. Ital J Zool 69: 195-203[18] Moncada S, Radomski MW, Palmer RMJ (1988) Endothelium-derived relaxing factor: identification as nitric oxide and role inthe control of vascular tone and platelet function. BiochemPharmacol 37: 2495-2501 [19] Quintarelli G, Tsuiki S, Hashimoto Y, Pigmanw W (1961)Studies of sialic acid containing mucin in bovine submaxillarand rat sublingual glands. J Histochem Cytochem 9: 176-183[20] Shepherd JT, Vanhutte PM (1991) Endothelin derived relaxing(EDRF) and contracting factors (EDCF) in the control of car-diovascular homeostasis: the pioneering observations. In: Car-diovascular Significance of Endothelium-Derived VasoactiveFactors. Rubanyi G [Ed], Futura, New York, pp 39-45 [21] Whittle BJR, Lopez-Belmonte J, Moncada S (1990) Regulationof gastric mucosal integrity by endogenous nitric oxide: inter-actions with prostanoids and sensory neuropeptides in the rat.Br J Pharmacol 99: 607-611[22] Zaccone G (1972) Comparative histochemical investigations onthe mucous cells of the branchial epithelium of Mugil cephalusL. and Anoptichthys jordani Hubbs and Innes. Acta Histochem44: 106-115[23] Zaccone G (1973) Morphochemical analysis of the mucous cellsduring the development of the respiratory tract in Mollienesiasphenops Cuvand Val (Teleostei Poeciliidae). Acta Histochem47: 233-243 [24] Zaccone G (1981) Effect of osmotic stress on the chloride andmucous cells in the gill epithelium of the freshwater teleostBarbus filamentosus (Cypriniformes, Pisces). A structural andhistochemical study. Acta Histochem 68: 147-159 [25] Zaccone G, Tagliaferro G, Goniakowska-Witalinska L, FasuloS, Ainis L, Mauceri A (1989) Serotonin-like immunoreactivecells in the pulmonary epithelium of ancient fish species. His-tochemistry 92: 61-63[26] Zaccone G, Goniakowska-Witalinska L, Lauweryns JM, FasuloS, Tagliaferro G (1989). Fine structure and serotonin immuno-histochemistry of the neuroendocrine cells in the lung of thebichirs Polypterus delhezi and P. ornatipinnis. Bas Appl His-tochem 33 : 277-287[27] Zaccone G, Fasulo S, Tagliafierro G, Licata A, Ainis L, RiccaMB (1990) Immunocytochemistry of neuroepithelial cells in thegills of teleost and lungfish species. Proc. 15th Conference ofESCE, Leuven (Belgium), p 100[28] Zaccone G, Lauweryns JM, Fasulo S, Tagliafierro G, Ainis L,Licata A (1992a) Immunocytochemical localization of sero-tonin and neuropeptides in the neuroendocrine paraneurons ofteleost and lungfish gills. Acta Zool 73: 177-183[29] Zaccone G, Wendelaar-Bonga S, Flik G, Fasulo S, Licata A, LoCascio P, Mauceri A, Lauriano ER (1992) Localization ofcalbindin D28K-like and immunoelectron histochemical study.Regul Pept 41: 195-208[30] Zaccone G, Fasulo S, Ainis L (1995) Neuroendocrine epithelialcell system in the respiratory organs of air breathing and teleostfishes. Int Rev Cytol 157: 277-314Glycoconjugates and nNOS in the gill of C. coelorhynchus                55[31] Zaccone G, Mauceri A, Ainis L, Fasulo S, Lo Cascio P, RiccaMB (1996) Localization of immunoreactive endothelin in theneuroendocrine cells of fish gill. Neuropeptides 30: 53-57[32] Zaccone G, Fasulo S, Ainis L, Licata A (1997) Paraneurons inthe gills and airways of fishes. Microsc Res Tech 37: 4-12[33] Zaccone G, Kapoor BG, Fasulo S, Ainis L (2001) Structural,histochemical and functional aspects of the epidermis of fishes.Adv Mar Biol 40: 255-347[34] Zaccone G, Mauceri A, Ainis L, Licata A, Fasulo S (2003) Nitricoxide synthase in the gill and air sac of of the Indian catfish,Heteropneustes fossilis,. In: Catfishes. Kapoor BG, Arratia G,Chardon, M, Diogo R [Eds], Oxford and IBH Publishing, NewDelhi-Calcutta, pp 771-788[35] Zaccone G, Ainis L, Mauceri A, Lo Cascio P, Lo Giudice F,Fasulo S (2003) NANC nerves in the respiratory air sac andbranchial vasculature of the Indian catfish, Heteropneustesfossilis. Acta Histochem 105: 151-163Received May 25, 2004Accepted after revision July 30, 200456 C. Calabro et al.

Morphological, histochemical and immunohistochemical study of the gill epithelium in the abyssal teleost fish Coelorhynchus coelorhynchus

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Morphological, histochemical and immunohistochemical study of the gill epithelium in the abyssal teleost fish Coelorhynchus coelorhynchus

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