The distribution of mitochondria-rich cells in the gills of air-breathing fishes

Respiration and ion regulation are the two principal functions of teleostean gills. Mainly found in the gill filaments of fish, mitochondria-rich cells (MRCs) proliferate to increase the ionoregulatory capacity of the gill in response to osmotic challenges. Gill lamellae consist mostly of pavement cells, which are the major site of gas exchange. Although lamellar MRCs have been reported in some fish species, there has been little discussion of which fish species are likely to have lamellar MRCs. In this study, we first compared the number of filament and lamellar MRCs in air-breathing and non-air-breathing fish species acclimated to freshwater and 5 g NaCl L(-1) conditions. An increase in filament MRCs was found in both air-breathing and non-air-breathing fish acclimated to freshwater. Lamellar MRCs were found only in air-breathing species, but the number of lamellar MRCs did not change significantly with water conditions, except in Periophthalmus cantonensis. Next, we surveyed the distribution of MRCs in the gills of 66 fish species (including 29 species from the previous literature) from 12 orders, 28 families, and 56 genera. Our hypothesis that lamellar MRCs are more likely to be found in air-breathing fishes was supported by a significant association between the presence of lamellar MRCs and the mode of breathing at three levels of systematic categories (species, genus, and family). Based on this integrative view of the multiple functions of fish gills, we should reexamine the role of MRCs in freshwater fish.     

Gill morphology and morphometry of the facultative air-breathing armoured catfish,Corydoras paleatus,in relation on aquatic respiration

The Neotropical armoured catfish Corydoras paleatus is a facultative air-breathing teleost commonly exported as ornamental fish. In this species, air breathing enables it to survive and inhabit freshwater environments with low oxygen levels. Therefore, it is important to analyse the gills from a morphological aspect and its dimensions in relation to body mass with reference to aquatic respiration. For that, the gills were analysed using a stereoscopic microscope for morphometric studies, and structural and ultrastructural studies were carried out to compare the four branchial arches. Furthermore, two immunohistochemical techniques were used to locate and identify the presence of a Na⁺/K⁺ pump. The characterization of the potential for cell proliferation of this organ was assessed using an anti-PCNA antibody. The results show that gills of C. paleatus present some characteristics related to its diet and lifestyle, such as the limited development of gill rakers and the abundance of taste buds. In addition, other special features associated with the environment and bimodal breathing were observed: scarce and absent mucous cells (MCs) in the gill filaments and branchial lamellae, respectively, and the localization of mitochondria-rich cells (MRCs) covering the basal third of the branchial lamellae, which reduces the gill respiratory area. A peculiar finding in the gill epithelium of this armoured catfish was the presence of mononuclear cells with sarcomeres similar to myoid cells, whose functional importance should be determined in future studies. Finally, in C. paleatus, the interlamellar space of gill filaments is an important site for cell turnover and ionoregulation; the latter function is also performed by the branchial lamellae.     

Fish gill morphology: inside out

In this short review of fish gill morphology we cover some basic gross anatomy as well as in some more detail the microscopic anatomy of the branchial epithelia from representatives of the major extant groups of fishes (Agnathans, Elasmobranchs, and Teleosts). The agnathan hagfishes have primitive gill pouches, while the lampreys have arch-like gills similar to the higher fishes. In the lampreys and elasmobranchs, the gill filaments are supported by a complete interbranchial septum and water exits via external branchial slits or pores. In contrast, the teleost interbranchial septum is much reduced, leaving the ends of the filaments unattached, and the multiple gill openings are replaced by the single caudal opening of the operculum. The basic functional unit of the gill is the filament, which supports rows of plate-like lamellae. The lamellae are designed for gas exchange with a large surface area and a thin epithelium surrounding a well-vascularized core of pillar cell capillaries. The lamellae are positioned for the blood flow to be counter-current to the water flow over the gills. Despite marked differences in the gross anatomy of the gill among the various groups, the cellular constituents of the epithelium are remarkably similar. The lamellar gas-exchange surface is covered by squamous pavement cells, while large, mitochondria-rich, ionocytes and mucocytes are found in greatest frequency in the filament epithelium. Demands for ionoregulation can often upset this balance. There has been much study of the structure and function of the branchial mitochondria-rich cells. These cells are generally characterized by a high mitochondrial density and an amplification of the basolateral membrane through folding or the presence of an intracellular tubular system. Morphological subtypes of MRCs as well as some methods of MRC detection are discussed.     

Gills of the medaka (Oryzias latipes): A scanning electron microscopy study

The general morphology and surface ultrastructure of the gills of adult and larvae medaka (Oryzias latipes) were studied in freshwater and seawater using scanning electron microscopy. The gills of all examined fish were structurally similar to those of other teleosts and consisted of four pairs of arches supporting (i) filaments bearing lamellae and (ii) rakers containing taste buds. Three cell types, specifically pavement cells, mitochondria‐rich cells (MRCs), and mucous cells, constituted the surface layer of the gill epithelium. Several distinctive characteristics of medaka gills were noted, including the presence of regularly distributed outgrowth on the lamellae, enlarged filament tips, the absence of microridges in most pavement cells in the filament and lamellae and the presence of MRCs in the arch at the filament base. A rapid mode of development was recorded in the gills of larval fish. At hatching, the larvae already had four arches with rudimentary filaments, rakers, and taste buds. The rudimentary lamellae appeared within 2 days after hatching. These results suggest the early involvement of larval gills in respiratory and osmoregulation activities. The responses of the macrostructures and microstructures of gills to seawater acclimation were similar in larvae and adult fish and included modification of the apical surface of MRCs, confirming the importance of these cells in osmoregulation. The potential roles of these peculiarities of the macrostructures and microstructures of medaka gills in the major functions of this organ, such as respiration and osmoregulation, are discussed.     

Structures and immunolocalization of Na+, K+ -ATPase, Na+ /H+ exchanger 3 and vacuolar-type H+ -ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas

The structure and immunolocalization of the ion transporters Na(+) ,K(+) -ATPase (NKA), Na(+) /H(+) exchanger (NHE3) and vacuolar-type H(+) -ATPase (VHA) were examined in the gills of teleosts of the family Blenniidae, which inhabit rocky shores with vertical zonation in subtropical seas. These features were compared among the following species with different ecologies: the amphibious rockskipper blenny Andamia tetradactylus, the intertidal white-finned blenny Praealticus tanegasimae and the purely marine yaeyama blenny Ecsenius yaeyamaensis. Light and electron microscopic observations indicated that thick gill filaments were arranged close to each other and alternately on two hemibranches of a gill arch in the opercular space of A. tetradactylus. Many mucous cells (MC) and mitochondrion-rich cells (MRC) were present in the interlamellar regions of the gill filament. An immunohistochemical study demonstrated that numerous NKA, NHE3 and some VHA were located predominantly on presumed MRCs of gill filaments and at the base of the lamellae. Analyses using serial (mirror image) sections of the gills indicated that only a few NKA immunoreactive cells (IRC) were colocalized with VHA on some MRCs in the filaments. In the gills of P. tanegasimae, NKA- and NHE3-IRCs were observed in the interlamellar region of the filaments and at the base of the lamellae. VHA-IRCs were located sparsely on the lamellae and filaments. In the gills of E. yaeyamaensis, the lamellae and filaments were thin and straight, respectively. MCs were located at the tip as well as found scattered in the interlamellar region of gill filaments. NKA-, NHE3- and VHA-IRCs were moderately frequently observed in the filaments and rarely on the lamellae. This study shows that the structure and distribution of ion transporters in the gills differ among the three blennid species, presumably reflecting their different ecologies.     

Fine structure of the gill epithelium of the terrestrial mudskipper, Periophthalmodon schlosseri

In this paper, we describe the fine structure of the branchial epithelium of the amphibious, air-breathing mudskipper Periophthalmodon schlosseri, and relate the observed structure to functions in gas exchange, and to the elimination of sodium chloride and ammonia. Also, we describe the fine structure of the opercular epithelimicrom. The gill lamellar epithelium is thickened by the presence of large mitochondria-rich (MR) cells. These MR cells are further characterized by an extensive tubular system that is continuous with the basolateral plasma membrane and by a deep apical crypt often lined with microvilli. There are very few specialized MR accessory cells, which are associated with NaCl excretion in marine teleosts. Instead, MR cells are commonly isolated from each other laterally by flattened cells rich in intermediate filaments. These filament-rich (FR) cells are interconnected by desmosomes and have unusual canaliculi. These branchial FR cells are unique to P. schlosseri and may have a structural role. Electron-dense pavement cells rich in vesicles and large vacuous mitochondria compose the superficial layer of the epithelium. The unusual morphology of P. schlosseri's gill lamellae may be related to the animal's ability to effectively eliminate ammonia during air exposure. The inner opercular lining and parts of the leading edge of the filament have intraepithelial capillaries, which provide a more suitable gas exchange surface than the thickened lamellae with its restricted interlamellar water spaces. The arrangement of respiratory and ion exchange epithelia is opposite to that found in all other fish in which the lamellae typically function in gas exchange and the gill filament in ion regulation.     

Scanning electron microscopy of the gills of a freshwater catfish,Rita Rita

Variations in the gross morphology and surface architecture of the gill filaments and secondary lamellae of a freshwater catfish (Rita rita) have been investigated using scanning electron microscopy. Heterogeneity of the gill has been correlated with the distribution of lamellar water-flow at different regions of a gill filament. Higher lamellar water flow (cc/pore/cmH₂O/sec) was estimated for the middle region of the filaments. The filaments are covered with epithelial cells whose surface is provided with well-developed microridges. The lamellae are generally covered with microvillous epithelial cells. The variations in surface architecture of the gill filaments and secondary lamellae have been correlated with their probable functions.     

鲻和鲮鳃丝的扫描电镜比较观察

对鲻（Mugil cephalus）和鲮（Cirrhina molitorella）的鳃丝表面结构进行了扫描电镜比较观察,结果表明,鲻鳃丝杆状部比鲮粗．鲻鳃小片高度比鲮低;两者鳃丝表面分泌孔洞口径和密度不同;鲻和鲮细胞外被不同,鲻细胞外被稀疏,鲮的则致密复杂;鳃小片细胞和鳃丝表皮细胞的表面形态存在差异,文章还描述了鳃丝表皮形态特异的细胞。     

Immunohistological classification of ionocytes in the external gills of larval Japanese black salamander,Hynobius nigrescens Stejneger

In this cytological and immunohistological study, we clarified the localization of the membrane transporters Na⁺, K⁺‐ATPase (NKA), vacuolar‐type H⁺‐ATPase (VHA), and epithelial sodium channel (ENaC) and distinguished ionocyte subtypes in the gill of the Japanese salamander (Hynobius nigrescens). In larvae (IY stages 43–65), NKA immunoreactivity was observed on the basolateral plasma membrane in more than 60% cells and less than 20% cells in the primary filaments and secondary lamellae of the external gills, respectively. VHA immunoreactivity was observed on the apical membrane of some epithelial cells in the secondary lamellae of the external gills. High ENaCα immunoreactivity was widely observed on the apical cell membrane of a population of squamous cells, presumably pavement cells (PVCs), and mitochondria‐rich cells (MRCs), in the primary filaments and secondary lamellae of the external gills. Using double immunofluorescence microscopy, epithelial cell types involved in ionic regulation were characterized and divided into three ionocyte types: NKA‐, NKA‐ and ENaC‐, and VHA‐positive cells. VHA‐immunoreactive cells as well as NKA‐positive cells were observed during IY stages 43–65 of the salamander larvae. During late stages of metamorphosis, NKA, VHA, and ENaCα immunoreactivities in the external gills decreased and finally disappeared during the completion of metamorphosis (IY stage 68). PVCs and MRCs in the external gills are probably involved in acid–base balance regulation and osmoregulation in urodele amphibian larvae. The results are discussed in relation to the ionocytes previously reported in fish gills and the frog skin epithelium. J. Morphol., 2011. © 2011Wiley‐Liss, Inc.     

Gill morphometrics in relation to gas transfer and ram ventilation in high‐energy demand teleosts: Scombrids and billfishes

This comparative study of the gill morphometrics in scombrids (tunas, bonitos, and mackerels) and billfishes (marlins, swordfish) examines features of gill design related to high rates of gas transfer and the high‐pressure branchial flow associated with fast, continuous swimming. Tunas have the largest relative gill surface areas of any fish group, and although the gill areas of non‐tuna scombrids and billfishes are smaller than those of tunas, they are also disproportionally larger than those of most other teleosts. The morphometric features contributing to the large gill surface areas of these high‐energy demand teleosts include: 1) a relative increase in the number and length of gill filaments that have, 2) a high lamellar frequency (i.e., the number of lamellae per length of filament), and 3) lamellae that are long and low in profile (height), which allows a greater number of filaments to be tightly packed into the branchial cavity. Augmentation of gill area through these morphometric changes represents a departure from the general mechanism of area enhancement utilized by most teleosts, which lengthen filaments and increase the size of the lamellae. The gill design of scombrids and billfishes reflects the combined requirements for ram ventilation and elevated energetic demands. The high lamellar frequencies and long lamellae increase branchial resistance to water flow which slows and streamlines the ram ventilatory stream. In general, scombrid and billfish gill surface areas correlate with metabolic requirements and this character may serve to predict the energetic demands of fish species for which direct measurement is not possible. The branching of the gill filaments documented for the swordfish in this study appears to increase its gill surface area above that of other billfishes and may allow it to penetrate oxygen‐poor waters at depth. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

Endothelin receptors in teleost fishes: cardiovascular effects and branchial distribution

By observing gill blood flow using epi-illuminating microscopy, in parallel with cardiovascular recordings and immunohistochemistry, we have tried to identify the receptor mediating endothelin (ET) type 1 (ET1)-induced pillar cell contraction in the lamellae of the Atlantic cod (Gadus morhua). Intra-arterial injection of the specific ET(B) receptor agonist BQ-3020 induced dose-dependent increases in ventral aortic blood pressure, gill vascular resistance, and pillar cell area (indicating contraction). The specific ET(A) receptor antagonist BQ-610 did not prevent either pillar cell contraction or increased gill vascular resistance induced by ET-1 injection. The cardiovascular responses were corroborated by the detection of ET(B) receptor-like immunoreactivity (IR) associated with pillar cells in the lamellar region and in neuroendocrine cells. ET(B) receptor-like IR was also found lining the muscle layer of lamellar arterioles and filament arteries. In contrast, strong ET(A) receptor-like IR was found on branchial nerves throughout the filaments. In addition, ET-like IR was concentrated in neuroendocrine cells in the filament and lamellae. We also present data suggesting that ET-mediated pillar cell contraction is widespread among teleost fish, including Atlantic cod, rainbow trout (Oncorhynchus mykiss), sculpin (Myoxocephalus scorpius), and mackerel (Scomber scombrus). Taken together, our results suggest that an ET(B)-like receptor mediates pillar cell contraction in fishes, whereas ET(A)-like receptors may serve another function in the gill, inasmuch as ET(A) receptor-like IR is found on branchial nerves.     

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.     

Gill lamellar pillar cell necrosis, a new birnavirus disease in Japanese eels.

Since the late 1980s, a birnaviral gill disease has been occurring in Japanese eels Anguilla japonica reared in warmwater ponds in western regions in Japan. Diseased eels mostly displayed marked formations of aneurysmal hematomas within gill lamellae and high mortalities. Histological examination revealed necrosis of pillar cells and subsequent aggregation of erythrocytes inside the lamellar capillaries, and proliferation of interlamellar epithelia onto the lamellae. Gastric gland cells were also necrotized. Electron microscopy revealed birnavirus infection in lamellar pillar cells. The causative birnavirus was isolated and cultured in fish cell lines and was found to be related to an infectious pancreatic necrosis virus (IPNV) Sp serotype by neutralization tests. The viral pathogenicity was confirmed by the results of histopathological examinations and infectivity experiments.     

Gross structure and dimensions of the gill in an air-breathing estuarine goby,Pseudapocryptes lanceolatus

The gills of the air-breathing estuarine goby,Pseudapocryptes lanceolatus, are reduced owing to the development of a specialized organ of O₂ uptake from air. In the first gill arch, the filaments of the outer hemibranch are reduced to nearly one-half in comparison to those of its inner hemibranch. A smaller number of secondary lamellae per mm (27.6) occurring on one side of the gill filament reduces the gill surface area. A bilogarithmic plot of the gill area and the body weight indicates a curve with two significantly different components, one (b = 0.924) related to the fish weighing up to 6 g and the other (b = 0.405) to the fish weighing 8 g and above.     

Gill microcirculation of the air-breathing climbing perch, Anabas testudineus (Bloch):relationships with the accessory respiratory organs and systemic circulation

The general macrocirculation and branchial microcirculation of the air-breathing climbing perch, Anabas testudineus, was examined by light and scanning electron microscopy of vascular corrosion replicas. The ventral aorta arises from the heart as a short vessel that immediately bifurcates into a dorsal and a ventral branch. The ventral branch distributes blood to gill arches 1 and 2, the dorsal branch to arches 3 and 4. The vascular organization of arches 1 and 2 is similar to that described for aquatic breathing teleosts. The respiratory lamellae are well developed but lack a continuous inner marginal channel. The filaments contain an extensive nutritive and interlamellar network; the latter traverses the filament between, but in register with, the inner lamellar margins. Numerous small, tortuous vessels arise from the efferent filamental and branchial arteries and anastomose with each other to form the nutrient supply for the filament, adductor muscles, and arch supportive tissues. The efferent branchial arteries of arches 1 and 2 supply the accessory air-breathing organs. Arches 3 and 4 are modified to serve primarily as large-bore shunts between the dorsal branch of the ventral aorta and the dorsal aorta. In many filaments from arches 3 and 4, the respiratory lamellae are condensed and have only 1-3 large channels. In some instances in arch 4, shunt vessels arise from the afferent branchial artery and connect directly with the efferent filamental artery. The filamental nutrient and interlamellar systems are poorly developed or absent. The respiratory and systemic pathways in Anabas are arranged in parallel. Blood flows from the ventral branch of the ventral aorta, through gill arches 1 and 2, into the accessory respiratory organs, and then returns to the heart. Blood, after entering the dorsal branch of the ventral aorta, passes through gill arches 3 and 4 and proceeds to the systemic circulation. This arrangement optimizes oxygen delivery to the tissues and minimizes intravascular pressure in the branchial and air-breathing organs. The efficiency of this system is limited by the mixing of respiratory and systemic venous blood at the heart.     

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.     

Gill surface area of water-breathing freshwater fish

Summary To provide a hitherto lacking review which focuses on gill surface area of freshwater fish, we collected and analysed morphometric data from the literature. The scaling exponent of gill area ranges from 0.36 to 1.13, with a mean value of 0.76. The absolute values for the largest gill areas are about 5 times as high as those of the smallest. This range resembles that of marine fish, if specially adapted steady swimmers, such as tunnies and some sharks, are excluded. Generally it appears that the gill areas of freshwater fish are smaller than those of comparable marine species. To establish whether a relationship exists between gill area and swimming activity or oxygen content of water, the activity of each species and the oxygen content of its habitat were estimated and checked against the gill area. ANOVA revealed that activity explains the presence of the smallest gill areas only, while oxygen content does not correlate with gill area at all. The morphometric variables determining gill area (total length of filaments, average lamellar density, average lamellar area) are highly correlated; total gill area correlates mainly with lamellar density and to a lesser degree with filament length; lamellar area varies independently. Different populations of the same species exhibit striking differences with respect to gill areas, total length of filaments, average lamellar density and average lamellar area. These differences point to a substantial morphological plasticity of the gill system.     

A scanning electron microscope study of the gills of the air-breathing catfish, Clarias batrachus L.

Using the scanning electron microscope, the gills of the air-breathing catfish, Clarias batrachus , have been studied. The overall morphology of the gills are similar to other teleosts. In contrast to water-breathing species, however, microridges are absent from the surfaces of the secondary lamellae and only short microvilli are present. Long, convoluted microridges are present on the epithelial cells of the gill filaments. The possible roles of these structures in relation to water flow are discussed.