Free radical production by the red tide alga, Chattonella antiqua.

Summary The red tide alga,Chattonella antiqua, was found to show a strong chemiluminescence, using luminol as the reagent, when exposed to ultraviolet irradiation. This luminescence was completely inhibited by ascorbate or catalase, suggesting that hydrogen peroxide was generated by the plankton. Red tide cells exposed to fish gill mucus from young yellowtail resulted in the release of a large number of mucocysts and a weak luminosity, and showed a strong reduction of cytochromec in the medium. Therefore, the discharge of mucocysts from the red tide, induced by the presence of gill mucus, may be accompanied by the release of active oxygen species. The active oxygen may be involved in depolymerization of mucus glycoproteins from the gill lamellae.     

A NEW METHOD FOR IDENTIFICATION OF INTER-AND INTRA-SPECIES OF THE RED TIDE ALGAE CHATTONELLA ANTIQUA AND CHATTONELLA MARINA (RAPHIDOPHYCEAE) BY MEANS OF MONOCLONAL ANTIBODIES1

Four monoclonal antibodies AT-83, AT-86, MR-18 and MR-21 were obtained from culture supernatants of hybridomas which had been established by cell fusion between myeloma and spleen cells of mice immunized with the raphidophycean algae Chattonella antiqua (H) O NIES-83 and 86 and Chattonella marina (S) H et C NIES-118 and 121, respectively. Antibody AT-86 was reactive with all ten strains of Chattonella antiqua tested but not with those of Chattonella marina. On the other hand, MR-18 was reactive with those of Chattonella marina but not with those of Chattonella antiqua. Species can thus be identified using these two antibodies. Antibody MR-21 was reactive with some of the strains of Chattonella antiqua and all six strains of Chattonella antiqua and all six strains of Chattonella marina. AT-83 was reactive with some strains of Chattonella antiqua By the selective reactivities of these four monoclonal antibodies, strains of the two species could be separated into two groups. AT-83 could distinguish between Chattonella marina isolated from Kagoshima prefecture (reactive) and the Seto Inland Sea region (non-reactive). Reactivities of MR-21 suggested that at least two groups of Chattonella antiqua caused red tides in Harima-Nada during 1977 and 1978. Monoclonal antibodies appear to be useful for the identification of inter-species and grouping of intra-species of these organisms.     

Fixation of mucus on rainbow trout (Oncorhynchus mykiss Walbaum) gills for light and electron microscopy

Fixation of mucus for the assessment of biofilms and surface associated pathogens often involves complex and expensive techniques. Rainbow trout killed by an overdose of MS 222 had their gills removed and immersion-fixed gently in buffered glutaraldehyde containing 2% Alcian blue. Control tissues consisted of gills fixed in Alcian blue-free fixative. Trout were also killed and directly immersed in liquid nitrogen and the gills freeze-dried then vapour fixed with osmium tetroxide at −50° C. Following fixation gill tissue was processed for light and electron microscopy. A continuous and intact mucous coat was not detected on tissue fixed by conventional methods but the addition of Alcian blue to the fixative preserved an extensive mucous coat trapped between the lamellae and overlying the epithelia. Electron microscopic examination revealed that mucus preservation with the conventional fixative was poor and intermittent whereas the addition of Alcian blue to the fixative greatly enhanced the preservation of the branchial mucous coat. Mucus appeared as interdispersed flocculant material between the epithelial microridges and formed extensive superficial sheets over the epithelium. Freeze-dried/vapour-fixed gill tissue also provides excellent preservation of the integrity of branchial mucous coats, the mucus appearing as a continuous sheet over the filament and secondary lamellae. However, freeze-dried tissue fails to preserve sufficient cellular integrity for this technique to be useful for light or transmission electron microscopy. The potential for use of glutaraldehyde-Alcian blue fixed-gill tissue diagnostically and in research are discussed.     

Plasma nitrate accumulation during the development of pacing-induced dilated cardiac myopathy in conscious dogs is due to renal impairment.

Heart failure is associated with an increase in plasma nitrate and nitrite (NOx). To date there is still some controversy regarding the causes of nitrate accumulation during the development of heart failure. The goal of this study was to analyze the underlying mechanisms that cause accumulation of plasma nitrates during the development of heart failure in dogs. Dogs were chronically instrumented for measurement of hemodynamics and renal function. Hearts were paced initially at 210 bpm for 3 weeks and then at 240 until the development of heart failure. Hemodynamics, renal function, renal blood flow, arterial blood gases, hemoglobin, plasma and urine NOx levels, and creatinine levels were measured weekly. Heart failure was assessed by hemodynamic alterations, physical signs such as lethargy, ascites, cachexia, and postmortem evidence of cardiac hypertrophy. LVSP (from 127 +/- 3 to 106 +/- 3 mmHg), LV dP/dt (from 2658 +/- 173 to 1439 +/- 217 mmHg/s), MAP (from 101 +/- 1.9 to 83 +/- 1.8 mmHg) fell, whereas LVEDP tripled (from 6.4 +/- 0.9 to 20 +/- 2.6 mmHg), and heart rate rose (from 101 +/- 4.2 to 117 +/- 6.3 bpm), all changes P < 0.05. RBF (from 146 +/- 10 to 96 +/- 9.9 ml/min), urine output (V) (from 0.26 +/- 0.02 to 0.16 +/- 0.02 ml/min), GFR (from 63 +/- 1.8 to 49 +/- 2 ml/min), and Na excretion (from 45 +/- 4.5 to 14 +/- 4.6 microEq/min) all decreased (P < 0.05), whereas RVR increased (from 0.68 +/- 0.05 to 0.94 +/- 0.1 mmHg/ml/min). These changes took place during a rise in plasma NOx (from 3.7 +/- 0.5 to 16+/-3.3 microM), a decrease in urine NOx (from 33 +/- 9.9 to 8.1 +/- 4.9 microM), and a concurrent increase in NOx reabsorption (from 221 +/- 31 to 818 +/- 166 nmol/min). There was a direct correlation between the increase in plasma NOx levels and an increase in filtered load (r(2) = 0.97, P = 0.02), a negative correlation between NOx levels and NOx excretion (r(2) = 0.65 P < 0.09), and a direct correlation between plasma NOx levels and NOx reabsorption (r(2) = 0.97, P = 0.02). These results indicate that elevated plasma NOx during heart failure are most likely the result of an impairment of the renal function and not increased NOx production. Furthermore, without knowing changes in renal function the measurement of plasma NOx in and of itself is a meaningless index of NO formation.     

Species characterization of plasma nitrite/nitrate (NOx) concentration

Experiments were designed to detect and determine differences between nitrite/nitrate concentration ([NOx]) in plasma across 15 species selected from seven classes of vertebrates. Blood collected in syringes was placed immediately into ethylenediaminetetraacetic acid (EDTA)-containing tubes and was centrifuged. Plasma [NOx] was determined by measurement of chemiluminescence. Across classes of vertebrates, baseline plasma [NOx] ranged from 0.6 to 171.3 nmol/ml. Mean +/- SD plasma [NOx] was highest in a fresh-water, jawless fish (lamprey, 95.5 +/- 9.1 nmol/ml) and lowest in a saltwater cartilaginous fish (skates, 1.1 +/- 0.4 nmol/ml). Both amphibians tested had a wide range in plasma [NOx], which was explained partly by temporal changes during the year. Within the mammalian class, plasma [NOx] ranged from 3.8 to 43.2 nmol/ml. Results of this study indicate that NO is detectable in plasma of all classes of vertebrates and that baseline concentration varies among species.     

Distribution and development of rodlet cells in the gills and pseudobranch of the bass, Dicentrachus labrax (L)

Rodlet cells in various stages of development were found in large numbers in the bass gill and pseudobranch. In the gill, rodlet cells were found in the epithelium at the base of the secondary lamellae and on the filament between adjacent lamellae, whilst in the pseudobranch they were found over the whole area of the secondary lamellae as well as in the filament epithelium.
During early development, rodlet cells are characterised by their amorphous cell inclusions, prominent supranuclear Golgi complex and network of granular endoplasmic reticulum. Later, with formation of a fibrous border the arrangement of the cell organelles undergoes reorganisation; the endoplasmic reticulum becomes distended, numerous vesicles appear and the mitochondria aggregate in the apical region of the cell. One of the most striking features is the development of club-shaped sacs containing electron dense cores, which are orientated towards the open apex of the cell.
Various staining properties of rodlet cells for light and electron microscopy were compared with those of mucous cells found in the same tissues. Possible functions of the cell are discussed.     

Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae)

Glycoproteins (GPs) were visualised histochemically in the secretory cells – the mucous goblet cells (the type A and the type B), the serous goblet cells, the club cells and the epithelial cells in the gill epithelium of Rita rita. The type A mucous goblet cells, the type B mucous goblet cells and the epithelial cells elaborate GPs with oxidizable vicinal diols and GPs with sialic acid residue without O-acyl substitution. In addition, GPs with O-sulphate esters are elaborated by the type A and GPs with O-acyl sugars by the type B mucous goblet cells. GPs are absent in the serous goblet cells and are with oxidizable vicinal diols in low moieties in the club cells. The analysis of the results elucidates interesting differences in the composition and concentration of GPs in the mucus elaborated by the epithelium of the gill arches and the gill rakers; and the gill filaments and the secondary lamellae indicating the potential importance of the glycoproteins at these locations. GPs elaborated on the surfaces of the gill arches and the gill rakers could be associated to assist in feeding activities and on the surfaces of the gill filaments and the secondary lamellae in the respiratory activity.     

不同NaHCO3碱度对瓦氏雅罗鱼鳃组织结构的影响

研究采用组织学方法观察和比较了达里诺尔湖瓦氏雅罗鱼(Leuciscus waleckii, 碱水种)和松花江瓦氏雅罗鱼(淡水种)在相同碳酸盐碱度胁迫下(C_A0、C_A30和C_A50)鳃组织结构的差异, 探究瓦氏雅罗鱼碱水种耐高碱特性与鳃组织结构微观调整的适应性关系。结果显示, 随着碱度增加, 碱水种鳃丝变宽、鳃小片变长、鳃小片间距变大(P<0.05); 淡水种鳃丝变宽、鳃小片间距变大(P<0.05), 鳃小片长度在C_A30时显著变长(P<0.05), 而在C_A50时与对照组无明显差异(P>0.05)。碱水种在C_A30和C_A50的氯细胞数量与对照组相比明显增加, C_A50的氯细胞排列更加紧密并且有叠加现象, 扁平上皮细胞变大, 细胞表面增厚; 淡水种在C_A30时的氯细胞数量明显多于C_A50, 但在碱度胁迫下, 鳃小片出现破损, 扁平上皮细胞、柱细胞和血细胞融合、脱落现象严重。另外, 在碱水种和淡水种鳃耙上皮细胞中发现了大量黏液细胞分泌, 随着碱度增加, 黏液细胞由大而稀疏变为小而密集, 其中碱水种的黏液细胞数量较淡水种多, 而且排列更为整齐、密集。鳃组织学研究结果表明, 瓦氏雅罗鱼碱水种通过保持鳃组织结构和生理功能的完整性达到对高碱环境的长期适应, 而淡水种则因鳃细胞融合、脱落造成生理功能丧失, 不能长期适应高碱环境。研究结果可为淡水鱼类在盐碱水的移植驯化提供依据和指导。     

Molecular Analysis of Ribosomal RNA Gene of Red Tide Algae Obtained from the Seto Inland Sea

Eleven clones from five species of the planktonic microalgae, (Chattonella antiqua, Chattonella marina, Heterosigma akashiwo, Alexandrium catenella, and Scrippsiella trochoidea), which were collected from the Seto Inland Sea in Japan and from Thailand, were subjected to nucleotide sequence analysis of the D1/D2 domain of the large subunit (LSU) of their ribosomal RNA genes. After amplification by polymerase chain reaction using degenerated primers, whole-nucleotide sequences for the D1/D2 domains of the LSU rRNA gene of 11 microalgae were analyzed. Phylogenic tree analysis using these nucleotide sequences showed each species located in a cluster corresponding to its morphological classification. The nucleotide sequence data for Chattonella spp. suggest that multiple clones of both Chattonella antiqua and Chattonella marina are present in the Seto Inland Sea and that red tide blooms of Chattonella spp. in different years may have contained different clones. Received September 6, 1999; accepted December 16, 1999.     

Occurrence of the polyamines caldopentamine and homocaldopentamine in axenic cultures of the red tide flagellates Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae)

The polyamines caldopentamine and homocaldopentamine were detected in axenic strains of Chattonella antiqua and Heterosigma akashiwo ( Raphidophyceae ), respectively, as well as spermidine, the most abundant polyamine in both phytoplankton species. Trace amounts of putrescine, diaminopropane and norspermine were also detected in both species. Spermine was detected only from C. antiqua . These long linear polyamines are characteristic components of thermophilic bacteria. The detection from two species of Raphidophyceae indicates that the occurrence of long linear polyamines is not restricted to thermophilic microorganisms.     

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.     

Cellular types in the gill arch of the teleost Cyprinus carpio (Cyprinidae, Pisces). A histochemical and ultrastructural study

On adult specimens of the common carp (Cyprinus carpio), we have carried out a histochemical and ultrastructural study of the epithelia which form the gill arch. Secondary lamellae have two cellular types, granular and mucous, which produce neutral carbohydrates and proteins rich in tryptophane, and another mucous cell type which contains glycosaminoglycans. In gill rakers, three cellular types show different histochemical behaviour: 1) granular cells elaborate neutral mucosubstances; 2) a second cell type contains glycosaminoglycans,, and 3) a third cell type secretes neutral and acid carbohydrates. We discuss the possible role of these cells according to their secretion. We describe pillar and chloride cells in secondary lamellae, and chloride and neuroendocrine-like cells in gill rakers.     

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.     

Lernanthropus kroyeri infections in farmed sea bass Dicentrarchus labrax: pathological features

Twenty sea bass Dicentrarchus labrax L. from a fish farm (floating cage) in Greece were examined for the presence of parasites. The gills of 7 (35%) fish were infected with adult female specimens of the parasitic copepod Lernanthropus kroyeri van Beneden, 1851, and the intensity of infection ranged from 1 to 24 parasites per host. The most infected portion of the gills appeared to be the primary lamellae. Erosion, desquamation and necrosis of the secondary lamellae were noticed near the site of copepod attachment; furthermore, the terminal claw of the second antennae lacerated tissue and vessels of infected gill. Parasitism by L. kroyeri affected the host's condition factor (mean +/- SE in uninfected vs parasitized; 1.88 +/- 0.04 vs 1.66 +/- 0.12; p < 0.05).     

Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na⁺/K⁺ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions     

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。     

Gross structure of the respiratory organs and dimensions of the gill in the mud–skipper, Periophthalmodon schlosseri (Bleeker)

In Periopkrlialnwdon scldosseri the respiratory organs consist of the gills, the suprabranchial and opercular chambers. The gills are more suited for aerial than aquatic respiration as is shown by the presence of the vascular papillae, blood sinusesand dilated blood vessels in their lamellae. The gill lamellae possess a surface coat of sulphated mucopolysaccharides that prevents water loss during exposure to the air. The filaments of the outer hemibranchs in the first gill arch are reduced to nearly one quarter of those of its posterior hemibranch. The gill area in relation to body weight shows a high slope value ( b =0·93).     

Measurement of non-steroid antiinflammatory drugs induced gastric microbleeding.

The damage of the mucous membranes in the gastrointestinal tract caused by non-steroid antiinflammatory drugs are well known. The gastrointestinal microbleeding was measured by the method of Fischer and Hunt before and after the intake of indomethacin (4 x 25 mg), naproxen-sodium (4 x 275 mg), diclofenac (3 x 50 mg) and azapropazone (2 x 600 mg). In the indomethacin group microbleeding increased from 0.91 +/- 0.12 ml/24 h to 7.30 +/- 1.20 ml/h. In the naproxen-sodium group from 1.22 +/- 0.16 ml/24 h to 3.56 +/- 0.40 ml/24 h, in the diclofenac group from 0.86 +/- 0.14 ml/24 h to 3.18 +/- 0.28 ml/24 h, in azapropazone group from 0.92 +/- 0.18 ml/24 h to 2.50 +/- 0.20 ml/24 h, respectively. All non-steroid antiinflammatory drugs increased the gastric microbleeding, however, there were considerable differences in the degree of enhancement. This can be explained by the different inhibitory activities of the drugs on the cyclooxygenase enzyme activity.     

Ichthyophthirius multifiliis (Ciliophora) Development in Gill Epithelium1

ABSTRACT. The development of Ichthyophthirius multifiliis trophonts in gill epithelium of channel catfish was studied on the first five days post-exposure (PE) by light and electron microscopy. Trophonts increased in average diameter from 48 μm at one day PE to 248 μm at five days PE. Although theronts invaded any part of the gill epithelium, at three days PE most parasites were found adjacent to major blood vessels, particularly the afferent vessel. From one to three days PE, 70–90% of trophonts were immediately adjacent to gill epithelium, but at four and five days PE only 50% of trophonts were closely apposed by host epithelial cells. Notable ultrastructural changes in the trophonts over the five-day period occurred in the mucocysts and lipid inclusions, both of which increased markedly in number. The few crystalline mucocysts present at one day PE were near the cell periphery but rarely attached to the plasma membrane. At three days PE, crystalline mucocysts were significantly more abundant, and at five days PE, they occurred in large numbers throughout the cytoplasm as well as at the periphery. At three days PE, secretory mucocysts were first observed. Contractile vacuoles were more prominent at five days PE than earlier in development. Development of mucocysts and lipid reserves is probably essential to survival and reproduction of the ciliate after it leaves the host.     

Lectin histochemistry of rainbow trout (Oncorhynchus mykiss) gill and skin

In order to characterize the glycoconjugate residues in skin and gills of the adult rainbow trout, the binding pattern of five biotinylated lectins with different carbohydrate specificities was examined. In the skin, mucous cells revealed binding sites for PNA and SBA; filament-containing cells were additionally labelled with Con A. However, the basal cell layer showed no reaction. In the gill, subpopulations of mucous cells reacted with Con A, PNA, SBA and UEA-I. This broader spectrum of glycoconjugates in gill mucous cells compared with the epidermal mucous cells could point to the additional function of gill mucus in ion and osmoregulation. Lectin binding sites were less common in the respiratory epithelial cells of the secondary lamellae than in those of the primary lamellae. Chloride cells revealed mannose, galactose and fucose residues. Immature chloride cells, as indicated by a comparison with Na⁺/K⁺ ATPase immunolabelling, reacted with Con A; subpopulations of them reacted with PNA, SBA and UEA-I. The results form the basis for further investigations in which these cell populations can be analysed under different environmental conditions This revised version was published online in November 2006 with corrections to the Cover Date.