大菱鲆水通道蛋白(AQP1、AQP3)以及离子通道蛋白(CFTR、NHE1)对低盐胁迫的响应

在鱼类适应环境盐度变化的过程中,鳃、肾、肠是主要的渗透调节器官,而水通道蛋白(Aquaporins, AQPs)、囊性纤维化跨膜传导调节因子(CFTR)、钠氢交换体(NHE)又是这些器官中重要的渗透调节基因。为研究AQP1、AQP3、CFTR、NHE1在大菱鲆(Scophthalmus maximus)低盐胁迫过程中的渗透调节功能,本研究采用荧光定量PCR技术,对4种基因在盐度5和盐度10下大菱鲆鳃、肾、肠中表达量随时间的变化进行检测。结果显示,AQP1表达量在鳃中极少(P<0.05),在肾和肠中较高,低盐胁迫下,盐度5组和盐度10组在鳃中的表达量无显著变化,在肾和肠中均显著上升(P<0.05)。AQP3表达量在肾中极少(P<0.05),在鳃中较高,在肠中较少,低盐胁迫下,盐度5组和盐度10组在肾中的表达量无显著变化,在鳃和肠中均显著上升(P<0.05)。CFTR表达量在肾中极少,在鳃中较高,在肠中较少,低盐胁迫下,盐度5组和盐度10组在肾中的表达量无显著变化,在鳃和肠中均显著下降(P<0.05)。NHE1在鳃和肠中表达量较少,在肾中较高,低盐胁迫下,盐度5组和盐度10组在鳃中的表达量无显著变化,在肾和肠中均显著上升(P<0.05)。这些结果表明,4种基因表达水平因组织、盐度和时间的不同而不同,反映了这4种基因的功能特异性;在低盐胁迫下,4种基因积极响应,表达量均发生不同程度的变化,表明AQP1、AQP3、CFTR和NHE1在大菱鲆低盐环境适应中可能具有潜在的重要作用。另外,本研究结果可为大菱鲆半咸水养殖和淡化养殖提供理论依据,同时为培育适应低盐环境大菱鲆良种提供理论和技术支撑。     

Natriuretic peptide inhibition of intestinal salt absorption in the Japanese eel: physiological significance

Electrophysiological studies in vitro demonstrated the significant inhibition by natriuretic peptides (NP) of short-circuit current across the eel intestine, an important osmoregulatory organ. Inhibitory potencies of several members of the NP family were assessed by voltage-clamp determination of net transepithelial salt absorption measured as the short-circuit current I_sc across the intestine of the freshwater-adapted (FW) and seawater-adapted (SW) Japanese eel (Anguilla japonica); the order of potency of synthetic eel peptides was: amidated atrial natriuretic peptide (ANP-NH₂) > ventricular natriuretic peptide (VNP) > atrial natriuretic peptide (ANP) >> C-type natriuretic peptide (CNP). Neither the order of potency nor the absolute potencies were effected by salinity adaptation. The observed potency sequence suggests that inhibition of intestinal absorption is mediated by A-type guanylyl cyclase-coupled NP receptors. The relatively low sensitivity of the intestinal response to NP compared with circulating NP concentrations suggests a role for intestinal regulation by NP which is independent of systemic delivery from cardiac sources. A novel model, incorporating the known immunohistochemical localization of NP-ergic cells and processes in the epithelial layer of the intestine and the dissipation of the Na⁺ electrochemical gradient along the alimentary tract, is developed in which local secretion of NP (in response to a bolus of food) inhibits salt absorption across the intestine regionally in favor of increased nutrient absorption.     

Physiology of seawater acclimation in the striped bass,Morone saxatilis (Walbaum)

Several experiments were performed to investigate the physiology of seawater acclimation in the striped bass, Morone saxatilis. Transfer of fish from fresh water (FW) to seawater (SW; 31–32 ppt) induced only a minimal disturbance of osmotic homeostasis. Ambient salinity did not affect plasma thyroxine, but plasma cortisol remained elevated for 24h after SW transfer. Gill and opercular membrane chloride cell density and Na⁺,K⁺-ATPase activity were relatively high and unaffected by salinity. Average chloride cell size, however, was slightly increased (16%) in SW-acclimated fish. Gill succinate dehydrogenase activity was higher in SW-acclimated fish than in FW fish. Kidney Na⁺, K⁺-ATPase activity was slightly lower (16%) in SW fish than in FW fish. Posterior intestinal Na⁺,K⁺-ATPase activity and water transport capacity (J_v) did not change upon SW transfer, whereas middle intestinal Na⁺,K⁺-ATPase activity increased 35% after transfer and was correlated with an increase in J_v (110%). As salinity induced only minor changes in the osmoregulatory organs examined, it is proposed that the intrinsic euryhalinity of the striped bass may be related to a high degree of “preparedness” for hypoosmoregulation that is uncommon among teleosts studied to data.     

Morphological changes in gill mitochondria-rich cells in cultured Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> acclimated to a wide range of environmental salinity

Morphological changes in gill mitochondria-rich (MR) cells were examined in cultured Japanese eel acclimated to deionized freshwater (DFW), freshwater (FW), 30%-diluted seawater (DSW), and seawater (SW). The gill Na⁺/K⁺-ATPase activity was higher in SW-acclimated eel than in those acclimated to DFW, FW, and DSW. Immunocytochemical observations revealed that MR cells in the gill filaments were most developed in SW, whereas MR cells in the lamellae were preferentially observed in DFW, suggesting that filament and lamellar MR cells are responsible for ion secretion and absorption, respectively. In scanning electron microscopic observations, the apical membrane of lamellar MR cells appeared as a flat or slightly projecting disk with a mesh-like structure on its surface. In contrast, the apical membrane of filament MR cells showed a slightly concave surface. Whole-mount immunocytochemistry revealed that most MR cells showed cystic fibrosis transmembrane conductance regulator immunoreaction in their apical region in fish in DSW and SW, but not in those in DFW and FW, indicating that MR cells developed in DSW and SW function as an ion-secreting site. In addition to MR cells, distinct Na⁺/K⁺-ATPase immunoreaction was observed in the outermost layer of gill epithelia, suggesting that pavement cells are an additional site of ion uptake in the gills.     

A field and laboratory study of the responses of cytoprotection and osmoregulation to salinity stress in mosquitofish (<Emphasis Type="Italic">Gambusia affinis</Emphasis>)

The mosquitofish (Gambusia affinis) naturally inhabits freshwater (FW; 1–3‰) and seawater (SW; 28–33‰) ponds in constructed wetland. To explore the physiological status and molecular mechanisms for salinity adaptation of the mosquitofish, cytoprotective responses and osmoregulation were examined. In the field study, activation of protein quality control (PQC) mechanism through upregulation of the abundance of heat shock protein (HSP) 90 and 70 and ubiquitin-conjugated proteins was found in the mosquitofish gills from SW pond compared to the individuals of FW pond. The levels of aggregated proteins in mosquitofish gills had no significant difference between FW and SW ponds. Furthermore, the osmoregulatory responses revealed that the body fluid osmolality and muscle water contents of the mosquitofish from two ponds were maintained within a physiological range while branchial Na⁺/K⁺-ATPase (NKA) expression was higher in the individuals from SW than FW ponds. Subsequently, to further clarify whether the cellular stress responses and osmoregulation were mainly induced by hypertonicity, a laboratory salinity acclimation experiment was conducted. The results from the laboratory experiment were similar to the field study. Branchial PQC as well as NKA responses were induced by SW acclimation compared to FW-acclimated individuals. Taken together, induction of gill PQC and NKA responses implied that SW represents an osmotic stress for mosquitofish. Activation of PQC was suggested to provide an osmoprotection to prevent the accumulation of aggregated proteins. Moreover, an increase in branchial NKA responses for osmoregulatory adjustment was required for the physiological homeostasis of body fluid osmolality and muscle water content.     

Acclimation of brackish water pearl spot (Etroplus suratensis) to various salinities: relative changes in abundance of branchial Na+/K+-ATPase and Na+/K+/2Cl− co-transporter in relation to osmoregulatory parameters

The present study was conducted to elucidate the osmoregulatory ability of the fish pearl spot (Etroplus suratensis) to know the scope of this species for aquaculture under various salinities. Juvenile pearl spot were divided into three groups and acclimated to freshwater (FW), brackish water (BW) or seawater (SW) for 15 days. The fish exhibited effective salinity tolerance under osmotic challenges. Although the plasma osmolality and Na⁺, K⁺ and Cl^? levels increased with the increasing salinities, the parameters remained within the physiological range. The muscle water contents were constant among FW-, BW- and SW-acclimated fish. Two Na+/K+-ATPase α-isoforms (NKA α) were expressed in gills during acclimation in FW, BW and SW. Abundance of one isoform was up-regulated in response to seawater acclimation, suggesting its role in ion secretion similar to NKA α1b, while expression of another isoform was simultaneously up-regulated in response to both FW and SW acclimation, suggesting the presence of isoforms switching phenomenon during acclimation to different salinities. Nevertheless, NKA enzyme activities in the gills of the SW and FW individuals were higher (p < 0.05) than in BW counterparts. Immunohistochemistry revealed that Na⁺/K⁺-ATPase immunoreactive (NKA-IR) cells were mainly distributed in the interlamellar region of the gill filaments in FW groups and in the apical portion of the filaments in BW and SW groups. The number of NKA-IR cells in the gills of the FW-acclimated fish was almost similar to that of SW individuals, which exceeded that of the BW individuals. The NKA-IR cells of BW and SW were bigger in size than their FW counterparts. Besides, the relative abundance of branchial Na⁺/K⁺/2Cl^? co-transporter showed stronger evidence in favor of involvement of this protein in hypo-osmoregulation, requiring ion secretion by the chloride cells. To the best of our knowledge, this is the first study reporting the wide salinity tolerance of E. suratensis involving differential activation of ion transporters and thereby suggesting its potential as candidate for fish farming under different external salinities.     

Insulin-like growth factor I of Japanese eel, Anguilla japonica: cDNA cloning, tissue distribution, and expression after treatment with growth hormone and seawater acclimation

In an attempt to understand growth regulation in the Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-I (IGF-I) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-I (eIGF-I) cDNAs encoding preprohormones, eIGF-I-Ea1and eIGF-I-Ea2, were cloned from the liver by polymerase chain reaction (PCR). The preproIGF-Is were identical in signal peptide and mature IGF-I, but different in the E domain—eIGF-I-Ea2 mRNA was 36 bp longer than eIGF-I-Ea1 mRNA. Eel IGF-I was 83–94% identical with that of teleosts, 71% identical with that of dogfish, 87% identical with that of bullfrog and chicken, and 83% identical with that of humans. In both males and females the highest eIGF-I-Ea1 mRNA levels were observed in the liver, with detectable levels also found in the gills, heart, stomach, spleen, kidney, intestine, swim-bladder, muscle, and gonads. eIGF-I-Ea1 mRNA levels in the liver were higher in females than in males whereas in the intestine they were lower than in males. eIGF-I-Ea2 mRNA was detected in all the tissues examined and at similar levels in males and females. In this experiment higher eIGF-I-Ea1 mRNA levels were observed in the liver of larger glass eels than in those of smaller fish. eIGF-I-Ea2 mRNA levels were also higher in larger eels, although they were lower than IGF-I-Ea1 mRNA levels. Both eIGF-I mRNA levels in liver were positively correlated with the body size of the␣glass eels. Intraperitoneal injection of recombinant eel GH (reGH), 0.25 μg g⁻¹ body weight, into glass eels resulted in a significant increase in both eIGF-I mRNAs in the liver 1 day after injection compared with control fish, but no elevation was observed 2 days after injection. Incubation of liver slices with reGH at concentrations of 10, 100, and 1,000 ng mL⁻¹ for 24 h resulted in a significant concentration-dependent increase in the levels of both eIGF-I mRNAs. Higher levels of eIGF-I-Ea1 and Ea2 mRNA were observed in the gills ofseawater-reared eels than in those of freshwater-reared fish, but no differenceswere observed in the whole kidney. These results suggest that IGF-I is involved in the regulation of somatic growth and also in adaptation of the Japanese eel to seawater.     

Osmoregulatory actions of growth hormone in juvenile tilapia (Oreochromis niloticus)

Growth hormone (GH) effectively promotes seawater (SW) adaptation in salmonids, but little is known of its effect in tilapias. Experiments were performed to investigate the effects of recombinant eel GH (reGH) on osmoregulatory actions and ultrastructural features of gill chloride cells in juvenile tilapia, Oreochromis niloticus. Tilapia showed a markedly improved SW survival, when directly transferred from freshwater (FW) to 62.5% SW 24h after a single reGH injection (0.25 or 2.5 µg g^-1) or 3 reGH injections (0.25 µg g^-1 every other day). Plasma Na+ and Mg²⁺ levels were significantly reduced by reGH (0.25 and 2.5 µg g^-1) compared with saline injections; Ca²⁺ concentrations were reduced significantly by high dose of reGH (2.5 µg g^-1) after SW transfer. However, fish failed to survive more than 24h when directly transferred to 70% SW, although the fish treated with reGH could survive longer than the controls. When examined by electron microscopy, the chloride cells were identified as mitochondrion-rich and an extensive tubular system was induced by GH treatment. The results of the present study suggest that, similar to its effect on salmonids, GH also exerts acute osmoregulatory actions and enhances SW adaptation in juvenile tilapia. GH also stimulates the differentiation of chloride cells toward SW adaptation.     

Histological and hormonal changes in the European eel (Anguilla anguilla) after exposure to environmental cocaine concentration

The aim of this study was the assessment of histological and hormonal changes induced in the European eel from environmental concentrations of cocaine. Silver eels were exposed to 20 ng L^?1 of cocaine during 50 days; at the same time, control, vehicle control and two post‐exposure recovery groups (3 and 10 days) were made. The general morphology of the skin and the intestine, and the plasma levels of prolactin, cortisol and dopamine were evaluated. In the skin, cocaine decreased the number and size of mucous cells, increased the thickness of the epidermis and altered the club cells and the basal lamina. In the intestine, cocaine increased the thickness of the epithelium and the number of mucous cells and reactivated the structure of the intestine and of the intestinal musculature. Moreover, cocaine increased plasma prolactin, cortisol and dopamine levels. These results suggest that cocaine induced histological changes, directly and/or through the hormonal changes observed. Considering the complex life cycle of the eel, the changes induced by cocaine in the skin, the intestine and the endocrine system could threaten the ability of the eel to successfully migrate and reproduce.     

Histochemical and immunohistochemical characterization of rodlet cells in the intestine of two teleosts,Anguilla anguilla and Cyprinus carpio

Rodlet cells (RC) are characterized by a distinctive cell cortex and conspicuous inclusions named “rodlets.” These cells are particularly abundant and large in size in intestine of eels. Histochemical, immunohistochemical and ultrastructural investigations were carried out on European eel Anguilla anguilla and Common carp Cyprinus carpio from Northern Italy. Eight biotinylated lectins were used to probe for specific carbohydrate residues in deparaffinized, hydrated intestinal sections of eel and carp. Five antibodies were tested on intestinal sections of both fish species: inducible nitric oxide synthase (i‐NOS), leu‐enkephalin, lysozyme, serotonin and tumour necrosis factor‐α. Lectin histochemistry revealed rodlet cells (RCs) of the eel intestine to react with two of the eight lectins tested, specifically Concanavalin A (ConA) and Sambucus Nigra Agglutinin (SNA). This contrasted to lectin staining of RCs in the intestine of common carp, where four of the eight lectins showed a positive reaction; Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), SNA and ConA. RCs in eel and carp intestine were immunoreactive with antibodies to lysozyme and i‐NOS. The occurrence of the inflammatory peptides lysozyme and i‐NOS in RCs of the eel and common carp poses in favour that these cells are involved in the mechanism of defence against pathogens.     

Biochemistry and physiology of a family of eel natriuretic peptides

Three natriuretic peptides with similar structures were isolated from eels and their amino acid sequences determined; atrial natriuretic peptide (ANP) from atria, ventricular natriuretic peptide (VNP) from ventricles, and C-type natriuretic peptide (CNP) from brains. All three hormones were circulating in eel blood, and their plasma levels invariably decreased when eels were transferred from fresh water (FW) to seawater (SW). Eel ANP and VNP inhibited drinking in FW and SW eels. Eel ANP inhibited water and Na⁺ absorption by the intestine of SW eels. The potency of these ANP effects was 2–3 orders greater than those of other hormones which are known to have similar effects. Eel ANP and VNP induced antidiuresis but not antinatriuresis in FW eels. Eel ANP increased plasma cortisol level in SW eels but not in FW eels. The antidiuretic effect and the stimulation of cortisol secretion in eels are opposite to the ANP effects reported in mammals. These data suggest that ANP plays a complex role in the eel osmoregulation.

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Résumé Trois peptides natriurétiques présentant des structures similaires ont été isolés chez l'anguille et leurs séquences en acides aminés a été déterminées; le peptide atrial natriurétique (ANP) dans l'atria, le peptide ventriculaire natriurétique (VNP) dans les ventricules et le peptide natriurétique de type C dans le cerveau. Ces trois hormones sont présents dans le sang d'anguille et leur niveau plasmatique decroit invariablement quand les anguilles sont transférées d'eau douce en eau de mer. L'ANP et le VNP d'anguille inhibent l'action de boire chez les anguilles d'eau douce et d'eau de mer. L'ANP d'anguille inhibe l'absorption d'eau et de Na⁺ par l'intestin d'anguille en eau de mer. Ces effets de l'ANP sont 2 à 3 fois plus grands que ceux observés avec d'autres hormones qui sont connues pour avoir des effets similaires. L'ANP et le VNP d'anguille induisent l'antidiurèse mais pas l'antinatriurèse chez les anguilles d'eau douce. L'ANP d'anguille augmente chez ce poisson les niveaux de cortisol plasmatique en eau de mer mais pas en eau douce. L'effet antidiurétique et la stimulation de la sécrétion de cortisol chez l'anguille sont contraire aux effets de l'ANP observés chez les mammifères. Ces résultats suggèrent que l'ANP joue un role complexe dans l'osmorégulation de l'anguille.

     

Ontogeny of the digestive tract and enzymes in rock bream Oplegnathus fasciatus (Temminck et Schlegel 1844) larvae

Histological development of the digestive tract and specific activities of three digestive enzymes (trypsin, alkaline phosphatase, and pepsin) were studied in rock bream Oplegnathus fasciatus from hatching to 50 days after hatching (DAH). At hatching, the digestive tract appeared as an undifferentiated straight tube and differentiated into the buccopharynx, esophagus, stomach, intestine, and rectum at mouth opening by 3 DAH. The taste bud and mandibular teeth were present in the buccopharyx at 8 DAH. The goblet cells appeared in the esophagus at 8 DAH and in the buccopharyx at 9 DAH. The stomach anlage was formed at 2 DAH and developed into cardia, fundus, and pylorus at 14 DAH. The gastric glands were visible at 16 DAH, and the pepsin was firstly detected on 22 DAH. At 2 DAH, the intestinal valve appeared and divided the intestine into anterior intestine (AI) and posterior intestine (PI). The rectum was differentiated from the PI at 3 DAH. The supranuclear vacuoles were visible in the rectum by 6 DAH, and the lipid inclusions were present in the AI at 8 DAH. The alkaline phosphatase was detected at 1 DAH, and the increase in its activity indicated the maturation of the intestine after 40 DAH. The hepatocytes and pancreatic cells were differentiated from the blast cells at 2 DAH, and the acidophilic zymogen granules in the exocrine pancreas were observed simultaneously. The trypsin was detected by 1 DAH and increased to the maximum at 19 DAH, followed by a decrease as the stomach became functional.     

Morphological and histochemical study of intestine in wild and reared European eel (Anguilla anguilla L.)

Diet-related differences between the ratio of intestine length to body size and the enzymatic activity in the intestinal tract of wild and reared European eel (Anguilla anguilla L.) were studied. Compared with reared eel, wild eel showed significantly shorter relative intestine length. For the purpose of histochemical examination, different parts (anterior, middle and posterior) of intestine proper were used. Activities of non-specific esterase, alkaline and acid phosphatase, and aminopeptidase were examined in each segment. All enzymes were present in the intestines of both wild and reared European eel. Fish from both groups showed similar enzyme distribution within the enterocytes, but distribution and intensity of enzyme activity along the intestine vary depending on the group. Generally, reared European eel showed highest enzymatic activity and wider distribution of enzymes throughout all parts of the intestine. These results suggest that different diets could be one of the reasons for observed changes.     

A new species of freshwater eel <Emphasis Type="Italic">Anguilla luzonensis</Emphasis> (Teleostei: Anguillidae) from Luzon Island of the Philippines

Anguilla luzonensis, a new species of freshwater eel, family Anguillidae, is described on the basis of 29 specimens collected from the Pinacanauan River system, which is a tributary of the Cagayan River on northern Luzon Island of the Philippines. The new species and A. celebesensis, which is distributed in the same region, both have variegated color marking and broad maxillary bands of teeth, but show statistically significant differences in the following characters of the new species: predorsal-fin length 28.6–33.1% of total length (TL); preanal length 39.6–44.8% of TL; trunk length 26.4–31.3% of TL; distance between the verticals through the anus and origin of the dorsal fin 9.3–13.9% of TL; length of gape 35.5–46.9% of head length; the total number of vertebrae 103–107; and number of abdominal vertebrae 40–42. These morphological differences, together with the present knowledge about anguillid eel taxonomy, genetics, and ecology, strongly support the presence of the new species A. luzonensis in the area of northern Luzon Island of the Philippines.     

Hyposmoregulatory ability and ion- and water-regulatory mechanisms during the leptocephalus stages of Japanese eel Anguilla japonica

We explored osmoregulatory ability and mechanisms of ion and water regulation in Japanese eel leptocephali. Tissue osmolality of leptocephali ranged from 360 to 540 mOsm/kg·H₂O. Immunocytochemical observations revealed that Na⁺/K⁺-ATPase-immunoreactive mitochondrion-rich (MR) cells were distributed over the entire body surface of leptocephali. Using a fluorescent sodium indicator and the chloride test, we localized Na⁺ and Cl^? secreting sites at the apical region of cutaneous MR cells. To further examine drinking behavior and water absorption in the intestine, leptocephali were exposed to seawater containing dextran labeled with Alexa Fluor. To calculate relative water absorption, fluorescent intensity was measured along the digestive tract. Whereas water was hardly absorbed in the stomach and intestine, water absorption predominantly took place in the rectum. Our findings indicate that Japanese eel exert hyposmoregulatory ability as early as during leptocephalus stages, secreting Na⁺ and Cl^? through cutaneous MR cells and primarily absorbing water from ingested seawater in the rectum.