Swimbladder gas gland cells cultured on permeable supports regain their characteristic polarity.
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A cell culture system has been developed in which swimbladder gas gland cells from the European eel (Anguilla anguilla) were cultured on a permeable support. Cells seeded on Anodisc 13 (Whatman) or Costar Transwell 13 mm membranes form a confluent cell layer within the first 2 or 3 days of culture but, on the basis of measurements of transepithelial resistance, it is a "leaky" cell layer. In a superfusion system, the apical and basal sides of the cells were superfused asymmetrically, with saline on the apical side and a glucose-containing cell culture medium on the basal side. Under these conditions, the cells continuously produced lactic acid, and approximately 60-70 % of this lactate was released at the basal side. To mimic the in vivo situation, the saline solution supplied to the apical side was replaced by humidified air in an additional series of experiments. Cells cultured in an air/liquid system produced even more lactate, and this lactate was only released to the basal side; there was no leakage of fluid to the apical side. After 4 or 5 days in the superfusion system, the cells were fixed for histological examination. The cells were columnar, similar to gas gland cells in vivo, and showed a clear polarity, with some small microvilli at the apical membrane and extensive membrane foldings at lateral and basal membranes. Immunohistochemical localization of Na+/K+ -ATPase revealed that this ATPase was present mainly in the lateral membranes; it was never found in the apical membranes. Cells cultured in the air/liquid system showed a similar structure and polarity. (+info)
Determinants of intracellular pH in gas gland cells of the swimbladder of the European eel Anguilla anguilla.
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Gas gland cells of the European eel (Anguilla anguilla) were cultured on collagen-coated coverslips, and intracellular pH was measured using the pH-sensitive fluorescent probe 2',7'-bis-(2-carboxypropyl)-5-(6)-carboxyfluorescein (BCPCF). The contributions of various proton-translocating mechanisms to homeostasis of intracellular pH (pHi) were assessed by adding specific inhibitors of the various proton-translocating mechanisms at a constant extracellular pH (pHe) of 7.4 and after artificial acidification of the cells using the ammonium pulse technique. The greatest decrease in pHi was observed after addition of 5-(N-ethyl-N-isobutyl)-amiloride (MIA), an inhibitor of Na(+)/H(+) exchange. Na(+)/H(+) exchange was active under steady-state conditions at an extracellular pH of 7.4, and activity increased after intracellular acidification. Incubation of gas gland cells with 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of anion exchange, also caused a decrease in pHi, but this decrease was not as pronounced as in the presence of MIA. Furthermore, at low pHi, the effect of DIDS was further reduced, suggesting that bicarbonate-exchanging mechanisms are involved in maintaining a steady-state pHi but that their importance is reduced at low pH. Bafilomycin A(1), a specific inhibitor of the V-ATPase, had no effect on steady-state pHi. However, recovery of intracellular pH after an artificial acid load was significantly impaired in the presence of bafilomycin. Our results suggest that Na(+)/H(+) exchange and anion exchange are important for the regulation of pHi at alkaline values of pHe. When pHi is low, a situation probably often encountered by gas gland cells during gas secretion, Na(+)/H(+) exchange continues to play an important role in acid secretion and a V-ATPase appears to contribute to proton secretion. (+info)
Primary structure and characteristics of a lectin from skin mucus of the Japanese eel Anguilla japonica.
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Two types of lactose-binding lectins, AJL-1 and AJL-2, were purified from the skin mucus extract of the Japanese eel Anguilla japonica by lactose affinity chromatography and subsequent gel filtration. The molecular masses of AJL-1 and AJL-2 were 16,091 and 31,743 Da, respectively. Intact AJL-1 was comprised of two identical 16-kDa subunits having blocked N termini and no disulfide bonds. AJL-2 was a homodimer with disulfide bonds. Based on the N-terminal amino acid sequence of the AJL-2 monomer, the nucleotide sequence of cDNA encoding this lectin was determined by 3'- and 5'-rapid amplification of cDNA ends. The deduced amino acid sequence showed approximately 30% homology with C-type lectins, which bind to carbohydrates in a Ca(2+)-dependent manner. In addition, AJL-2 exhibited highly conserved consensus amino acid residues of the C-type carbohydrate recognition domain, although this lectin showed Ca(2+)-independent activity. Gene expression of AJL-2 was detected only in the skin by Northern blot analysis, and this lectin localization was demonstrated in the club cells by immunohistochemistry. These results indicate that AJL-2 is secreted on the body surface and function as a component of skin mucus. AJL-2 agglutinated Escherichia coli and suppressed its growth, suggesting that this lectin is involved in host defense. (+info)
Complete sequence and structure of ribosomal RNA gene of Heterosporis anguillarum.
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The ribosomal RNA (rRNA) gene region of the microsporidium Heterosporis anguillarum has been examined. Complete DNA sequence data (4060 bp, GenBank Accession No. AF402839) of the rRNA gene of H. anguillarum are presented for the small subunit gene (SSU rRNA: 1359 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA: 2664 bp). The secondary structures of the H. anguillarum SSU and LSU rRNA genes are constructed and described. This is the first complete sequence of an rRNA gene published for a fish-infecting microsporidian species. In the phylogenetic analysis, the sequences, including partial SSU rRNA, ITS, and partial LSU rRNA sequences of the fish-infecting microsporidia, were aligned and analysed. The taxonomic position of H. anguillarum as suggested by Lom et al. (2000; Dis Aquat Org 43:225-231) is confirmed in this paper. (+info)
Branchial expression of an aquaporin 3 (AQP-3) homologue is downregulated in the European eel Anguilla anguilla following seawater acclimation.
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A cDNA encoding the homologue of mammalian aquaporin 3 (AQP-3) was isolated by reverse transcription-polymerase chain reaction from the gill of the European eel. The derived amino acid sequence shares 67-70% homology with other vertebrate AQP-3 homologues. Northern blot analysis revealed two AQP-3-specific mRNA species of 2.4 kb and 7 kb. AQP-3 mRNA is expressed predominantly in the eye, oesophagus, intestine (as found in mammals) and the gill; no expression could be demonstrated in the stomach and only low and sporadic levels in the kidney. Quantitative studies demonstrated that, following the 3-week acclimation of freshwater (FW)-adapted yellow and silver eels to seawater (SW), transcript abundance in the gill was reduced by 76% and 97%, respectively. The half time of branchial AQP-3 mRNA downregulation in yellow eels was approximately 10 h, with a maximal 94% decrease in expression after 2 days in SW (compared to time-matched FW controls). However, in fish acclimated to SW for more than 4 days, the fall in AQP-3 mRNA abundance recovered slightly, such that after 3 weeks, expression was 16% of that in time-matched FW controls. The potential roles for this aquaporin isoform in water or solute transport in the eel gill are discussed. (+info)
Immunolocalisation of aquaporin 3 in the gill and the gastrointestinal tract of the European eel Anguilla anguilla (L.).
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The expression of a putative water channel protein, aquaporin 3 (AQP-3), has been localised within branchial and intestinal tissues from the 'silver' life stage of the European eel Anguilla anguilla, using a specific polyclonal antibody directed against the C-terminal of the amino acid sequence. Western blots using the AQP-3 antiserum identified the presence of a major immunoreactive protein of 24 kDa in extracts of gills from both freshwater (FW) and 3 week seawater (SW)-acclimated eels. SW acclimation induced a 65 % reduction in AQP-3 protein abundance in the gill extracts. AQP-3 immunoreactivity was apparent throughout the branchial epithelium from both FW and SW-acclimated fish, but especially so within the chloride cells, which also stained heavily with specific antisera for the beta-subunit of the Na, K-ATPase. AQP-3 immunoreactivity not only colocalised with Na, K-ATPase within the basolateral tubular network but also stained the apical regions of the chloride cell where Na, K-ATPase was absent. Although there were no obvious differences in expression between the chloride cells of FW and SW-acclimated fish, considerably higher intensities of immunoreactivity were apparent near the periphery of the non-chloride cells of FW fish, especially within cells forming the base of the primary filaments and the branchial arch. AQP-3 immunoreactivity was also detected in intra-epithelial macrophage-like cells within the intestine of FW and SW-acclimated eels and in the mucous cells of the rectal epithelium of SW-acclimated fish. These results suggest that AQP-3 may play an important functional role in osmoregulation the teleostean gill but is unlikely to be responsible for the increases in intestinal water absorption that occur following SW acclimation. (+info)
Isolation and molecular characterization of herpesvirus from cultured European eels Anguilla anguilla in Taiwan.
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A herpesvirus has been isolated for the first time from a population of European eels Anguilla anguilla cultured in a recirculated system in Taiwan. Syncytia formation was detected in EP-1 (eel epidermis) cell cultures inoculated with cell-free homogenates prepared from both integument and visceral organs of moribund fish. Inoculation of homogenates onto EK (eel kidney) cell cultures induced giant cell formation. Subsequent passages produced a consistent and progressive cytopathic effect (CPE) in cell cultures. In this study, EP-1 cell cultures infected with EEHV (European eel herpesvirus) were examined using an electron microscope. Numerous nucleocapsids of about 100 nm in diameter were found within the nucleus of infected cells, whereas enveloped particles were observed within the cytoplasm. The mature viral particle, about 235 nm in diameter, had an electron-dense core with a hexagonal nucleocapsid surrounded by a coarse capsule. Histopathological examination of moribund fish showed epithelial hyperplasia with intracytoplasmic metabolic inclusions in the skin. Macrophage aggregates were found in liver, spleen, and kidney. A pair of primers designed from channel catfish virus and salmonid herpesvirus 1 was used in a polymerase chain reaction. A 402 bp fragment was amplified and cloned from genomic DNA of EEHV. The nucleotide homology was 99% (298 of 300) with DNA polymerase of eel herpesvirus (anguillid herpesvirus). EEHV nucleic acids were detected within melanomacrophages in the skin, liver, spleen and kidney by in situ hybridization (ISH). (+info)
Spatial and temporal variation in Anguillicola crassus counts: results of a 4 year survey of eels in Mediterranean lagoons.
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We present the results of a survey of anguillicolosis in the Rh6ne River delta. From January 1997 to December 2000, a total of 13,319 eels (Anguilla anguilla from elver to silver phase) were examined, in which we found 22,227 swimbladder nematodes (Anguillicola crassus adults and pre-adults). A generalised linear model (GLM) framework was used to explore the relative contribution of various factors to the occurrence, intensity and abundance of the parasite. We reveal a major influence of the month of sampling, and we document the existence of a seasonal pattern with regular peaks in early summer and late winter. In contrast, the year of sampling is of secondary importance, and no particular trend in the development of the infection can be detected. More than a decade after the first record of A. crassus in the Rhjne River delta, anguillicolosis has thus attained a constant infection rate of nearly 50%, with a mean number of 3 or 4 macroscopic lumen worms per infected eel. The eel length strongly influences the intensity and the abundance of the nematode, but has little if any effect on the probability of being infected. There exists a linear relationship between eel size and the number of parasites, but not between eel size and prevalence. We observe a decrease in the proportion of infected individuals among elver eels. We discuss this result in relation to the possible mortality of heavily infected individuals and/or a change in the eels' alimentary diet. (+info)