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Coastal and transitional water ecosystems are amongst the most productive and valuable ecosystems on Earth. For their ecological characteristics they have been largely used for fish farming. The lagoons, in particular, are also important as nursery habitats for fishes and for their role in supporting the economically valuable fishes stocks. Fish farming affects the environmental quality with risks of eutrophication, which can lead to the destruction of the same stock bred and to severe anoxic conditions. Such changes in the physical and chemical characteristics in the sediment can have a strong impact on the structure of the benthic macroinvertebrate communities. Therefore, this work deals with the evaluation of the impact of cage rearing of Sparus aurata, farmed at low and high density, in a Mediterranean coastal lagoon located in Apulia (South Italian coastline). Here the changes in macroinvertebrates community structure and in most common ecological indicators were analyzed. Sediment samples were collected with a box-corer between November 2014 and February 2015 at five stations of the lagoon: two located nearly the cage rearing (high density and low density of S. aurata) while three control sites were located at increasing distance from the rearing cages in areas not impacted by fish farming. In parallel, 75 leaf-bags were submerged in the same five stations (15 leaf-bags per site) and retrieved after 5, 15, 30, 60 and 90 days of exposure. At each sampling occasion, some physical-chemical parameters (temperature, conductivity, pH, dissolved O2, salinity, TDS) were also recorded. Macroinvertebrates collected from box-corer and leaf-bags were identified at lowest possible level and following ecological indicators AMBI, M-AMBI, BENTIX and BITS were calculated. The most evident changes of cage rearing were a significant effect on benthic macroinvertebrate community and an accumulation of nutrients in the sediments of the stations directly interested by fish farming.

The aim of this research is the substitution of fish meal with rice protein concentrate (RPC) in the nutrition of Siberian sturgeon (Acipenser baerii) diet. 240 sturgeons of initial mean body weight of 19.1±6.7 g were used. 3 isonitrogeonous (CP 42.4%) and isoenergetic (19.9 KJg-1 DM) diets were formulated with 20% of RPC inclusion (R20), 35% inclusion (R35), 53% inclusion (R53) against a control diet, fish meal based (FM); feeding ratio was 1.5% of BW. All the diets and fish fillets were analyzed to determine the proximate composition. The diet amino acid composition was measured and fillet colour was detected at the end of experimentation. Fish growth showed very good performances of sturgeon fed with high RPC inclusion, but these data were not considered conclusive because of technical problems during the experimentation. However, considering that the highest level of RPC in the sturgeon diets corresponded to an almost complete substitution of fish meal in the fish diet, the result obtained from fish quality analysis showed no adverse effect of experimental diets, with the only limitation of high muscle lipid storage, in fish fed with highest level of RPC inclusion.10.4194/1303-2712-v15_2_1310.4194/1303-2712-v15_2_13

In oviparous vertebrates such as the marine teleost gilthead seabream, water and fluid homeostasis associated with testicular physiology and the external activation of spermatozoa is potentially mediated by multiple aquaporins. To test this hypothesis, we isolated five novel members of the aquaporin superfamily from gilthead seabream and developed paralog-specific antibodies to localize the cellular sites of protein expression in the male reproductive tract. Together with phylogenetic classification, functional characterization of four of the newly isolated paralogs, Aqp0a, -7, -8b, and -9b, demonstrated that they were water permeable, while Aqp8b was also permeable to urea, and Aqp7 and -9b were permeable to glycerol and urea. Immunolocalization experiments indicated that up to seven paralogous aquaporins are differentially expressed in the seabream testis: Aqp0a and -9b in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and sperm. In the efferent duct, only Aqp10b was found in the luminal epithelium. Ejaculated spermatozoa showed a segregated spatial distribution of five aquaporins: Aqp1aa and -7 in the entire flagellum or the head, respectively, and Aqp1ab, -8b, and -10b both in the head and the anterior tail. The combination of immunofluorescence microscopy and biochemical fractionation of spermatozoa indicated that Aqp10b and phosphorylated Aqp1ab are rapidly translocated to the head plasma membrane upon activation, whereas Aqp8b accumulates in the mitochondrion of the spermatozoa. In contrast, Aqp1aa and -7 remained unchanged. These data reveal that aquaporin expression in the teleost testis shares conserved features of the mammalian system, and they suggest that the piscine channels may play different roles in water and solute transport during spermatogenesis, sperm maturation and nutrition, and the initiation and maintenance of sperm motility.

The aim of the present study was to evaluate the effect of sea cucumber meal on gilthead sea bream growth. Two diets were used: a fishmeal based diet (control) and a diet containing 18% of sea cucumber meal inclusion (HM). A 100-days growth trial was carried out (120 fish, initial mean body weight of 35.28±9.31 g). The experimental plan used was monofactorial, balanced with three replicates for two experimental treatments (fish diet). The diets were isolipidic (CL 15.80 ±0.5%), isonitrogenous (CP 49.70±1.0%) and isoenergetic (GE 20.00±1.0 MJ kg-1) and feeding rate was 1.5% of biomass. At the end of the trial, growth performances parameters, digestive enzymes (liver and intestinal), haematological parameters, Na+/K+ ATPase activity were measured. Oressigenic and anoressigenic brain neuropeptides were also analyzed in order to investigate other effects of food intake in fish. Productive parameters, blood plasma parameters, enzyme activities and neuropeptides expression were not affected by the introduction of sea cucumber meal in fish feed thus showing that HM can be used as a partial substitute of fish meal in sea bream nutrition. © Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey in cooperation with Japan International Cooperation Agency (JICA), Japan.

9 pages,5 figures, 1a table, The expression of aquaporins in the spermatozoa of the marine teleost gilthead sea bream (Sparus aurata) and their involvement in the motility activation process were investigated. Sperm motility was activated by a hyperosmotic shock, but it was completely inhibited by 10 lM HgCl2, such inhibition being partially recovered by beta-mercaptoethanol (ME). Conventional RT-PCR using primers specific for S. aurata aquaglyceroporin (glp) and aquaporin 1a (aqp1a) demonstrated the presence of both mRNAs in spermatozoa. Heterologous expression in Xenopus laevis oocytes showed that 10 and 100 lM HgCl2 equally inhibited water and solute transport through S. aurata aquaporin 1a and S. aurata aquaglyceroporin, but treatment with ME only recovered aquaporin 1a-mediated water permeability. Western blot analysis using isoform-specific antisera on protein extracts from spermatozoa revealed bands that corresponded to the predicted molecular mass of S. aurata aquaglyceroporin (31 kDa) and S. aurata aquaporin 1a (28 kDa). The antisera also demonstrated that both aquaporins were localized in the head and flagellum of the spermatozoa. However, the immunoreaction at the plasma membrane of the spermatozoa head was more intense after the hyperosmotic activation, suggesting the translocation of both aquaporin 1a and aquaglyceroporin into the plasma membrane after the osmotic shock. This study therefore provides the first direct demonstration for the presence of aquaporins in fish sperm. The different sensitivities of S. aurata aquaporin 1a and S. aurata aquaglyceroporin to ME may explain the failure of this reducing agent to fully recover the mercurial inhibition of sperm motility, suggesting that these aquaporins may play different physiological roles during the activation and maintenance of sperm motility in sea bream., 1Supported by the Ministry of Agriculture and Aquaculture of the Region Puglia (Italy) under the program between the Region of Puglia and the University of Salento, and by Spanish Ministry of Education and Science grant AGL2007-60262/ACU to J.C. F.C. was supported by the Marie Curie Research Training Network Aqua(glycero)porins grant MRTN-CT-2006-035995 from the European Commission.

We have previously demonstrated that in sea bream Sparus aurata motility initiation determined changes in the phosphorylation state of some proteins. This paper describes an investigation of the effect of a freezing–thawing procedure on the protein phosphorylation/dephosphorylation pattern. Proteins extracted from fresh and cryopreserved spermatozoa (before and after motility activation) were separated on SDS–PAGE, blotted on nitrocellulose membrane and treated with anti-phosphotyrosine, antiphosphothreonine, or anti-phosphoserine antibodies. The results obtained demonstrate that the cryopreservation protocol has a strong effect on the phosphorylation state of proteins. In general, compared to fresh sperm, phosphorylated proteins are most numerous in both activated and non-activated cryopreserved sperm, and in particular we observed a dramatic increase in threonine phosphorylation. However, frozen–thawed sperm showed a minor number of proteins that changed their phosphorylation state after motility activation. Among these, we identified the acetyl-coenzyme A synthetase that plays a role in sperm motility initiation in both fresh and cryopreserved sperm.

In this study we examined the fluctuations of the intracellular calcium concentration in isolated hepatopancreatic R cells during the four molting stages of the prawn Marsupenaeus japonicus. In addition, we used the Fura-2-AM fluorescence technique to investigate the release of calcium from mitochondria and ATP-sensitive calcium stores (endoplasmic reticulum (ER), Golgi, and nucleus) into cytoplasm during the molting cycle. Results demonstrate that both the cytosolic free calcium concentration and the total cell calcium (free, bound to calcium-binding proteins, and stored in amorphous form) in the R cells strictly depend upon the molting cycle. Interestingly, the total cell calcium was higher (approximate to 10 mmol 1(-1)) in postmolt than in premolt (approximate to 1 mmol 1(-1)) and intermolt (approximate to 0.3 mmol 1(-1)). The calcium released from mitochondria was higher during premolt than during postmolt and intermolt, but the amount of calcium released from ATP-sensitive calcium stores was similar during all four stages. All together, our results suggest that the mitochondria-ATP-sensitive calcium stores system does not play a key role in calcium storage during the molting cycle but that it is involved in transcellular calcium flux. We hypothesize that lysosome or membraneclad concretion vacuoles could represent the main site of calcium storage in hepatopancreatic R cells.

Aim: Sperm of Sparus aurata, like those of other teleosts, become activated when spawned into the external medium. Several extracellular factors (ions, osmotic pressure, O2/CO2, sperm activating peptides) have been reported to control sperm motility activation. These factors act on the flagellar motile apparatus, the axoneme, through signal transduction across the plasma membrane and determining the dynein-mediated sliding of the axonemal outer-doublet microtubules through protein phosphorylation. In the present study we have investigated the role of different proteins involved in this signal transduction cascade using cryopreserved sperm of Sparus aurata. Methods: Studies have been performed by using cryopreserved sperm (Fabbrocini et al., Cryobiology 40:46-53, 2000). To investigate the role played by different proteins involved in the signal transduction cascade we evaluated: (1) the effect of specific protein inhibitors on sperm motility (assessed by CASA), (2) quantitative and qualitative evaluation of phosphorylated proteins (tyrosine-phosphorylated, serine-phosphorylated and threonine-phosphorylated) by western blot analysis in activated and non-activated sperm. Results: Results obtained indicated that: (1) calcium and potassium were not involved in sperm motility activation; (2) osmolality values

In these decades, we are assisting to the impoverishment of our marine ecosystem and the conservation of genetic diversity is becoming very important for the long-term interest of any species. In addition, the intensification of fish culture practises has increased the need to study genetic structure in cultured and wild fish stocks. Molecular markers have been very useful for analysis of genetic diversity. Among the several marker systems, amplified fragment lenght polymorphism (AFLP) is highly reliable for the assessment of genetic variation among and within populations. Until now, RAPD, microsatellites and mitochondrial haplotype analysis has been used to study the genetic diversity of sea bass. AFLP analysis was used to evaluate the effect of the sowing of fry obtained from intensive fish farm on the genetic structure of lagoon (Alimini, Otranto, LE) sea bass populations. As control we analysed the genetic structure of a sea bass lagoon population (Acquatina, Frigole, LE) where no fry sowing occurred. A group of 40 genotypes belonging to 2 populations was screened using 6 different AFLP primer combinations. A total of 366 loci were produced. Mean proportion of polymorphic loci of wild and extensive populations was 0.92 and 0.93, while mean expected heterozigosity was 0.37 and 0.36, respectively (Table 1). The genetic similarity valued based on Nei’s coefficient revealed an average population similarity of 92%. The individual-based similarity trees and the principal coordinate analysis revealed that the 2 populations were separated into two clusters. AMOVA analysis shows that a relatively high proportion of total genetic diversity was attributed within the groups (91.29%). AMOVA and Bayesian analysis was performed to evaluate FST and the two different analysis gave concordant results showing low genetic differentiation among Acquatina and Alimini populations (FST = 0.091; B = 0.0719, Table 2).

A new interesting species for the diversification of national aquaculture is Argyrosomus regius. It is a very promising species, presenting interesting biological features (growth and larval rearing). The application of DNA markers has allowed rapid progress in aquaculture investigations of genetic variability and inbreeding, parentage assignments, species and strain identification, and the construction of high-resolution genetic linkage maps for aquaculture species. Among the several marker system, amplified fragment length polymorfism (AFLP) is highly reiable for the assessment of genetic variation among and within populations. In this work we evaluated the genetic variation, by using AFLP analysis, of a broodstock of Argyrosomus regius.

The present paper reported postmortem changes in muscle proteins on sea bream (Sparus aurata), during ice storage. The postmortem evolution of protein patterns in farmed sea bream was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimension electrophoresis (2DE). Matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used for identification of proteins. Results of the bio-informatics analysis demonstrate that: (1) the normalized volume of skeletal alpha-actin and tropomyosin is not affected by the time of storage; (2) the normalized volumes of myosin light chain 3 and of major histocompatibility complex (MHC) class II beta1 increase; (3) the normalized volumes of Sec 13-like and parvalbumin significantly decrease.

Four cell types compose the crustacean hepatopancreas: E (embryonic), R (resorptive), F (fibrillar) and B (blister-like) cells. The physiological activity played by these cells seems to be affected by different physiological/environmental conditions such as the molting cycle, nutritional state, osmoregulation, etc. The aim of the present study was to evaluate whether the activities of different enzymes, i.e. acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), plasma membrane calcium ATPase (PMCA), sarco-endoplasmic calcium ATPase (SERCA) were expressed in the R cells and then affected by the molting cycle. ACC and FAS are involved in the de novo fatty acid synthesis being ACC generally considered the rate limiting step of this metabolic pathway. The characteristics of ACC and FAS have been extensively studied in both mammalian and fish liver. In this respect, however, no information is so far available in marine invertebrates. The calcium pump activities were investigated since the epithelial cells of the crustacean hepatopancreas play an important role in Ca2+ balance. ACC and FAS activities were assayed at different temperatures in fresh cells coming from hepatopancreas of shrimps at the pre-moult stage. The highest activities of both ACC and FAS were found at 25 °C. When these enzymatic activities were measured as dependence of moult stage, intermoult was the stage in which both ACC and FAS activities showed their maximum value. SERCA and PMCA activities were detected only in early premoult stage. The results obtained demonstrated that the molting cycle affects important metabolic pathways and are in agreement with the role carried out by the R cells mainly represented by lipid and calcium storage.

In the present study we used the two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to compare the protein expression in two cell lines (PC-Cl3 and PC-E1A+raf). PC-Cl3 is a immortalized rat thyroid cell line which in vitro retain biochemical characteristics of differentiated thyroid cell. PC-E1A+raf originates by stable transfection with E1A and v-raf oncogenes of the PC-Cl3 cells, and displays a malignant phenotype. The protein profiles differed between the two cell lines as revealed by visual inspection and by image analysis software. We identified 414 ± 10 (n = 10) spots in PC-Cl3, among these 11 significantly decreased, eight were absent and six increased in 2-D gel obtained with PC-E1A+raf. Five of these spots were analyzed with MALDI-TOF, but only three showed a significant match in the databases used in the bioinformatics analysis (Profound, Mascot, ad MS-fit). In particular, one of the spot showed homology with a GAPDH (glyceraldehydes-3-phosphate dehydrogenase, one with H(+) transporting ATP-synthase and one with PEBP (phoshatidylethanolamine-binding protein). The present work shows that the transfection with E1A and v-raf oncogenes of the PC-Cl3 cells causes the change of protein expression of PC-Cl3. Two out of three of these identified spots (ATP-synthase and GAPDH) seems to be involved in the cell apoptosis.

This study demonstrated that sea breams cultured both in a semi-intensive and intensive system, present a physiological state comparable to that of wild individuals coming from extensive aquaculture. Fish reared with different culture systems can compete with wild specimens in terms of flesh quality and safety for food consumption.

This study demonstrated that under our maturating condition we obtained a microbial maturated water that was able to buffer the perturbations due to the addition of larvae of the system. The addition of nitrifying bacteria did not ameliorate the microbial water maturation.

The most common parameters used to evaluate sperm quality are motility rate and duration and fertilization ability. In this study, chemical and biochemical parameters of sea bass (Dicentrarchus labrax) sperm were investigated to find an alternative method for evaluating sperm fertilization ability before and after cryopreservation. The biochemical and chemical analyses were performed with fresh and frozen-thawed sperm and seminal plasma. To cryopreserve sperm, 250-mul straws were used. Fertilization ability was evaluated by inseminating eggs (obtained from hormonally stimulated females) with fresh and cryopreserved sperm. The results revealed a linear relationship (P < 0.05) between semen fertilization capacity and some seminal plasma (beta-D-glucuronidase activity, potassium concentration) and sperm (ATP concentration, aspartate aminotransferase activity) parameters. Variations in semen fertilization rate could be best described by two multiple regression models: one including the sperm parameters and another including the seminal plasma parameters. For practical application, the use of simple regression models is of value. Fertilization rate in both fresh and cryopreserved sperm was reliably predicted by determining the ATP concentration or the beta-D-glucuronidase activity or both.

The results obtained in this study indicate that in immature sea bass, hCG (LH-like) stimulates spermatogenesis and spermiation probably acting as maturational gonadotropin. Its ability in inducing spermatogenesis is affected by individual differences in the clearance rates.

In the present study we used two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-associated laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry to verify whether the protein expression of sea bass sperm was affected by the cryopreservation procedure. The protein profiles differed between fresh and frozen-thawed semen as revealed by visual inspection and by image analysis software. We identified 163 spots in fresh sperm; among these, 13 were significantly decreased and 8 were absent in two-dimensional gel obtained with cryopreserved sperm. Five of these spots were analyzed with MALDI-TOF, but only three showed a significant match in the databases used in bio-informatics analysis (Pept- Ident, Mascot, and MS-Fit). In particular, spot 5 showed homology with a novel protein of zebrafish (similar to SKB1 of human and mouse), spot 13 showed homology with amphibian G1/S-specific cyclin E2, and spot 20 showed homology with the hypothetical protein DKFZp566A1524 of Brachidanio rerio. The present work shows that the use of the cryopreservation procedure causes the degradation of sperm proteins and among these, two could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm.

Background/Aims: In thyroid cells, the intracellular pH plays a key role in the control of the iodide uptake, since iodide accumulation is associated with an intracellular acidification. In the present paper we studied the kinetic proprieties of the Na+/H+ antiporter (NHE) and the molecular expression of different NHE isoforms in rat thyroid PC-CI3 cells. In addition the intracellular buffer capacity was also evaluated. Methods: pHi was measured using the pH sensitive fluorescent dye BCECF-AM. Amiloride, 5-(N,N-Dimethyl) hydrochloride was used to inhibit NHE activity. RT-PCR and western blot analyses were used to study the expression of NHE mRNA and protein isoforms. Results: PC-CI3 cells shown a resting pHi, in the absence of CO2/HCO3-, of 6.94 +/- 0.1; after an acid load PC-CI3 cells recovered toward resting pHi value, using a Na-dependent H+ extrusion mechanisms which was amiloride sensitive (K-i = 23 muM). The kinetic parameters were K-(Na)app = 10 +/- 2 mM and V-max = 0.23 +/- 0.02 DeltapH/min x 10(5)cells. NHE1, NHE2 and NHE3 were expressed at the mRNA level as well as at the protein level. Conclusion: PC-CI3 cells express a functional Na/H exchange activity and different isciforms (NHE1, NHE2 and NHE3) are expressed in the plasma membrane.

17 pages, 9 figures, 1 table, In oviparous vertebrates such as the marine teleost gilthead seabream, water and fluid homeostasis associated with testicular physiology and the external activation of spermatozoa is potentially mediated by multiple aquaporins. To test this hypothesis, we isolated five novel members of the aquaporin superfamily from gilthead seabream and developed paralog-specific antibodies to localize the cellular sites of protein expression in the male reproductive tract. Together with phylogenetic classification, functional characterization of four of the newly isolated paralogs, Aqp0a, -7, -8b, and -9b, demonstrated that they were water permeable, while Aqp8b was also permeable to urea, and Aqp7 and -9b were permeable to glycerol and urea. Immunolocalization experiments indicated that up to seven paralogous aquaporins are differentially expressed in the seabream testis: Aqp0a and -9b in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and sperm. In the efferent duct, only Aqp10b was found in the luminal epithelium. Ejaculated spermatozoa showed a segregated spatial distribution of five aquaporins: Aqp1aa and -7 in the entire flagellum or the head, respectively, and Aqp1ab, -8b, and -10b both in the head and the anterior tail. The combination of immunofluorescence microscopy and biochemical fractionation of spermatozoa indicated that Aqp10b and phosphorylated Aqp1ab are rapidly translocated to the head plasma membrane upon activation, whereas Aqp8b accumulates in the mitochondrion of the spermatozoa. In contrast, Aqp1aa and -7 remained unchanged. These data reveal that aquaporin expression in the teleost testis shares conserved features of the mammalian system, and they suggest that the piscine channels may play different roles in water and solute transport during spermatogenesis, sperm maturation and nutrition, and the initiation and maintenance of sperm motility, Supported by the Spanish Ministry of Science and Innovation (MICINN; AGL2010-15597 to J.C.), with additional financial support from the Research Council of Norway (204813/F20 and 224816/E40 to R.N.F.), and from the Ministry of Agriculture and Aquaculture of the Puglia Region (Italy) under the program between the Region of Puglia and the University of Salento (to S.V.). F.C. and M.B. were supported by a postdoctoral (Juan de la Cierva Programme) and predoctoral (FPI) fellowship, respectively, from MICINN

Changes in the plasma membrane lipid composition have been related to a decrease in sperm quality during cryopreservation. Antifreeze proteins (AFPs) have been tested in different species because of their ability to depress the freezing point and their potential interaction with membranes, but controversial effects were reported. In the present study we analyzed separately the lipid composition of two sperm membrane domains, head plasma membrane (HM) and flagellar membrane (FM), after cryopreservation with an extender containing 5% dimethyl sulfoxide (DMSO) either alone or with AFPI or AFPIII (1 μg/ml). We used sperm from a teleost, Sparus aurata, because the lack of acrosome avoids changes of lipid profiles due to capacitation process or acrosomal losses during freezing/thawing. Comparing with the control (cryopreservation with 5% DMSO alone), the addition of AFPIII increased the velocity, linearity of movement, and percentage of viable cells. In addition, freezing with DMSO alone increased the phosphatidyl-serine content as well as the saturated fatty acids and decreased the unsaturated ones (mainly polyunsaturated) both in HM and FM. These changes in the lipid components were highly avoided with the addition of AFPIII. HM had a higher amount of saturated fatty acids than FM and was more affected by cryopreservation without AFPs. The percentage of viable cells was positively correlated with the amount of unsaturated fatty acids in the HM, whereas the motility parameters were positively correlated with both FM and HM amount of unsaturated fatty acids. AFPs, especially AFPIII, seem to have interacted with unsaturated fatty acids, stabilizing the plasma membrane organization during cryopreservation and contributing to improve sperm quality after thawing., Supported by the project Petri (PTR 95.1026.OP.01). J.B.S. was supported by a Ph.D. grant from the Portuguese Ministry of Science and Technology (SFRH/BD/31990/2006) cofounded by POPH–QREN–Tipologia 4.1 (FEDER and MCTES).

This study has demonstrated the existence of an L-proline-dependent (Na independent) proton flux at the apical membrane level of the eel intestinal absorbing cells. Using isolated eel enterocytes and the pH-sensitive fluorescent dye 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxy-fluorescein, acetoxymethyl ester (BCECF), it was shown that a 20 mM concentration of the imino acid L-proline in the extracellular medium determined an intracellular acidification of similar to0.28 pH units. However, neither sucrose nor other amino acids were able to significantly acidify the resting intracellular pH. A hyperbolic relationship between extracellular proline concentration and intracellular proton accumulation was observed. Using both isolated brush-border and basolateral membrane vesicles, it was demonstrated that this proline-proton cotransport mechanism was located at the apical membrane level only. In addition, the existence of a coupling mechanism between proline and proton fluxes was demonstrated by the observation that, in brush-border membrane vesicles, the presence of a pH gradient (pH(in) > pH(out)) stimulated the uptake of L-proline.

Trabajo presentado en el 3rd International Workshop on Biology of Fish Gametes, celebrado en Budapest del 7 al 9 de septiembre de 2011., Changes in the plasma membrane lipid composition during cryopreservation have been related to a decreasein sperm quality (Muller et al., 2008). In the present work we tested the addition of antifreeze proteins(AFPs) to the cryopreservation extender because of their ability to depress the freezing point and their potential interaction with membranes, This work was supported by the project Petri (PTR 95.1026.OP.01). J. Beirão was supported by a PhD grant from the Portuguese Ministry of Science and Technology (SFRH/BD/31990/2006) co-foundedby POPH - QREN (FEDER and MCTES).

Transposable elements are genetic elements capable of moving from one chromosomal location to another. They carry a gene encoding the enzyme for their own transposition (transposase) and flanking sequences required for recognition by the transposase. Transposable elements of the Tc1/mariner family are widely distributed in the animal kingdom, probably due to horizontal transmission among different species. To increase the efficiency of foreign DNA integration in fish, we are using one of these elements, namely Bari-1 from Drosophila melanogaster, to set up new transformation vectors for gene delivery into fish genomes

An electroneutral Na+/H+ exchange mechanism (dimethylamiloride inhibitable, Li+ sensitive, and Ca2+ insensitive) was identified in brush-border membrane vesicles (BBMV) from Kuruma prawn hepatopancreas by monitoring Na+-dependent H+ fluxes with the pH-sensitive dye acridine orange and measuring Na-22(+) uptake. Kinetic parameters measured under short-circuited conditions were the Na+ concentration that yielded one-half of the maximal dissipation rate (F-max) of the preset transmembrane Delta pH (K-Na) = 15 +/- 2 mM and F-max = 3,626 +/- 197 Delta F.min(-1).mg protein(-1), with a Hill coefficient for Na+ of similar to 1. In addition, the inhibitory constant for dimethylamiloride was found to be similar to 1 mu M. The electroneutral nature of the antiporter was assessed in that an inside-negative transmembrane electrical potential neither affected kinetic parameters nor stimulated pH-dependent (intracellular pH > extracellular pH) Na-22(+) uptake. In contrast, electrogenic pH-dependent Na-22(+) uptake was observed in lobster hepatopancreatic BBMV. Substitution of chloride with gluconate resulted in increasing K-Na and decreasing Delta F-max, which suggests a possible role of chloride in the operational mechanism of the antiporter. These results indicate that a Na+/H+ exchanger, resembling the electroneutral Na+/H+ antiporter model, is present in hepatopancreatic BBMV from the Kuruma prawn Penaeus japonicus.

Using labelled ligand-binding methods, previous studies have identified specific angiotensin II receptors (ANG II-Rs) in eel liver, kidney and intestine membranes. Isoelectric focusing on polyacrylamide gels also showed that there are two Ang II-R isoforms in eel liver, focusing at isoelectric points (pI) 6.5 and 6.7. These may have different functions. In contrast, eel enterocyte plasma membrane and renal brush border membranes contain only the pI 6.5 form. To characterize the eel receptors more fully, a newly developed monoclonal antibody (6313/G2) which selectively recognizes the AT(1) subtype of mammalian Ang II-R was used. In ligand-binding experiments, the preincubation of eel liver membranes with 6313/G2 antibody eliminated the specific [3,5-H-3]Tyr(4)-IIe(5)-Ang II binding. Moreover, Ang II-receptor complexes from solubilized liver membranes, which were immunoprecipitated by 6313/G2-coated beads, had a pI of 6.5. In immunoblotting experiments, the antibody recognized the isoform focusing at pI 6.5 in eel intestine and liver preparations, but not the liver pI 6.7 isoform. Immunoblotting of SDS gels showed that the antibody bound to a single protein of molecular mass of 75 kDa in eel liver, gill and kidney and to a doublet of molecular mass of about 74 and 75 kDa intestinal membrane preparations. Immunocytochemistry of paraffin-embedded and cryostat sections of eel liver, kidney, intestine and gill showed that antibody 6313/G2 bound to uniformly distributed intracellular sites and cell surface membrane in proximal tubular cells, absorptive intestinal cells, hepatocytes and chloride cells. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine. The data suggest that the previously described Ang II-R from eel liver, kidney and intestine may be similar to the mammalian AT(1) subtype.