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Identification of alkaline proteases produced during larval stages of gilthead sea bream, Sparus aurata was achieved using SDS-PAGE and specific inhibitors. Such techniques were also applied to determine proteases existing in rotifers, Brachionus plicatilis, and Artemia nauplii, which are used as live food for these larvae, as well as proteases of adult fish. The results show a great prominence of trypsin-like proteases during the 4 weeks after hatching, but the number of enzyme species was reduced in adult fish. Alkaline proteases present in the rotifers and Artemia showed clear differences when compared with those of the larvae and were not detected in extracts obtained from fed larvae. The results obtained provide information about the role of exogenous enzymes in larval feeding of sea bream.

The effect of delayed initial feeding on the ingestion ability of gilthead seabream Spams aurata larvae was studied. The percentage of larvae with visible gut contents was maximal when food was first provided on the 5th day after hatching and decreased to half this value when provision of food was delayed until Day 8, indicating the point at which starvation became irreversible. Morphological and histological changes were studied in growing and starving larvae. Sizes of different organs increased in actively feeding larvae but decreased in unfed larvae, particularly in the case of the liver. In unfed larvae, development due to yolk reserves attained a maximum on Day 5. The digestive tract was functional in 4-day-old larvae, which had food in the gut. The absorption process was obvious from Day 6, although it could be detected in 5-day-old larvae. Starving larvae exhibited widespread histological degeneration, especially in the digestive tract and associated glands.

11 páginas, 4 figuras, 1 tabla., Vitrification could provide a promising tool for the cryopreservation of fish embryos. In order to achieve successful cryopreservation, several parameters should be taken into account in the design of a vitrification protocol. In the present study some relevant factors were investigated (permeable and non-permeable cryoprotectant toxicity, toxicity of vitrificant solutions, adequate container for embryo loading and temperature of thawing) using two gilthead seabream embryonic development stages (tail bud and tail-bud-free). Permeabilized embryos were incubated in dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and 1,2-propanediol (PROH) in concentrations ranging from 0.5 to 6 M for 10 and 30 min and in 5%, 10% and 15% polyvinyl pyrrolidone (PVP), 10%, 15% and 20% sucrose or 0.1%, 1% and 2% X1000® for 2 min. After treatment, embryos were washed and incubated in seawater until hatched. The toxicity of permeable cryoprotectants increased with concentration and exposure time. EG was best tolerated by the embryos. Exposure to non-permeable cryoprotectants did not affect the hatching rate except at F stage. Six vitrificant solutions (DMSO—V1, V2 and V3 and EG—V1, V2 and V3) were tested using a stepwise incorporation protocol. The DMSO-based solutions contained 5 M DMSO + 2 M MeOH + 1 M EG plus 5% PVP, 10% sucrose or 2% X1000® and the EG-based solutions contained 5 M EG + 2 M MeOH + 1 M DMSO plus 5% PVP or 10% sucrose. Before loading the embryos into 0.5 ml straws or 1 ml macrotubes, toxicity tests were effected with these solutions. Our results demonstrated that DMSO-based solutions were better tolerated by seabream embryos than EG-based solutions. After thawing (water bath, 0 or 25 °C), embryos were evaluated by stereoscopic microscopy and the percentage of embryos with intact morphology was registered. The highest percentage of embryos with intact morphology (28%) was observed in samples frozen in macrotubes and thawed at 25 °C. Several malformations associated with ice crystal formation inside the embryos were detected. None of these embryos achieved hatching. Our results suggest that the absence of a proper incorporation of cryoprotectants prior to vitrification is the main problem that must be overcome. This procedure should be optimized in order to avoid ice crystal formation inside embryo compartments., This work was supported by the project INIA/MCYT ACU02-002-C2-2 and by a post-doctoral grant SFRH/BPD/6461/2001 from the Portuguese Ministry of Science and Technology.

11 páginas, 6 figuras, 6 tablas., The toxicity of sediments from two littoral ecosystems of the Gulf of Cadiz was tested using juveniles of the fishSparusaurata(seabream). Concentrations of total carbon and nitrogen, organic carbon, 14 heavy metals (Fe, Mn, Cu, Zn, Pb, Cd, Ag, Hg, As, Sn, V, Ni, Co, Cr), and the surfactant linear alkyl benzenesulfonate (LAS) in the sediments were measured. Chemical analysis was performed in the stations to determine the degree and nature of contamination. Four different endpoints were selected in the toxicity test: survival, superficial alteration, hematocrit analysis, and histological damage. After 14 days, survival, superficial alteration, and hematocrit analysis did not reveal effects of the different sediments tested. The histological and cellular damage revealed a more sensitive response to measured chemicals in sediments and they were found to be a powerful tool to evaluate sedimenttoxicity effects. Semiquantitative evaluation of the histological damage demonstrated correlation with sediment concentrations of some of the heavy metals (Cr, Cd, Pb, Ag, Cu) and the surfactant (LAS). Data derived from chemical concentrations and toxicity tests were assembled by multivariate statistical techniques (principal components analysis) to identify the ranges of chemical concentrations associated with an adverse effect. The results obtained, as suggested by site-specific sediment quality values, were the following: Cr≥90.2; Cd≥1.24; Pb≥52.5; Ag≥0.68; Cu≥71.2; LAS≥8.7 mg kg−1of dry sediment. These results are mainly in concordance with studies performed in other areas of the world and therefore support wide application of the method., This study was performed as a part of Contract AMB94-0291 granted by the Spanish Science and Technology Interministerial Commission (CICYT).

14 páginas, 3 figuras, 1 tabla., Resorption of the yolksac and development of the digestive tract and associated organs, including the swim bladder, were studied in Sparusaurata larvae from hatching until day 30 using histological and histochemical procedures. At the onset of exogenous feeding three regions could be easily distinguished in the gut: the foregut including the oesophagus and a primordial stomach, the midgut and lastly the hindgut. At this time, the digestive tract was functional even though the stomach was not yet completely developed; gastric glands, for instance, were not present in the period studied. Glycogen and zymogen grains were stored in the liver and pancreas, respectively between days 4 and 6 after hatching. Proteins were observed in the pancreas, hepatic vascular system and to a lesser extent the hepatocytes. Once feeding had commenced, the anterior intestinal epithelium developed the capacity for the absorption of lipids, most of which were included in large lipid droplets. Simultaneously, acidophilic supranuclear inclusions containing proteins were observed in the posterior intestinal epithelium. All oesophageal mucous cells, intestinal goblet cells, epithelial columnar cells in the stomach, and enterocytes of the digestive epithelium were rich in carboxylated, sulphated and/or neutral mucosubstances. Proteins were not present in digestive goblet cells., This work was supported by the Comision Interministerial de Ciencia y Tecnologia, Spain (CICYT Projects MAR88-0196 and MAR91-0548).

Two basic types of protein-walled microcapsules were developed with the aid of using different preparation techniques. One type (Type A) was spherical and hard-walled, whereas the other (Type G) was irregularly shaped and soft-walled. The present work examined the larval growth and development of Sparus aurata reared either with these microcapsules as the sole food source or with a mixed diet of microcapsules and rotifers from the start of feeding. Larvae fed on Type A microcapsules evacuated them practically intact. These larvae showed strong degeneration of the gut epithelium, liver and pancreas and died within two to three days after the onset of feeding. In contrast, larvae fed from first feeding with Type G microcapsules alone ingested and broke down the particles from the onset of feeding. No larval growth was observed, but some larvae were still alive at the end of the experiment (Day 13). Larvae reared with a mixed diet, including Type G microcapsules and a small amount of rotifers (0.5 rotifer mL-1), showed in general normal development of gut epithelium. These larvae had good growth but survival was reduced to one-half of that obtained under routine rearing of larvae fed on rotifers alone. Type G microcapsules appear to represent an adequate departure point for the development of an inert diet able to support larval growth in marine fish.

The evolution of the digestive enzyme equipment in seabream from hatching to 30 days old larvae was studied; there was a progressive increase in the activity of protease, amylase and acid and alkaline phosphatase from day 15 onwards. The use of specific inhibitors, and SDS-PAGE provided evidence to suggest that most of the proteases belonged to the serine group. A high α-amylase activity was also denoted. Zymograms of larval extracts indicated that exogenous food has more a qualitative than a quantitative role in the secretion of digestive enzymes in this species.