F.J. Alarcón

Characterization of digestive enzyme activity during larval development of gilthead seabream (Sparus aurata)

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.

Characterization and functional properties of digestive proteases in two sparids; gilthead seabream (Sparus aurata) and common dentex (Dentex dentex)

Digestive proteases present in two sparids, seabream (Sparus aurata) and common dentex (Dentex dentex), have been characterized using both biochemical and electrophoretic techniques. Although optimum pH and temperature for maximum activity of both acid and alkaline proteases were similar in the two species, important differences in total activity, as well as in thermal and pH stability were found. Specific inhibitors and SDS-PAGE zymograms were used to clarify such differences. Evidences support the existence of a more active and complex protease set in dentex. Results are discussed from the perspective of their application to the formulation of feeds for each species.

Substrate-SDS-PAGE determination of protease activity through larval development in sea bream

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.

Digestive proteases during development of larvae of red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)

The evolution of digestive proteases during larval development of Rhynchophorus ferrugineus (Olivier, 1790) has been studied. A progressive increase of protease activity has been found. The optimum pH for proteolytic activity against azocasein was determined. Caseinograms revealed an active complex of alkaline proteases from the early stages of the development. From the apparent molecular masses, three groups of proteases have been found - high molecular-mass proteases, medium molecular-mass proteases, and low molecular-mass proteases. Studies using specific protease inhibitors showed the major presence of serine proteases in gut extracts. The results obtained from larvae reared on different substrates have made possible a comparative assessment of the influence of diet on the development of the digestive enzymatic system. Larvae fed on an artificial diet showed a complete pattern of digestive proteases. Data suggest that this diet seems to be suitable for future research with this insect pest.

Use of the probiotic Shewanella putrefaciens Pdp11 on the culture of Senegalese sole (Solea senegalensis, Kaup 1858) and gilthead seabream (Sparus aurata L.)

The application of probiotics on aquatic animals is increasing for a better fish welfare status as well as an environment-friendly activity which are actual demands of modern aquaculture industry. A bacterium from skin mucus of healthy gilthead seabream (Sparus aurata L.) has been isolated and identified as Shewanella putrefaciens Pdp11. Different studies have been done to know its application as probiotic in the Senegalese sole and gilthead seabream farming. This article reviews the studies carried out with this probiotic microorganism focusing on the current knowledge of its in vitro and in vivo mechanisms of action. The results suggested that the probiotic S. putrefaciens Pdp11, due to its beneficial effects, could be used in the aquaculture activity of both species.

Improved detection of amylase activity by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with copolymerized starch

An improved method, based on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) for detection of amylase activity is described. This method will allow better characterization of certain amylases than that obtained by the Davis technique. The main features of the technique are: (i) identification of amylase bands and molecular mass determination are possible in the same gel; (ii) the hydrolysis of copolymerized substrate during electrophoretic separation is prevented using very low temperatures instead of inactivating agents such as chelating agents; and (iii) the technique is applicable to reveal amylase activity in a wide range of biological samples. The method is not useful for enzymes sensitive to SDS and for high molecular mass amylases.

Effect of dietary protein level and source on digestive proteolytic enzyme activity in juvenile Senegalese sole, Solea senegalensis Kaup 1850

The effect of dietary protein level and protein source on growth and proteolytic activity of juvenile Solea senegalensis was studied. In Experiment 1, fish were fed on four experimental diets containing increased protein levels (36, 46, 56 and 67%). In Experiment 2, Senegalese soles were fed on five diets with partial substitution of fish meal by soybean meal, soybean protein concentrate, soybean protein isolate, wheat gluten meal or pea protein concentrate. Results prove that growth and proteolytic activity in the distal intestine of fish were affected by the quantitative increase in dietary protein. The origin of protein source used in the elaboration of experimental diets affected both the amount and composition of the alkaline proteases secreted into the intestinal lumen; however, it did not decrease animal growth. Juvenile Senegalese sole showed capability to modulate digestive protease secretion when the concentration and/or source of dietary protein were modified. Quantity and quality of dietary protein affected protein hydrolysis in Senegalese sole intestine. This study establishes that 30% fish meal protein can be replaced by soybean derivatives without affecting intestinal proteases. Replacement with wheat gluten meal or pea protein concentrate should be taken cautiously, but further research is needed to establish whether growth performance and digestive enzyme physiology of Senegalese sole are affected by plant protein-supplemented diets in a long-term trial.

Characterization of digestive carbohydrase activity in the gilthead seabream (Sparus aurata)

The presence of digestive carbohydrases was determined in seabream intestines, and the main activity identified was amylase. Optimum activity for this enzyme was found at pH 8.0 at 40 °C. Amylase activity was highly sensitive to extreme pH, and temperatures exceeding 50 °C. The use of SDS–PAGE zymograms allowed identification of amylase in the form of a high molecular mass fraction exceeding 100 kDa. Results confirm the existence of a well developed amylase equipment in this species which supports the possibility of increasing the amount of carbohydrates in the formulation of its commercial feeds.

Use of SDS-page in the assessment of protein hydrolysis by fish digestive enzymes

Inthe field of fish nutrition, the preliminary evaluation of feedstuffs usingin vitro techniques may be an alternative to invivo assays. Degradation of the protein fraction in feedstuffs byseabream (Sparus aurata) digestive enzymes was studiedunder different conditions simulating either acid, alkaline, or acid + alkalinedigestion using a modified pH-stat technique. In addition, a sequential sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the releasedproducts was used both to visualise and to quantify the level of proteinhydrolysis. Gels were analysed by optical densitometry and results wereexpressed as a Coefficient of Protein Degradation (CPD). Values of CPD showedclear differences related to the type of protein digested or the steps followedin the digestion. CDP index provides information based on the enzymaticbreakdown of the proteins visualised in SDS-PAGE gels. The pH-stat evaluation(Degree of Hydrolysis) supplies measurement of the total number of peptidebondsbroken down in a given protein. CDP index mainly focuses on the action ofendoproteases, whereas degree of hydrolysis (DH) includes both endo- andexoproteases. CPD and DH are two complementary indexes capable of measuring theprotein hydrolysis by fish enzymes of a given feedstuffs or diet. Both methodswere in agreement with respect to assessing the hydrolysis of protein. Thesignificance of the SDS-PAGE techniques in the assessment of aquafeeds by fishdigestive enzymes is discussed.

Feed pellets containing chitosan nanoparticles as plasmid DNA oral delivery system for fish: In vivo assessment in gilthead sea bream (Sparus aurata) juveniles

&NA; The aim of this study was the assessment of preloaded feed pellets as a delivery system for plasmid DNA (pDNA), with the purpose of evaluating the potential administration of DNA vaccines orally in aquacultured fish. Pellets were made up by usual feed ingredients, which were mixed with chitosan nanoparticles entrapping a model plasmid (pCMV&bgr;) expressible in eukaryotic cells before being elaborated. The plasmid is characterized by the insertion of the reporter gene lacZ, encoding for the bacterial enzyme &bgr;‐galactosidase (&bgr;‐gal). The possible in vivo expression of the exogenous gene was measured in different fish tissues of gilthead sea bream (Sparus aurata) juveniles by two different procedures. On the one hand, the activity of the enzyme &bgr;‐gal was detected and quantified in muscle, liver and intestine; on the other, specific IgM against &bgr;‐gal antigen was titrated in blood samples. Intramuscular (i.m.) injection of equal amounts of plasmid was also carried out for the purpose of comparison with oral administration. The expression of the reporter gene was detected in fish tissues following both oral and i. m. administration of pDNA up to 60 days. However, organ distribution of the gene expression was more evident after oral (&bgr;‐gal activity measured in gut, liver and muscle) than after parenteral administration (restricted to adjacent muscle tissues). In agreement, specific IgM titration indicated that humoral immune response was more intense and sustained throughout the experimental period after oral than after i. m. delivery of equal amounts of pDNA. These results suggest that feed pellets containing chitosan nanoparticles might enable efficient oral delivery of pDNA, a fact that might imply valuable applications in terms of on‐farm mass immunization purposes, especially with regard to DNA‐based vaccines and small size fish, in which i. m. administration remains unfeasible. Graphical abstract Figure. No Caption available. HighlightsOral administration is the ideal route for mass vaccination in fish.Plasmid‐based vaccines are readily inactivated within the digestive tract of fish.If protected in adequate nanoparticles, plasmids can resist feed processing.Feed‐delivered plasmids exhibited remarkable in vivo biological activity.Feed pellets enable non‐disturbing, efficient oral delivery of plasmid DNA.