Search

The aim of this research was to evaluate the possible positive effects of feeding Nile tilapia (Oreochromis niloticus) with a diet containing a commercial probiotic AQUA-PHOTO® [Lactobacillus plantarum (1.51 × 106 CFU g−1) and Bacillus subtilis (1.34 × 107 CFU g−1)] on digestive enzymes’ activity and gut microbiome profile. The experimental design was completely randomized, with two treatments [control and probiotic diets (0.08 g.100 g−1 of feed)] and six replicate tanks per group (n = 8 animals per experimental unit, mean weight 8.18 g ± 1.85 g). The 96 fish were distributed into 12 aquaria and fed with the probiotic and control diets for 7 days. After this period, intestine samples (anterior, middle, and posterior portions) were collected from two individuals per tank. The samples were analyzed for assessment of DGGE profile and digestive enzymes’ activity (amylase, alkaline protease, and alkaline phosphatase). The results were subjected to analysis of variance (ANOVA) and the means compared by t test (P

Understanding the daily digestive function could lead to more efficient feeding practices in the farmed fish species. In this work, the effect of several daily feeding protocols was assessed on the daily expression of the genes related to the circadian system (arntl1, clock, per1, and cry1), appetite (cck), nutrients hydrolysis (try1, ctrb1, pga, atp4a, cel, and amy2a), peptides transport (slc15a1), pH, and activity of key digestive (trypsin, chymotrypsin, amylase, 4C- and 7C-like lipases) and absorptive (alkaline phosphatase) enzymes in the gastrointestinal tract (GIT) of Senegalese sole (Solea senegalensis) juveniles. Some of the tested feeding protocols demonstrated a significant influence on the entrainment of the peripheral oscillator. The synchrony of slc15a1 and some digestive proenzymes with per1 in some of the experimental treatments could underline the role of clock genes in the regulation of these biological functions. Unlike gene expression of the digestive proenzymes wherein feeding protocol occasionally affected the temporal organization, the influence of this factor was determinant on the daily average production and/or circadian pattern of their activities. Daily pH was maintained at approximately neutral levels in all the GIT segments, and no pepsin activity was detected. Fish fed a single diurnal meal showed the lowest activity for all the studied enzymes, except for amylase, suggesting an insufficient utilization of the ingested nutrients. Higher amylase activity in this group, on the other hand, implies the possible activation of alternative compensatory metabolic pathways. Overall, our results pointed to the suitability of higher feeding frequencies, especially during dark hours, for the aquaculture production of this species., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by projects EFISHDIGEST (AGL2014-52888-R) with FEDER/ERDF contribution granted to MY and WISEFEED funded by the European Union's H2020 Program (Marie Skłodowska-Curie Grant No. 691150). NG was supported by a doctoral fellowship (BES-2015-071662) from MINECO (Spain).

Fishmeal (FM) possesses one of the highest buffering capacities in comparison to most alternative vegetable aquafeed ingredients and its decreasing content in current formulations might affect the ideal gastrointestinal environment for digestive enzyme action and gut bacterial community of carnivorous fish species. A study was undertaken, therefore, to assess growth response, gastrointestinal pH, digestive enzyme activity and gut bacterial community of European sea bass (Dicentrarchus labrax L.) juveniles fed low FM diets (10% FM) with or without a feed buffering additive, calcium carbonate (FM10 + B and FM10, respectively) in comparison to a standard FM diet (20% FM, FM20). Three isonitrogenous and isolipidic extruded diets were fed to triplicate fish groups of 80 individuals (initial weight: 23 g) to overfeeding over 64 days. No significant differences due to low FM dietary levels were observed in final body weight, specific growth rate, feed intake, feed and protein efficiency. Low FM diet did not affect gastrointestinal pH in the stomach, anterior intestine, mid-intestine and hindgut at 0, 4, 8 and 12 hours post meal (hpm) while the inclusion of calcium carbonate in low FM diet seems to slightly increase the pH in the hindgut at 12 hpm. The absence of significant differences of pepsin, chymotrypsin, amylase and lipase activity suggests a comparable digestive efficiency among treatments although trypsin activity was slightly reduced in low FM diets after 4 hpm. Decreasing FM content seems to exert an effect on the overall gut bacterial community analysed by next-generation sequencing even if no significant effects on specific bacterial component were detected. The gut bacterial community in all the treatments was particularly rich in lactic acid bacteria such as Lactobacillus spp. which may provide important beneficial functions for the host and be associated with a healthy intestinal epithelium. According to the results, increasing the feed buffering capacity does not seem to improve digestive conditions while it is feasible to include 10% FM dietary level in practical formulation for European sea bass juveniles without negatively affecting growth, feed efficiency and digestive luminal conditions. info:eu-repo/semantics/publishedVersion

A 121-day feeding trial was undertaken to test the effects of two dietary lipid levels (16 and 21% L16, L21) in triplicated gilthead sea bream groups (initial weight: 67.5 g) reared at two different water temperatures (high, H 23°C and low, L 17°C) in the same recirculation system but exposed to a switch in temperature after 58 days. Fish kept at H were transferred to L (HL transition, autumn shift), and the fish kept at L were exposed to H (LH transition, summer shift), while continuing to receive the same diet to apparent satiation in each group. At the end of the trial, no significant diet effect on specific growth rate (SGR), feed intake (FI), and feed conversion rate (FCR) were detected in fish exposed to HL transition compared with those exposed to LH transition, while gross lipid efficiency (GLE) and lipid efficiency ratio (LER) were higher in L16. After temperature changes, L16 displayed higher SGR, FI, GLE, and LER, while mesenteric fat index was reduced. After temperature changes, the combined effects of low lipid diet and low temperature conditions resulted in higher pepsin activity, while trypsin, chymotrypsin, and lipase activities were generally higher at high lipid content. The combined effect of diet and temperature did not alter the metabolic plasma profile, except for the observed final higher aspartate aminotransferase (AST) and alkaline phosphatase (ALP) values when combining high dietary lipid (L21) and temperature changes. Different diets showed a significantly different gut microbiome layout, only at high temperature with L16 diet resulting in a higher load of Lactobacillus. On the contrary, no dietary impact on ecosystem diversity was observed, independently from the temperature. In addition, L16 diet in the HL transition favored an increase in Weissella and Bradyrhizobium genera in the gut microbiome, while in the final condition of LH transition, L21 diet favored a significant increase in Streptococcus and Bacillus. According to the results, the utilization of 16% dietary lipid levels in gilthead sea bream should be preferred during seasonal temperature changes in order to optimize feed utilization and gut health., This research was undertaken under the MedAID (Mediterranean Aquaculture Integrated Development) project, which has received funding from the European Union’s Horizon 2020 Research and Innovation Programme, Call H2020-SFS-23-2016, Grant agreement no. 727315 (http://www.medaid-h2020.eu/). MY and NG received support from the Spanish Ministry of Science, Innovation and Universities (MCIU), the State Research Agency (AEI), the European Regional Development Fund (FEDER/ERDF), and project Thermodigest (RTI2018-096134-B-I00) granted to MY.

A better knowledge of daily digestive function may help to improve the feeding efficiency in farmed fish species. In this study, daily expression profiles of key genes related to the circadian system (arntl1, clock, per3 and cry1), nutrients hydrolysis (pga, atp4a, try, ctrb, cel1b, pla2, and amy2a), appetite control and food intake (ghrl and cck), and nutrient transport (slc15a1) were assessed in the gastrointestinal tract (GIT) of gilthead seabream (Sparus aurata) juveniles, under four different daily feeding frequencies (one, three, five diurnal meals and continuous diurnal feeding). Additionally, the activity of two key proteolytic enzymes (pepsin and trypsin) and variations in luminal pH within the different GIT segments were determined. Clock genes showed significant circadian patterns, being highly synchronized to the light-dark cycle and first daily meal in all the groups. In contrast, most of the genes codifying digestive enzyme precursors did not exhibit relevant changes in expression along the daily cycle. A correlation was detected between the expression of slc15a1 and per3 and, to a lesser extent between arntl1/clock and ghrl, supporting the role of clock genes in daily organization of some digestive functions. Feeding frequency had a poor influence on the expression of all the analysed genes. In contrast to the results obtained for the expression of genes encoding digestive enzymes, the proteolytic activities exhibited circadian rhythms, and their temporal distribution were significantly affected by some of the experimental feeding protocols, being mostly synchronized with the presence of food in the corresponding GIT segment. Fish fed three times a day did not exhibit a significant daily rhythm for trypsin activity, but the daily average of activated trypsin was similar in all feeding frequencies. Gastric pH was maintained neutral, and acidification occurred only after food ingestion although showing different daily pattern for each feeding protocol. Overall, these results indicate that in gilthead seabream the majority of modulations for the hourly profile of digestive functionality occur post-transcriptionally to adapt to different feeding frequencies., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by projects EFISHDIGEST (AGL2014-52888-R) with FEDER/ERDF contribution granted to M.Y. and WISEFEED funded by the European Union's H2020 programme (Marie Skłodowska-Curie grant No 691150). The authors would like to thank Dr. Tomé Silva for his valuable help in revision of the results and statistical advices. Neda Gilannejad was supported by a doctoral fellowship (BES-2015-071662) from MINECO (Spain).

The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture, This work was funded by project AGL2010-14876 from MICINN to JMM.

Larvae and early juveniles of fast-growing fish species show tremendous growth potential, leading to higher requirements for protein, high-unsaturated fatty acids, and other nutrients. Several commercial weaning diets with relatively high success have been developed for low/moderate-growing species; however, additional challenges are outlined to meet growth potential and energy requirements of such fast-growing species. The objective of the present study was to evaluate two commercial microdiets for marine fish, one having simultaneously a very high protein and high lipid contents (HIGH), and another (MOD) with a high protein and moderate lipid content, in a growth performance trial with greater amberjack post-larvae from 33 to 78 days after hatching. Moreover, histological preparations of liver, anterior and posterior intestine were assessed for hepatic and intestinal lipid inclusions quantification and gut epithelial brush height measurement. Activities of the digestive enzymes: pepsin, trypsin, chymotrypsin, lipase and amylase were also analyzed. Post-larvae fed HIGH microdiet exhibited higher final weight and lower feed conversion ratio that those fed on MOD microdiet. Liver displayed a higher level of lipid inclusions for the MOD diet than for HIGH diet. Moreover, enterocytes of posterior intestine presented a much higher level of supranuclear vacuoles for the HIGH diet compared to MOD diet. The lower Trypsin/Chymotrypsin ratio observed at the end of the experiment in larvae fed on MOD diet may indicate a deficiency in protein of this diet. Together, these results support that larvae of greater amberjack, and likely other fast-growing marine fish species, require high protein–high lipid microdiets. The use of microdiets developed targeting slower growing marine species may lead to sub-optimal performances in fast-growing larvae., This study was undertaken by Project WEANTUNA supported by Eurostars (E! 7994) and co-financed by FEDER through PO Algarve 21 in the framework of QREN 2007-2013 (30200).

The morphological and functional development of the digestive system in meagre ( Argyrosomus regius, Asso, 1801) was described by means of histological and enzymatic approaches in order to provide insight into the digestive physiology of the species at early life stages of development and evaluate whether the acquisition of digestive capacities matched the morphological development of digestive organs. For this purpose, A. regius were reared from hatching to the juvenile stage at 18 °C using a standard feeding sequence of enriched rotifers (2–15 days posthatching, dph), Artemia metanauplii (14–51 dph) and microdiet (35–51 dph). Histological and enzymatic analyses indicated that A. regius larvae had at the onset of exogenous feeding (2 dph, 3.2 ± 0.1 mm in standard length, SL) a well differentiated exocrine pancreas, and bile salt-activated lipase and alkaline proteases being the main pancreatic enzymes involved in food digestion. Ontogenic changes in pancreatic enzymes occurred between 3.2 and 3.9 mm in SL, coinciding with the resorption of the oil globule and the complete transition to exogenous feeding, and between 4.2 and 5.8 mm in SL (20–25 dph). Regarding acid digestion, pepsin was not detected in A. regius until 6.0 and 6.8 mm in SL (31 dph), coinciding with notochord flexion and the progressive decrease in the activity of alkaline protease and leucine-alanine peptidase (intestinal cytosolic enzyme) activities, indicating a shift in the mode of digestion. The detection of pepsin activity in A. regius occurred after the appearance of the first gastric glands (4.6 and 5.8 mm SL). Thus, authors need to be cautious when extracting conclusions about the beginning of acid digestion and the onset of weaning of larvae just using histological data, since it has been shown that morphology does not match functionality with regards to the stomach. The comparison of present results with other studies conducted in this species using different rearing conditions ( e.g. mesocosm technique, larval density, water temperature, feeding sequence) indicated that the functional development of the digestive system in A. regius assessed by the activity of alkaline and acid proteases is a well-conserved process that generally occurs within a range of body size regardless of larval age and rearing conditions. Thus, authors recommend using morphometric and/or morphological variables ( e.g. length, developmental stage) together with the age of larvae expressed in days after hatching for comparative purposes among different studies. Statement of relevance The morphological and functional development of the digestive system in meagre ( Argyrosomus regius ) was described by means of histological and enzymatic approaches in order to provide insight into the digestive physiology of the species at early life stages of development. Combining histological and enzymatic analytical procedures we evidenced that stomach's functionality did not match its morphological organization, since pepsin activity was not detected until some days after the appearance of the first gastric glands. This indicates that authors need to be cautious when extracting conclusions about the beginning of acid digestion and the onset of weaning of larvae just using histological data.

In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24 h cycle in larvae reared under a 12 h light/12 h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60 days post-hatch. In each sampling day, larvae were sampled every 3 h during a complete 24 h cycle. The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by project RIDIGEST (AGL2011-23722) with FEDER/ERDF contribution. J.A. Mata-Sotres was supported by a doctoral grant (ID 215473) from the Mexican National Council for Science and Technology (CONACYT).

Activities of pepsin, trypsin, chymotrypsin, alpha-amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick-lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS-PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha-amylase and no lipase activity recorded in pre-adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.

The impact of deoxynivalenol (DON) on rainbow trout, Oncorhynchus mykiss, is mainly characterised by impaired growth performance and reduced feed intake, usually with the total absence of any visible clinical signs. Despite the high concentrations of DON in the present study (up to 11,412 ± 1141 μg kg−1), no clinical signs (except anorexia at the higher DON dosage) were observed, which confirms the difficulties of diagnosing DON ingestion. Compared to the control group, the proteolytic enzyme activities (pepsin, trypsin and chymotrypsin) in trout were altered by DON ingestion. However, it was not clear if the observed impact on digestive enzymes was due to the direct action of DON, or a consequence of the lower feed intake determined for DON-treated animals. The impact of DON on the abundance of specific measured mRNA transcripts was unexpected with higher expression levels for insulin-like growth factors, igf1 and igf2, which are directly related to elevated insulin levels in plasma. This can also in part be influenced by the trypsin activity and by npy, given its higher mRNA expression levels. The apparent digestibility of dry matter, protein and energy was not affected by dietary levels of DON, however, nutrient retention, protein, fat and energy retention were significantly affected in animals fed DON. Adenylate cyclase-activating polypeptide (PACAP) expression seems to play an important role in controlling feed intake in DON fed trout. In the present study, we have shown for the first time that DON is metabolized to DON-3-sulfate in trout. DON-3-sulfate is much less toxic than DON, which helps to explain the lack of clinical signs in fish fed DON. Being a novel metabolite identified in trout makes it a potential biomarker of DON exposure. Suppression of appetite due to DON contamination in feeds might be a defense mechanism in order to decrease the exposure of the animal to DON, therefore reducing the potential negative impacts of DON., The present work was performed under the WiseFeed project (European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 691150) being Biomin Holding GmbH, a partner of the project, represented by Rui A. Gonçalves in his secondment to Instituto de Ciencias Marinas de Andalucía, Consejo Superior de Investigaciones Científicas (ICMAN-CSIC), Puerto Real, Cádiz, Spain.

There is a large amount of scientific literature on digestive enzymes activity during fish larval development. Nevertheless, most of this information refers to descriptive aspects of the ontogeny of hydrolyzing capacities, and only few studies looked into functional aspects and its relation with feeds and feeding activity. A comparison of published data normalized by days-degrees reveals at least three groups of species (precocious, medium and late) in relation to the time required for reaching maximum activity of digestive enzymes capacities, irrespective of the taxonomic group. Recent progress has allowed enlarging our knowledge on fish larvae digestive function. Such progress is partially based on new methodologies, but also on extended experimental designs and sampling protocols. On one side, the molecular basis of the onset of the digestive functionality has been examined in several species, cloning the mRNAs codifying the precursors of the main digestive enzymes and determining the gene expression profiles. On the other side, the daily rhythms coupling feeding and digestive enzyme production activities started to be investigated, giving a new dimension to our understanding of the digestive capacity during these early stages. Comparison between molecular expression and actual activity of the different enzymes is providing new insights on the regulation mechanisms. The connection between feeding behavior and digestive response both at genomic and biochemical levels is a required step towards the rationalization of feeding protocols with both live and inert diets, as a way to increase the nutrients utilization and the developmental performance at these early stages.

Gilthead seabream is a fish species of great importance in Mediterranean aquaculture, attracting many studies on nutrition and chronobiology, although nothing is known about the effect of feeding frequency on the daily rhythms of the gastric digestion process. In this article, we investigated daily rhythms in stomach fullness, gastric and intestine pH, as well as pepsin activity and expression of pepsinogen and proton pump in juvenile fish under three different feeding protocols: (A) one daily meal at 9:00, (B) two daily meals at 9:00 and 17:00 and (C) continuous feeding during the daytime. The results revealed that feeding protocol affected significantly the rhythm of gastric pH and the pepsin activity pattern. The gastric pH exhibited significant daily rhythms in the three cases with the acrophase located at night in the regimes A and B and during daytime, in the regime C. In the regimes A and B, the pepsin activity peaked few hours after the meals, although the afternoon meal in B produced a higher peak. In the regime C, the peak occurred in the middle of the feeding period. Lowest total pepsin activity was observed in regime A, and the highest activity with the regime C. In contrast, the pepsinogen gene expression remained low along the daily cycle, with an expression peak just before or after the morning meal in regimes A and C, respectively. The proton pump gene expression was also practically constant with a peak right after the morning meal in the regime C. On the other hand, intestinal pH showed a postprandial increase after the first morning meal in all the three treatments, recovering the resting values in the dark period. Two meals and continuous feeding allowed a better and prolonged gastric digestion and consequently the juveniles exhibited better growth with the same daily ration of food. In short, while the gastric digestion pattern is mainly driven by pH changes induced by the time of food ingestion, the regulation of the intestinal digestion seems to be more independent of the feeding protocol., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by projects RIDIGEST (AGL2011-23722) and AQUAGENOMICS (Consolider-Ingenio Program CDS2007-0002) with FEDER/ERDF contribution.

To assess to what extent addition of phytase to a plant-based diet results in spatio-temporal changes of phytate, available P, soluble protein, total amino acids and the activity of the main digestive proteases in gilthead sea bream, fish were fed two plant-based diets with or without phytase. Stomach, proximal intestine and distal intestine contents were monitored for these parameters at 0, 1, 2, 4 and 6 h after feeding. A reduction (P < 0.0001) of the soluble P–IP6 in the stomach when phytase was added to the diet was observed. Within stomach, most of the total P–IP6 was precipitated (86%), possibly due to the low acidification capacity of the sea bream (pH > 4), but 57% of the dietary P–IP6 was dephosphorylated, suggesting that phytase could have the capacity to dephosphorylate insoluble IP6 at such pH. An increment (60%) (P < 0.01) in total gastric protease activity was observed by phytase addition, this being the first demonstration of the in vivo effect of IP6 on the pepsin activity in fish stomach. Gastric pH and residence time of the digesta inside the stomach are critical factors for an efficient phytase action and improve P and N bioavailability in plant-based diets used in fish aquaculture. Fil: Morales, Gabriel Alejandro. Universidad de Almería; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marquez, L.. Universidad de Almería; España Fil: Saenz de Rodrigañez, M.. Universidad de Almería; España Fil: Bermúdez, L.. Fundación Centro Tecnológico de Acuicultura de Andalucía; España Fil: Robles, R.. Fundación Centro Tecnológico de Acuicultura de Andalucía; España Fil: Moyano, F. J.. Universidad de Almería; España

Cholecystokinin (CCK) is an important regulator of pancreatic enzyme secretion in adult mammals and teleosteans. Although some studies have focused on the interaction between CCK and trypsin in marine fish larvae, little is known about the circadian patterns of the regulatory mechanism involving these two digestive components. In this study, we took advantage of the characteristic change from a diurnal to a nocturnal feeding habit that occurs in Senegalese sole (Solea senegalensis) post-larvae, to conduct an experiment where larvae and postlarvae were submitted to three different feeding regimes from mouth opening: continuous feeding, diurnal feeding and nocturnal feeding. The aim was to establish different daily feeding scenarios to uncover the operating mechanisms of CCK and tryptic enzyme activity over the 24-hour cycle to better understand the regulation of digestion in developing fish larvae. Results show a prevalence of simultaneous and opposing trends of CCK level and tryptic activity as a function of the postprandial time. This finding supports the existence of a regulatory loop between these two digestive components in pre- and post-metamorphic Senegal sole larvae. In addition, CCK level was also modulated by the gut content, tending to be lower when the gut is full and higher when is being emptied. Furthermore, larvae were able to synchronize digestive functions to very different feeding regimes, although it seems to be important having a diurnal feeding phase during pre-metamorphic stages for a proper development., This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by project RIDIGEST (AGL2011-23722) with FEDER/ERDF contribution granted to M. Yúfera. C. Navarro-Guillén was supported by a doctoral fellowship (BES-2012-051956) from MINECO (Spain).

12 páginas, 2 tablas, 3 figuras, Differences in survival and growth rates in seahorse Hippocampus guttulatus juveniles feeding on Artemia sp. or copepods have been related to specific digestive capacities of seahorse newborn, which are capable of actively forage on available prey from the first day of live. Other seahorse species, such as H. abdominalis and H. hippocampus, show high success feeding on Artemia nauplii suggesting species-specific differences in the digestibility of prey among seahorses. In this study, the profiles of digestive enzyme activity during the initial 15 days after release (DAR) were very low for trypsin, chitinase and α-amylase. In contrast, higher activities towards any of the assayed substrates for lipase (butyrate, octanoate and oleate) were evident from 0 DAR onwards. From 15 DAR onwards, the effect of diet composition became evident in juveniles previously fed on a mixed diet (Artemia + copepods), which showed a clear increase in all the assayed enzymes when compared with juveniles fed on Artemia as a sole prey. As a practical applicability of this study, a feeding schedule ensuring an adequate digestibility of the prey is proposed based on ontogenetic enzymatic activities of seahorse juveniles fed on different prey, The study was financed by the Spanish Government (Plan Nacional, Project CGL2009-08386) and the Regional Government of Galicia (Xunta de Galicia, Project 09MDS022402PR). A. Blanco was supported by PhD JAE-Pre grants (Junta para la Ampliación de Estudios Program) from the Spanish National Research Council (CSIC), co-financed by the European Social Fund

This study aimed to find a way for increasing the water content after rehydration of microdiet particles for fish larvae and to assess whether such increase contributes to a better disaggregation and digestion of particles within the gut of gilthead seabream larvae (4–15 days after hatching, dph). Four microdiets were prepared with increasing amounts of carboxymethylcellulose (CMC): 0, 20, 40 and 80 g kg−1. The inclusion of CMC resulted in an effective increase of the hydration capacity of particles. Water content after immersion ranged from 800 g kg−1 in the microdiet without CMC to 900 g kg−1 with 80 g kg−1 CMC. Larvae of different ages were fed during 10 h with these microdiets. The microparticles containing 80 g kg−1 CMC were more degraded in the gut of the younger larvae. No clear differences were observed from 10 dph onwards. Differences in enzymatic activities were not evident except for α‐amylase in 6 dph larvae. Gene expression of the enzyme precursors did not show any relation to the water content of microdiets. Overall, the results indicate that increasing the water content up to 900 g kg−1 may enhance the disintegration of food particles within the gut of younger larvae and to a lesser extent affect the response of digestive enzyme activities., This work has been funded by project ARRAINA – Advanced Research Initiatives for Nutrition & Aquaculture (EU seventh Framework Programme contract No. 288925).

The aim of the present study was to determine the optimum dietary levels of krill phospholipids (KPL) for sea bream (Sparus aurata) larvae, and its influence on larval development and digestive enzymes activity. Larvae were fed five formulated microdiets with five different levels of KPL. Complete replacement of live preys with the experimental microdiets for seabream larvae produced high survival and growth rates, particularly in fish fed the highest levels of KPL. In the present study, increase in dietary KPL up to 120 g kg−1 (100 g kg−1 total PL) significantly improved larval survival and growth, whereas further increase did not improve those parameters. An increase in alkaline phosphatase, trypsin and lipase activity with the elevation of KPL up to 120 g kg−1 was also found denoting a better functioning of digestive system. Besides, there was a linear substrate stimulatory effect of dietary KPL on phospholipase A2 activity. Finally, increasing dietary KPL lead to better assimilation of n-3 HUFA especially eicosapentaenoic acid, reflected in the higher content of these fatty acids in both neutral and polar lipids of the larvae. In summary, KPL were found to be an excellent source of lipids for seabream larvae. Optimum inclusion levels of this ingredient in microdiets to completely substitute live preys at this larval age were found to be 120 g kg−1 KPL.

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6 days after hatching: dah), larvae were fed Artemia nauplii until 15 dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60 dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up to 60 dah. Most digestive enzymes were present from yolk absorption (5-6 dah), except for the specific acid proteases activity (pepsin-like), which increase rapidly from 8 dah up to 20 dah. Three alkaline proteases isoforms (24.0, 24.8, 84.5 kDa) were detected at 8 dah using SDS-PAGE zymogram, corresponding to trypsin, chymotrypsin and probably leucine aminopeptidase enzymes, and only one isoform was detected (relative electromobility, Rf = 0.54) for acid proteases (pepsin-like) from 3 dah onwards using PAGE zymogram. We concluded that C. urophthamus is a precocious fish with a great capacity to digest all kinds of food items, including artificial diets provided from 13 dah.

Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed.

The effect of food deprivation on larval performance of the spider crab Maja brachydactyla was studied in terms of survival, moulting capacity, size, weight and enzymatic activities. Five feeding treatments that differed in the initial age of first feeding larvae (fed from hatching, 2, 4 and 6 days post-hatching and unfed) were tested for 20 days. Newly hatched larvae kept without food supply lasted for 10 days and did not moult; with 50% survival observed at 6 days post-hatching. Larvae (zoea I stage) were only able to tolerate 2 days of food deprivation after the onset of exogenous feeding without their performance being compromised. Multivariate analyses suggest that digestive enzyme activities may be good indicators of the nutritional condition of larvae.

Ontogenetic changes in digestive capabilities were analyzed in larvae and first juveniles of the spider crab Maja brachydactyla. Activities of five proteinases (total proteases, trypsin, chymotrypsin, pepsin-like and aminopeptidase), three carbohydrases (amylase, maltase and chitinase), an esterase and an alkaline phosphatase were studied to evaluate digestive enzyme profiles of the species. Both quantitative (spectrophotometry and fluorometry) and qualitative (SDS-PAGE) approaches were used. All assayed enzymes were active from hatching (zoea I-ZI) throughout larval development and in first juveniles. Significant variations during ontogeny were found only in total activities likely as a consequence of digestive system development. Specific activity varied little over ontogeny, being significant only for chitinase. Total proteases, trypsin and pepsin-like activities showed a similar pattern of increase as larval ontogeny advanced, decreasing significantly in juveniles. Chymotrypsin continued to increase, showing maximum activity after metamorphosis. Proteinase zymograms confirmed strong proteolytic activity in first zoeas, with increasing bands over the course of ontogeny, decreasing after metamorphosis. A group of bands with high molecular mass was specific to larval stages. Amylase and maltase showed a parallel pattern of continuous increase of total activity as development advanced. Gel-SDS-PAGE showed unchanged patterns of amylase activity in first zoeas of different ages and the most complex set of bands during larval ontogeny in second zoea. Esterase total activity increased significantly as ZI's aged likely reflecting introduction of a lipid-enriched diet. The importance of lipid accumulation at the beginning of ontogeny was also confirmed by the protease/esterase and amylase/esterase activity ratios, which decreased from hatch to late ZI and might be explained as an adaptation, ensuring the next molt. The results suggest that larvae of M. brachydactyla are capable of digesting a variety of dietary substrates as soon as they hatch.

Changes in major digestive enzymes through developmental and molt stages were studied for the spiny lobster Panulirus argus. There were significant positive relationships between specific activity of trypsin and amylase enzymes and lobster size, whereas esterase and lipase specific activities decreased as lobsters aged. No relationship was found between amylase/trypsin ratio and lobster size. Positive trends were found, however, for trypsin/lipase and amylase/lipase ratios. Results suggest that changes in enzyme activity respond to the lobsters' physiological needs for particular dietary components although multivariate analysis suggested that enzyme activities could be not totally independent of diet. On the other hand, the pattern of changes of major enzyme activities through molt cycle was similar for most enzymes studied. Following molt, trypsin, chymotrypsin, amylase, and lipase activities gradually increased to maximal levels at late intermolt (C4) and premolt (D). There were no variations in the electrophoretic pattern of digestive enzymes through developmental and molt stages and thus, it is demonstrated that regulation is exerted quantitatively rather than qualitatively. Further studies on the effect of other intrinsic and extrinsic factors on digestive enzyme activities are needed to fully understand digestive abilities and regulation mechanisms in spiny lobsters.

The potential use of fluorescent substrates for measuring digestive enzyme activities has been adapted to decapod crustacean larvae and compared to traditional analytical techniques based on spectrophotometry. The ontogeny of three digestive enzymes (trypsin, amylase, and esterase) from the hepatopancreas of Maja brachydactyla has been selected to illustrate the advantages and disadvantages of both spectrophotometric (SP) and fluorometric (FL) methods. The three enzyme activities showed the same pattern with either of the techniques used. Standardised discriminant function coefficients indicated that the stage of development in spider crab larvae was most strongly discriminated by the activity of amylase, whichever method of analysis was considered. The FL method allows the detection of up to three enzyme activities in a single larva (

The potential use of digestive activities as indicators of the nutritional status in bivalves is discussed in relation to the results obtained in two clam species exposed to starvation and refeeding. Activities of some digestive enzymes (amylase, laminarinase, cellulase, and protease) were measured in juveniles of two commercially interesting species of clams, Ruditapes decussatus and Venerupis pullastra. The specimens were fed normally, being after subjected to a 15-days starvation and a further refeeding period. Samples were obtained at different moments of such feeding schedule to evaluate enzymes as well as weight (live, dry and organic) and length, in order to calculate growth rates and feeding efficiencies. Starvation led to a major decrease in clam growth as measured by dry weight and a negative growth as measured by organic weight, this coinciding with a certain degree of growth of the shell and a consumption of soft tissue. This response occurred more rapidly in R. decussatus but was of a lower magnitude than in V. pullastra. Activity of carbohydrases decreased rapidly in both species with starvation, although protease activity was maintained in R. decussatus. Recovery after the end of starvation was not similar in both species; while R. decussatus attained similar growth rates and enzyme activities to those measured prior to nutritional stress, V. pullastra only recovered 50% of its initial values. For both species of bivalves it can be concluded that digestive enzymes, and more specifically amylase, could be used as indicative of their nutritional condition.

The present study was aimed to evaluate differences in physicochemical characteristics (pH and temperature optima) of the main digestive enzymes (amylase, cellulase, laminarinase, and protease) in two commercially important bivalves, the intertidal clam, Ruditapes decussatus, and the subtidal clam, Venerupis pullastra, as well to assess individual variability of such activities. The assays were performed on extracts obtained from the digestive glands and crystalline styles of individuals collected in the wild and laboratory-conditioned during 1 week. In both species, maximum activity for amylase and cellulase was measured at pH 6 and that of laminarinase at pH 5.5, while pH profiles of protease activity showed important differences between them. Temperature profiles also showed interspecific variations, mainly in laminarinase. Carbohydrase activities were comparatively much higher in R. decussatus and proteolytic activities in V. pullastra. The observed differences both in physicochemical characteristics and relative importance of digestive enzymes are discussed in relation to the amount and type of food available in their respective habitats., Sí

The objective of this study was to develop novel type of protein walled microparticles suitable for using in early feeding of fish larvae.The microparticles were made of casein and protamine through complex coacervation and did not require further cross-linking or use of environmentally problematic reagents. The methodology was oriented to generate microparticles with an appropriate size range for easy recognition and ingestion by fish larvae (50-200 microm), adequate floating properties in saline, sufficient stability in terms of protein leakage and appropriate digestibility by the gut enzymes of fish larvae.Desired particle size and stability against protein leakages over 8 h were successfully achieved by optimizing the coacervation process conditions. The floating properties under static conditions were considered appropriate as a main particle fraction remained in suspension during at least 10 min. Very importantly, an enzyme extract from larval gut readily digested the particles. The digestibility of the casein-protamine particles was similar to that measured for Artemia nauplii and for two previously developed casein-based microparticles produced by interfacial polymerization and ionic gelation; the latter microparticle type had previously achieved good results of digestibility in early feeding of marine fish larvae.The in vitro evaluation of the newly developed casein-protamine microparticles revealed promising characteristics as artificial larval feed. Thus, these particles merit further development with respect to entrapping nutrients and testing them in larval cultures for their nutritional value.

The effects of starvation and re-feeding on metabolites and tissue composition, GH/IGF-I axis, and digestive enzyme activities in juvenile thick-lipped grey mullet (Chelon labrosus) were evaluated. Fish were divided into three feeding groups (n = 72, 82.00 ± 4.09 g initial body mass). The control group was fed 1% of their body mass once a day throughout the experiment with commercial pellets. The other two groups were deprived of feed for 21 days (starved), or re-fed for 7 days after 14 days of food deprivation (re-fed). Full-length cDNAs from pituitary GH and hepatic IGF-I were cloned by screening a cDNA library or by PCR techniques. Furthermore, changes in their mRNA expressions were assessed by real time PCR in specimens maintained under the different feeding patterns. Results showed a negative growth in starved and re-feeding groups. Starvation induced a significant increase in plasma triglycerides as well as a decrease in liver glucose and glycogen. Re-feeding increased plasma glucose, lactate and protein, as well as liver glucose and glycogen. In addition, starvation significantly increased pituitary GH expression, while re-feeding down-regulated it. No significant changes were observed in hepatic IGF-I expression in any dietary treatment. Digestive enzyme activities were not significantly affected either by starvation or by re-feeding. The results of the present work suggest that juveniles of the thick-lipped grey mullet may easily adjust their metabolism under situations characterized by a short-term starvation and re-feeding., This work was funded by project AGL2010-14876 from MICINN to JMM.

This study describes the digestive capacities of 10-day-old white shrimp postlarvae (PL10) and how some inhibitors of proteinases affect the digestion of protein in aquafeeds by using in vitro hydrolysis techniques. Biochemical data showed eight active proteinases in the PL10 hepatopancreas extract. Enzymes belong to the metallo- and serine-proteinase classes. The effect of inhibitors present in protein ingredients and aquafeeds on PL10 proteinases showed that ovalbumin alone and in commercial microcapsules yielded a significant inhibition in proteolytic activity of PL10 hepatopancreas enzymes. The capacity of PL10 proteinases to hydrolyse the protein fraction within different sources and microcapsules was demonstrated by two in vitro approaches, the pH-stat (degree of hydrolysis, DH) and electrophoresis (coefficient of protein degradation, CPD). It was shown that PL10 proteinases hydrolyse, in different extent protein sources and microcapsules. Casein, cuttlefish meal and feeds containing these ingredients are quickly hydrolysed. By contrast, ovalbumin and microcapsules containing ovalbumin are not hydrolysed.

Activity of some of the main enzymes involved in protein digestion and absorption (acid and alkaline professes, leucine-aminopeptidase, acid and alkaline phosphatases) as well as those of amylase and lipase, was assessed during larval development of while sea bream Diplodus sargus. All enzyme activity was detected at the moment of mouth opening. The variations observed in the activity profiles of the digestive enzymes were correlated either to developmental events. such as the functional start of the stomach (22 days after hatching), or to changes in the nature of the diet. The early and noticeable development of digestive enzyme activities was linked to a high survival after weaning.

The pH-stat system has been used to assess the effect of tannic acid (TA) on solubility and in vitro enzyme hydrolysis of different proteins. Added TA (from 10 to 50 g kg−1) decreased the extent of hydrolysis of bovine serum albumin. Enzymic hydrolysis of casein, pea meal, soybean meal, and haemoglobin (HB) was increased, as measured by total amino acids released and by the degree of hydrolysis. SDS-PAGE confirmed the results of the in vitro enzymatic hydrolysis. These findings suggest that, under in vitro conditions, when simulating the gastrointestinal environment of domestic mammals, the negative effects of TA described from in vivo experiments are not necessarily due to reduced hydrolysis of proteins. Copyright © 2003 Society of Chemical Industry

In the present study, amylase activity existing in the gut of different species of sparid fish was measured and characterized. The study was conducted on five species of Mediterranean sparids, (F. Sparidae), some of which coexist in the same coastal waters; Pagrus pagrus, Pagellus erytrhinus, P. bogaraveo, Boops boops and Diplodus annularis. Main differences were found in the optimal pH for amylase activity as well as in sensitivity to temperature, with resistance to heating very low in B. boops and very high in D. annularis. PAGE zymograms revealed the presence of two isoforms in all the species, while the analysis of extracts using IEF showed a range from one to three active bands. It is suggested that differences in amylase activity are related to variations in feeding habits of the studied fish, and could also represent an ecological advantage for those species showing a more diversified amylase equipment.

A sensitive and quick method was developed to determine the presence of α-amylase in the gut of aquatic organisms, as well as its sensitivity to inhibitors. The assay is based on the utilization of Petri dishes filled with starch–agarose gel as a substrate for the enzyme solution, which is placed in small wells punched in the surface. Circular zones produced by the action of amylase remain colourless after staining with lugol. Pure commercial porcine amylase was used to fit the better conditions for developing the assay (1 g L–1 starch in the gels, 4 h of incubation). The diameter of the cleared zones were related to the activity of enzyme and the method detected linearly amylase activity in a range of 2–20 U well–1, so it was used to reveal the presence of amylase in digestive extracts obtained from different sparid fish. The method was also used to evaluate the effect produced by a specific inhibitor on fish amylases, showing a linear response when the ratio inhibitor:enzyme (in units) changed from 20:1 to 2:1. Comparison of the cleared zones produced by amylases of sparid fish in the presence or absence of inhibitor, revealed differences in their sensitivity to inhibition, which ranged from 15 to 50% of total activity. The assay is proposed for a preliminary evaluation of possible inhibitors contained in feedstuffs used in fish feeding.

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.

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.

This study was conducted to evaluate the effect that feeding on differently treated soybean meals may have on digestive protease activity of the Nile tilapia (Oreochromis niloticus). Untreated full fat soybean (USB), heated fullfat soybean (HSB), soaked fullfat soybean (SSB) and commercial defatted soybean meal (DSB) totally replaced fish meal (FM) in test diets (30% crude protein) fed to fish juveniles (mixed sexes) for 30 days. After this period, total activity of digestive proteases was determined in each group and the ability of such proteases to hydrolyse the different protein sources and formulated feeds was tested in vitro. Significant differences were observed between treatments, with lower values obtained for fish fed on USB, suggesting a modification in the composition of secreted proteases. In addition, an inhibitory effect produced by wheat bran included in the test diets on protease activity of fish was also shown. The usefulness of using in vitro assays for a preliminary assessment of the nutritive value of aquafeed ingredients is discussed.

The presence of protease inhibitors in different ingredients used in fish feeds may negatively affect their digestive utilisation. The effect of inhibitors present in several protein sources on the activity of digestive proteases of sea bream was assessed using `in vitro' assays. Inhibition produced by extracts of plant proteins ranged from 25 to 50 % of total activity, whereas that obtained using animal protein sources ranged from 1 to 20 %. The exception was ovoalbumin, which showed the highest measured value (62 %). A plot of the inhibition values obtained by changing the relative concentrations enzyme/inhibitor resulted in different curves for different protein sources. The use of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) zymograms allowed visualisation of the aforementioned differences in inhibition. The importance of measuring protease inhibition in the preliminary evaluation of fish feed ingredients is discussed.

The development of artificial feeds for marine fish larvae is demanding and, taking into account the diAculties and costs associated with the rearing of the larvae of some fish, preliminary evaluation of feed ingredients using in vitro techniques may be an alternative to in vivo assays. Some proteins used in microfeeds for marine fish were tested in vitro for examination of their eAect on the proteases of seabream larvae. Casein and cuttlefish meal did not aAect protease activity of crude larval extracts, whereas ovalbumin 1 produced a 60% inhibition. The use of a pH-stat for the assessment of the degree of hydrolysis (DH) of proteins confirmed the low nutritive value of ovalbumin, since a much lower value was obtained for microcapsules prepared using this protein source when compared with those prepared using casein or cuttlefish meal (3.2 vs. 7.3 and 7.6, respectively). Products resulting from such hydrolysis were analysed using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐ PAGE). Image analysis of such gels led to the formulation of an index named the coeAcient of protein degradation (CPD) which expressed the extent to which the main protein fractions were hydrolysed by larval proteases in a given time. A significant correlation (r 2 a 0.98, P < 0.05) was obtained between DH and CPD values measured for either protein sources or microcapsules. The combination of protease inhibition assays with measurements of DH and CPD is proposed as a preliminary evaluation of protein ingredients used in the formulation of artificial feeds for larval fish.

The activities of the main digestive enzymes (proteases, amylase, lipase) as well as those of acid and alkaline phosphatases were assessed during the larval development of the Senegal sole, Solea senegalensis. Important variations in specific activities of all the enzymes were observed during the period of study and were mostly related to the beginning or the end of metamorphosis. Both acid and alkaline protease activities were identified at early stages of development. Acid proteases accounted for only 10% of total protease activity in a 9 days-old larva, but exceeded 75% in a 33 days-old individual. Maximum lipase activity was related to the development of exocrine pancreas (days 6 to 10) and to metamorphosis. It is suggested this can be related to the metabolism of lipid reserves taking place during this morphological change. Activity of alkaline phosphatase decreased from day 5 to day 20 but therafter revealed a sharp increase, possibly linked to the development of enterocytes. The pattern of development of the main digestive enzymes found in S. senegalensis is similar to that described in other flatfish species.

The present study was aimed to establish the relationship between the functionality of the digestive gland and physiological rates including SFG (scope for growth) in wild mussels, Mytilus galloprovincilis. The experimental set-up consisted in the evaluation of changes in the morphology of the gland, as well as in the activity of some key digestive enzymes (amylase, laminarinase, cellulase and protease) within a broad range of SFG obtained through manipulation of food ration. The higher SFG values were correlated to an increase in both the size of the digestive gland and the activities of enzymes when expressed in relation to individual. In contrast, no clear relations were observed when the activity of enzymes was expressed in relation to soluble protein, with the exception to amylase. The higher protease activities measured in mussels showing lower SFG may reflect an initial stage of catabolic processes intended to compensate the energy deficit produced by food restriction. The potential use of parameters measured in digestive glands in studies of marine pollution was discussed.

Two different modes for regulation of stomach acid secretion have been described in vertebrates. Some species exhibit a continuous acid secretion maintaining a low gastric pH during fasting. Others, as some teleosts, maintain a neutral gastric pH during fasting while the hydrochloric acid is released only after the ingestion of a meal. Those different patterns seem to be closely related to specific feeding habits. However, our recent observations suggest that this acidification pattern could be modified by changes in daily feeding frequency and time schedule. The aim of this study was to advance in understanding the regulation mechanisms of stomach digestion and pattern of acid secretion in teleost fish. We have examined the postprandial pattern of gastric pH, pepsin activity, and mRNA expression for pepsinogen and proton pump in white seabream juveniles maintained under a light/dark 12/12 hours cycle and receiving only one morning meal. The pepsin activity was analyzed according to the standard protocol buffering at pH 2 and using the actual pH measured in the stomach. The results show how the enzyme precursor is permanently available while the hydrochloric acid, which activates the zymogen fraction, is secreted just after the ingestion of food. Results also reveal that analytical protocol at pH 2 notably overestimates true pepsin activity in fish stomach. The expression of the mRNA encoding pepsinogen and proton pump exhibited almost parallel patterns, with notable increases during the darkness period and sharp decreases just before the morning meal. These results indicate that white seabream uses the resting hours for recovering the mRNA stock that will be quickly used during the feeding process. Our data clearly shows that both daily illumination pattern and feeding time are involved at different level in the regulation of the secretion of digestive juices., Spanish Ministry of Science and Innovation (Projects AGL2007-64450-C02-01/02 and Consolider Ingenio 2010 Program (Project Aquagenomics CSD2007-0002) + European Regional Development Fund supported this work.

After the development of the gastric function in juvenile fish, dietary proteins enter a two-phase digestive process comprising an acidic gastric phase followed by an alkaline intestinal phase. However, the main gastric protease, pepsin, is strictly dependent on the existence of a low-enough environmental pH. In 20-g gilthead sea bream, Sparus aurata, the mean minimal gastric pH is close to 4.5, while the mean pH in the duodenal portion of the intestine was nearly fixed at 6.5. The mean maximal gastric content of HCl was approximately 20 microEq for a low-buffering diet. Gastric proteases were more severely affected than intestinal proteases when assayed at actual sub-optimal pH values, 4.5 and 6.5, respectively. When the gastric proteases of juvenile fish were pre-incubated with a citric acid buffer at pH 6.0, the activity at pH 4.5 was very low, whereas when they were pre-incubated with the same buffer at pH 3.0, the activity at pH 4.5 was significantly increased; this fact suggests a deficient activation of zymogens during the gastric digestion and points to a potential approach to improve protein digestion in juvenile gilthead sea bream.

Common snook (Centropomus undecimalis) is one of the most important marine species under commercial exploitation in the Gulf of Mexico; for this reason, interest in developing its culture is a priority. However, larviculture remains as the main bottleneck for massive production. In this sense, our objective was to determine the changes of digestive enzymes activities using biochemical and electrophoretic techniques during 36 days of Common snook larviculture fed with live preys (microalgae, rotifers, and Artemia). During larviculture, all digestive enzymatic activities were detected with low values since yolk absorption, 2 days after hatching (dah) onwards. However, the maximum values for alkaline protease (6,500 U mg protein(-1)), trypsin (0.053 mU × 10(-3) mg protein(-1)), and Leucine aminopeptidase (1.4 × 10(-3) mU mg protein(-1)) were detected at 12 dah; for chymotrypsin at 25 dah (3.8 × 10(-3) mU mg protein(-1)), for carboxypeptidase A (280 mU mg protein(-1)) and lipase at 36 dah (480 U mg protein(-1)), for α-amylase at 7 dah (1.5 U mg protein(-1)), for acid phosphatases at 34 dah (5.5 U mg protein(-1)), and finally for alkaline phosphatase at 25 dah (70 U mg protein(-1)). The alkaline protease zymogram showed two active bands, the first (26.3 kDa) at 25 dah onwards, and the second (51.6 kDa) at 36 dah. The acid protease zymogram showed two bands (RF = 0.32 and 0.51, respectively) at 34 dah. The digestive enzymatic ontogeny of C. undecimalis is very similar to other strictly marine carnivorous fish, and we suggest that weaning process should be started at 34 dah.

Several samples of P. splendida larvae were obtained from eggs until day 60 after hatching (dah) to determine acid and alkaline proteases, trypsin, chymotrypsin, leucine aminopeptidase, α-amylase, lipase, and acid and alkaline phosphatase activities using biochemical techniques. Additionally, SDS–PAGE alkaline protease zymogram and PAGE acid protease zymogram were carried out to identify active isoforms during larviculture. Alkaline protease and chymotrypsin were present at the moment of hatching, increased gradually reaching the maximum values at 35 dah. Trypsin and leucine aminopeptidase activities were low from hatching, increasing gradually as larvae grew. Alkaline protease zymogram showed four zymogens, which appears at different days, remaining present until the end of the larviculture (95.2 kDa at 11 dah, 26.4 kDa at 9 dah, 21.4 kDa at 3 dah, and 23.3 kDa at hatching). Pepsin activity was present at day 7 after hatching and increased progressively until the end of the larviculture. Acid protease zymogram only showed one zymogen (0.65 rf), which appear at 6 dah. Lipase was high at the time of hatching and increased until 15 dah, after which decreased gradually. Amylase was high from the beginning and until 15 dah and then decreased rapidly to almost nothing onward. Alkaline and acid phosphatases presented a high activity at the egg stage, fell slightly during the first feeding and increased again from 20 to 30 dah. Results obtained in this study show that larvae can be fed artificial diets starting on day 10 after hatching.

The present study develops an experimental procedure aimed to estimate the efficiency of protein digestion in fish by measuring both gut transit rate and total amount of the main intestinal proteases (trypsin and chymotrypsin). The selected species was the Nile tilapia (Oreochromis niloticus). Total time for digestion, calculated through the estimation of gut transit rate using differently colored feeds, was 7.15 h. Mean production of trypsin and chymotrypsin was 15.94 and 24.11 mU in the proximal intestine and much lower (2,39, 4.90 mU) in the distal intestine. The enzyme efficiency, calculated from the average enzyme activity and time of residence of the digesta in each intestinal section, points to the major role of proximal intestine in protein digestion for this species. Results are discussed in relation to the main features characterizing digestion in stomachless fish.

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17–21 kDa) and isochymotrypsin-like enzymes (23–38 kDa) were identified. Amylases (38–47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus . Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.

International audience; The aim of the present work was to evaluate to what extent variation in the activity of either trypsin or chymotrypsin could be a sensitive and reliable indicator of nutritional deficiencies during post-weaning stages in a marine fish. Sea bass Dicentrarchus labrax larvae obtained from two different spawns classified as high quality (HQ) or low quality (LQ) on the basis of their hatching variables, were used to evaluate if enzyme production could be conditioned by factors other than those related to the feeding regime. In addition, larvae in each group were either fed normally or received a ration reduced by 60%. Trypsin and chymotrypsin activities were separately measured in larvae at days 3 and 15 after weaning. Changes in mass and final survival were also recorded. Food limitation resulted in a lower final mass only in the HQ larvae. Survival was significantly higher in HQ larvae, but also in larvae suffering food restriction, irrespective of the quality of the spawn. In both groups of larvae, lower food availability was correlated to increased values of trypsin, and particularly, of chymotrypsin. Variations were high enough to support the usefulness of such enzymes as indicators of a modified nutritional status. It is suggested that the noticeable increase in the activity of both enzymes observed in the restrictedly fed larvae could compensate initial deficiencies and explain the good results in growth and survival obtained in LQ larvae.

Heat shock proteins (HSPs) expression is commonly used as indicators of cellular stress in animals. However, very little is known about either the expression patterns of HSPs or their role in the stress-tolerance phenomenon in early life stages of fish. To this end, we examined the impact of food-deprivation (12 h), reduced oxygen levels (3.5 mg/L for 1 h) and heat shock (HS: +5 degrees C for 1 h) on HSP70 and HSP90 protein expression in early life stages of the gilthead sea bream (Sparus aurata), a warm-water aquaculture species. Also, we investigated HSP70 and HSP90 response to food-deprivation (7 days) in early life stages of rainbow trout (Oncorhynchus mykiss), a cool-water aquaculture species, and the tolerance of this larvae to heat shock (either +5 or +10 degrees C for 1 h). Our results clearly demonstrate that food-deprivation enhances HSP70 and HSP90 protein expression in larvae of both species. In gilthead sea bream larvae, the stressors-induced HSP70 and HSP90 (only in the reduced oxygen group) protein expression returned to unstressed levels after 24 h recovery. In fed trout larvae, a +5 degrees C heat shock did not elevate HSP70 and HSP90 expression, whereas 100% mortality was evident with a +10 degrees C HS. However, food-deprived trout larvae, which had higher HSP70 and HSP90 protein content, survived HS and showed HS-dependent increases in HSP70, but not HSP90 expression. Overall, HSP70 and HSP90 protein expression in early life stages of fish have the potential to be used as markers of nutritional stress, while elevation of the tissue HSPs content may be used as a means to increase stress tolerance during larval rearing.

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.