A meta-analysis for assessing the contributions of trypsin and chymotrypsin as the two major endoproteases in protein hydrolysis in fish intestine.

For the majority of fish species, regardless of being gastric or agastric, trypsin and chymotrypsin are known as the two main alkaline proteases responsible for the initial stage of protein hydrolysis in the fish intestine. Although the critical role of these proteases for protein hydrolysis in fish intestine is without doubt, the relative input of each enzyme in protein hydrolysis is still unclear. Data used in the present study has been retrieved from a bibliographic search using the Dimensions application (https://app.dimensions.ai/discover/publication tool). Retrieved articles were carefully inspected to identify whether they contained the description of the development of ontogenetic activities for trypsin, chymotrypsin, and total alkaline proteases in fish intestine. From the list of consulted articles, 21 studies were chosen based on correlation coefficients (Pearson correlation test), and four groups of fish were identified with high significant correlation between 1) the activity of chymotrypsin and total alkaline proteases; 2) the activity of trypsin, chymotrypsin, and total alkaline proteases; 3) the activity of trypsin and total alkaline proteases, and 4) mainly negative correlation between trypsin, chymotrypsin, and total alkaline proteases. These results indicated that the relative inputs of trypsin and chymotrypsin in protein hydrolysis may vary significantly among different fish species, which is a crucial point for proper understanding of species-specific digestive traits in both natural and aquaculture scenarios.

Impact of dietary porcine blood by-products in meagre (Argyrosomus regius) physiology, evaluated by welfare biomarkers and the antibacterial properties of the skin mucus.

Tools are required for quick and easy preliminary evaluation of functional feeds efficiency on fisheries. The analysis of skin mucus biomarkers is a recent alternative approach providing a faster feed-back from the laboratory which is characterized by being less invasive, more rapid and with reduced costs. The effect of replacing fishmeal and fish protein hydrolysates by means of two porcine by-products, the porcine spray-dried plasma (SDPP) and pig protein hydrolysate (PPH), in compound diets (50.4% crude protein, 16.2% crude protein, 22.1 MJ/kg feed) was evaluated in juvenile meagre (Argyrosomus regius) during a two-months period. To determine the impact of these dietary replacements, growth and food performance were measured together with digestive enzymes activities and filet proximal composition. Additionally, skin mucus was collected and characterized by determining main mucus biomarkers (protein, glucose, lactate, cortisol, and antioxidant capacity) and its antibacterial properties, measured by the quick in vitro co-culture challenges. In comparison to the control group, the inclusion of PPH and SDPP, in meagre diets reduced growth (7.4-8.8% in body weight), increased feed conversion ratios (9.0-10.0%), results that were attributed to a reduction in feed intake values (24.2-33.0%) (P < 0.05). Porcine blood by-products did not modify the activity of gastric and pancreatic digestive enzymes as well as those involved in nutrient absorption (alkaline phosphatase) nor liver oxidative stress condition (P > 0.05). In contrast, a reduction in fillet lipid content associated to an increase in fillet protein levels were found in fish fed SDPP and PPH diets (P < 0.05). As compared to the control diet, the dietary replacement did not alter the levels of the skin mucus biomarkers related to stress (cortisol and antioxidant capacity) or nutritional status (soluble protein, glucose and lactate) (P > 0.05). Interestingly, regardless of the worst performance in somatic growth, meagre fed diets containing both tested porcine by-products showed a significantly improved antibacterial capacity of their skin mucus. This enhancement was more prominent for fish fed with the PPH diet, which may be attributed to a higher content of immunomodulatory bioactive compounds in PPH. Further research will be necessary to provide insights on how the inclusion of SDPP and PPH, at the expense of dietary fishmeal and fish protein hydrolysates, affects feed intake and growth performance in meagre. However, the use of skin mucus biomarkers has been demonstrated to be an excellent methodology for a preliminary characterization of the functional feeds, in particular for their prophylactic properties by the study of mucus antibacterial activity.

Soybean protein concentrate as a protein source for totoaba (Totoaba macdonaldi) juveniles: Effect on intermediary metabolism and liver histological organization.

This study aimed to investigate the effects of replacing fish meal (FM) with soybean protein concentrates (SPC) on the intermediary metabolism and health of Totoaba macdonaldi juveniles. Fish (initial weight 50 ± 1 g) were fed for 60 days with eight diets: a reference diet (RD) and seven experimental diets where FM was replaced gradually with 15 to 100% SPC (SPC15, SPC30, SPC45, SPC60, SPC75, SPC90, and SPC100, respectively). Hexokinase (HK), glucokinase (GK), and alanine aminotransferase (ALT) enzyme activities showed highly significant differences (p < 0.01) between fish fed RD (0% SPC) compared to fish fed the diets with 60, 75, 90, and 100% SPC. The ALT enzyme shows a highly significant (p < 0.01) decrease in activity for fish fed 75, 90, and 100% SPC inclusions compared to fish fed the RD. The aspartate aminotransferase AST/ALT ratio showed a significant increase in activity for fish fed 100% soybean compared only with fish fed the control diet. The histological organization of the liver in totoaba juveniles fed RD, SPC15, SPC30 and SPC45 diets were similar. Totoaba fed with SPC90 and SPC100 showed histological alterations in hepatic and pancreatic parenchyma. Overall, according to the findings in this study, 45% of dietary FM could be replaced by SPC without causing adverse changes in metabolism, histological organization of liver, and health of juveniles of totoaba when cultured for 60 days. However, levels greater than 60% of SPC could compromise the health status of fish.

Larval rearing and ontogeny of digestive enzyme activities in yellowfin seabream (Acanthopagrus latus, Houttuyn 1782).

The present research was conducted to provide insight into digestive larval capacity in Acanthopagrus latus larvae from hatching up to 30 days after hatching (DAH). Newly hatched larvae were stocked into six 300-L cylindrical polyethylene tanks at a density of larvae 50 larvae/L and reared by means of the green water system using Nannochloropsis oculata (0.5 × 106/mL). After mouth opening, larvae were fed with rotifers (5-16 individual/mL) from 2 to 20 DAH; then, Artemia nauplii (0.5-3.0 individuals/mL) were offered to larvae from 18 to 30 DAH, meanwhile a commercial microdiet was offered to larvae from 25 to 30 DAH. Larval performance in terms of growth and survival, and the assessment of the activity of selected digestive enzymes ontogeny of digestive enzymes activities was evaluated in larvae sampled at 0 (hatching), 7, 15, 22 and 30 DAH. Larvae showed an exponential growth characterized by two different growth stanzas, a first one characterized by slow growth rates comprised between hatching to 15 DAH (4.7 ± 0.2 mm), followed by a period of faster growth rates between 16 and 30 DAH (7.5 ± 0.6 mm). The activities of the brush border (alkaline phosphatase, ALP) and cytosolic (leucine-alanine peptidase, LAP) enzymes, as well as those of the pancreatic ones like total alkaline proteases, bile salt-activated lipase and α-amylase were detected from the mouth opening stage. Total activities of pancreatic and gastric enzymes increased with larval growth showing an enhancement of digestive capacities with larval age and size. The intestinal maturation in A. latus as assessed by the ratio of AP to LAP did not occur as expected by end of the first month of life suggesting the complete establishment of digestive luminal processes may take place at older ages. This study related to the growth patterns and ontogenic changes in activity of pancreatic, gastric and intestinal enzymes in A. latus and their nutritional regulation may be considered as the first step for improving the larviculture, as well as assessing and refining the nutritional requirements during the larval and early juvenile stages of this sparid species.

The onset of piscivory in a freshwater fish species: analysis of behavioural and physiological traits.

The onset of piscivory in fish, resulting in a shift from zooplankton or invertebrate to fish prey, was studied on pikeperch (Sander lucioperca) larvae displaying and not displaying piscivorous behaviour at different ages (23, 30, 37, 44 and 52 days post-hatching) using behavioural (attack, capture and swimming activity), morphological (allometry) and digestive enzymatic (trypsin, α-amylase and pepsin) analyses. The shift from zooplanktonic food items (Artemia nauplii) to a piscivorous diet did not occur at the same time for all individuals within the same cohort. Predation tests, conducted under controlled conditions (20°C; ad libitum feeding), showed that some larvae attacked fish prey as early as the age of 3 weeks [11.0 ± 1.3 mm total length (TL)], whereas others did not start until the age of 6 weeks (16.6 ± 1.9 mm TL). Piscivorous individuals were bigger, with larger heads, longer tails, higher acid protease and lower alkaline protease activities, than non-piscivorous conspecifics. In conclusion, high interindividual variability in morphological and digestive system developments linked to the development of predatory abilities could induce cannibalism in fish.

Gene expression analysis of the innate immune system during early rearing and weaning of meagre (Argyrosomus regius).

The present study is the first report of some representative innate immune genes in meagre (Argyrosomus regius) larvae. This study has specifically focused on the growth period from hatching to the juvenile stage, a critical time in marine fish development when reliance on innate immune mechanisms are required for survival. We report molecular cloning of partial open reading frames and expression patterns for some innate immune genes (c3, cox2, met, lyzc, mxp, myd88, nod2, nod3). In addition, phylogenetic analyses of some of the sequences obtained was performed where confusion among closely allied isoforms may have existed. These results show the met isoform from meagre is met II, an isoform more similar to a homolog described in Larimichthys crocea; lysozyme (lyzc) corresponds to the c-type and NOD isoforms (nod2, nod3) separate into different clades confirming their distinctness within a common evolutionary history. Gene expression profiles of innate genes were investigated, for nine developmental stages, from 8 days post-hatching (dph) to 120 dph. Present results demonstrated that c3, cox2, met II, lyzc, mxp, myd88, nod2, and nod3 were expressed in all stages of larval development and displayed distinct expression profiles in separate tissues (kidney, spleen gut and gill). Moreover, expression patterns suggested theses innate immune genes may be influenced by feeding practices, i.e. switching from live prey (rotifer and Artemia) and weaning onto an inert commercial diet. In addition to evaluating changes in gene expression during early development, this study evaluated the modulation of gene expression by means of in vivo trials in juveniles that were stimulated with PAMPs (LPS, poly I:C, ß-glucan). These results revealed significant changes in mRNA levels of target genes in the kidney, spleen, gut and gills. However, expression profiles differed in magnitude depending on the stimulant and/or tissue. These results are discussed in terms of their relevance and potential application in aquaculture practices.

Ontogeny of lymphoid organs and mucosal associated lymphoid tissues in meagre (Argyrosomus regius).

This study investigates the development of lymphoid organs and mucosal tissues in larval and juvenile meagre, Argyrosomus regius. For this purpose, meagre larvae were reared from hatch to the juvenile stage, under mesocosm conditions at 18-19 °C, using standard feeding sequences with live prey and artificial food. The kidney was evident upon hatch and included a visible pronephros, with undifferentiated stem cells and excretory tubules at 1 dph (3.15 ±â€¯0.1 mm SL). The thymus was first detected 8 dph (4.49 ±â€¯0.39 mm SL) and was clearly visible 12 dph (5.69 ±â€¯0.76 mm SL), 33 dph (15.69 ±â€¯1.81 mm SL) an outer thymocytic zone and inner epithelial zone were visible. The spleen was present 12 dph, located between exocrine pancreas and intestine and by 26 dph (11.84 ±â€¯1.3 mm SL) consisted of a mass of sinusoids filled with red blood cells. Melanomacrophage centers were found 83 dph (66.25 ±â€¯4.35 mm SL) in the spleen. Between 14-15 dph (6.9 ±â€¯1.1 mm SL), goblet and rodlet cells appear in the gill and intestinal epithelium. The lymphoid organs, which appear in the order of pronephric kidney (1 dph), thymus (8 dph) and spleen (12 dph) remarkably increase in size during the post-flexion stage. While functional studies are needed to confirm the activity of the immune response, the morphology of the lymphoid organs suggest that meagre is not immuno-competent until 83 dph.

Weaning European glass eels (Anguilla anguilla) with plant protein-based diets and its effects on intestinal maturation.

Weaning glass eels with compound diets (36% proteins, 16% lipids) differing in their fishmeal (FM) level (50, 75 and 100% FM replaced by a blend of plant proteins, PP) was compared to a group fed cod roe. Weaning lasted for 20 days and then, eels were fed compound diets for 70 days, whereas the other group was only fed cod roe (90 days). Diets were tested with four replicates and evaluated in terms of growth, survival, glass eels metamorphosis into elvers, oxidative stress status and activity of digestive enzymes. Although glass eels are traditionally fed with fish roe and progressively weaned onto compound diets, results revealed that this strategy should not be prolonged for a long time, since feeding glass eels with cod roe for 90 days negatively affected their growth (2 times lower than fish fed compound diets), delayed their metamorphosis, as well as the maturation of their digestive function as the ratio of alkaline phosphatase and leucine-alanine peptidase indicated. Weaning glass eels onto compound diets differing in their FM levels did not affect their growth, metamorphic stage nor the activity of pancreatic enzymes (total alkaline proteases, trypsin, bile salt-activated lipase and α-amylase), although 75% FM replacement by PP sources delayed the level of intestinal maturation in eels. In comparison to glass eels fed the 100% FM diet, survival was negatively affected in groups fed diets with 50 and 75% FM replacement by PP ingredients, which indicated that further improvement is needed in diet formulation for this stage of development.

The effect of algal turbidity on larval performance and the ontogeny of digestive enzymes in the grey mullet (Mugil cephalus).

A study comprised of two trials determined the effects of water turbidity produced by live microalgae and inert clay particles on the larval rearing of grey mullet (Mugil cephalus). Trial 1 evaluated the effect of microalgae produced water turbidity on grey mullet larval performance and digestive tract (DT) enzyme ontogeny. Two microalgae (Nannochloropsis oculata and Isochrysis galbana) water turbidity levels (0.76 and 1.20 NTU, respectively) and a non-microalgae control (0.26 NTU) were investigated on 2 to 23 dph grey mullet larvae. The higher turbidity (1.2 NTU) larvae (5 dph) consumed markedly (P < .05) more rotifers than other treatment fish, independently of the microalgae type. There was no clear effect of the turbidity treatments on DT enzyme ontogeny. However, in all treatments lipase and alkaline proteases appeared to be modulated by the diet. Alkaline phosphatase activity was ca. 8 times higher and α-amylase activity increased 5.3 times in 79 dph fish compared to 40 dph individuals. The ratio of alkaline phosphatase and leucine-alanine aminopeptidase indicated gut maturation occurred around 61 dph. Trial 2 compared the most effective N.occulata produced turbidity level (1.2 NTU) with the identical water turbidity produced by inert clay on larval performance. M. cephalus larvae exposed to high algal turbidity demonstrated superior performance (P < .05), in terms of rotifer ingestion, dry weight gain and survival, compared to cohorts reared under the clay treatment and the lower microalgae produced turbidity. These findings suggested that water algal turbidity is not the dominant factor determining improved grey mullet larval performance.

Dietary aquaculture by-product hydrolysates: impact on the transcriptomic response of the intestinal mucosa of European seabass (Dicentrarchus labrax) fed low fish meal diets.

BACKGROUND: Aquaculture production is expected to double by 2030, and demands for aquafeeds and raw materials are expected to increase accordingly. Sustainable growth of aquaculture will require the development of highly nutritive and functional raw materials to efficiently replace fish meal. Enzymatic hydrolysis of marine and aquaculture raw materials could bring new functionalities to finished products. The aim of this study was to determine the zootechnical and transcriptomic performances of protein hydrolysates of different origins (tilapia, shrimp, and a combination of the two) in European seabass (Dicentrarchux labrax) fed a low fish meal diet (5%), for 65 days. RESULTS: Results were compared to a positive control fed with 20% of fish meal. Growth performances, anterior intestine histological organization and transcriptomic responses were monitored and analyzed. Dietary inclusion of protein hydrolysates in the low fish meal diet restored similar growth performances to those of the positive control. Inclusion of dietary shrimp hydrolysate resulted in larger villi and more goblet cells, even better than the positive control. Transcriptomic analysis of the anterior intestine showed that dietary hydrolysate inclusion restored a pattern of intestinal gene expression very close to the pattern of the positive control. However, as compared to the low fish meal diet and depending on their origin, the different hydrolysates did not modulate metabolic pathways in the same way. Dietary shrimp hydrolysate inclusion modulated more metabolic pathways related to immunity, while nutritional metabolism was more impacted by dietary tilapia hydrolysate. Interestingly, the combination of the two hydrolysates enhanced the benefits of hydrolysate inclusion in diets: more genes and metabolic pathways were regulated by the combined hydrolysates than by each hydrolysate tested independently. CONCLUSIONS: Protein hydrolysates manufactured from aquaculture by-products are promising candidates to help replace fish meal in aquaculture feeds without disrupting animal metabolism and performances.

Effects of dietary soybean lecithin on growth performance, blood chemistry and immunity in juvenile stellate sturgeon (Acipenser stellatus).

An eleven weeks feeding trial was conducted to determine the effects of different levels of dietary soybean lecithin (SBL) on growth performance, blood chemistry and immunity in juvenile stellate sturgeon (Acipenser stellatus). Fish were fed seven isoproteic (44% crude protein) and isolipidic (17% crude fat) diets containing graded levels of SBL: 0 (control), 1, 2, 4, 6, 8 and 10%. Results showed that dietary SBL supplementation significantly improved the final body weight (BW) and weight gain (WG). Fish fed 6% SBL showed the highest BW and WG values in comparison to fish fed the control diet (P < 0.05), whereas increasing SBL levels above 6% had little practical benefit in terms of somatic growth performance. The inclusion of SBL in diets significantly improved the immune response as data from lysozyme, total Ig levels, alternative complement, phagocytic and bactericidal activities indicated (P < 0.05). The broken-line regression analysis of immunological variable revealed that depending on the parameter considered, the optimal SBL levels in diets for stellate sturgeon juveniles varied. In particular, dietary SBL levels requirements in stellate sturgeon when considering the phagocytic activity rate were determined at 3.3%, whereas 4.1-4.2% were recommended when considering data from lysozyme, alternative complement and bactericidal activities. In contrast, the highest minimum dietary SBL content was estimated at 6.9% when data from total Ig levels were considered. These results indicated that dietary PLs are required for boosting innate immunity in stellate sturgeon, although their minimal level changed depending on the immunological parameter considered. Therefore, we assume that SBL levels comprised between 3.3 and 6.9% may be used as a prophylactic measure to improve the health status in stellate sturgeon. Red blood cell count, hemoglobin and hematocrit levels increased with increasing dietary SBL levels, especially in those sturgeons fed the diet with 6% SBL (P < 0.05). In addition, white blood cell counts significantly increased as dietary SBL levels increased from 4 to 8% in comparison to the control group. Blood biochemistry was also affected by different dietary SBL levels. In particular, significantly higher levels of glucose, cholesterol, HDL and triglycerides were detected in fish fed >6%, >4%, >2% and 2% SBL, respectively (P < 0.05). Based on somatic growth parameters, blood chemistry and systemic immunity parameters, diets containing ca. 6% SBL are recommended for juvenile stellate sturgeon.

Thermal imprinting modifies bone homeostasis in cold-challenged sea bream (Sparus aurata).

Fish are ectotherms and temperature plays a determinant role in their physiology, biology and ecology, and is a driver of seasonal responses. The present study assessed how thermal imprinting during embryonic and larval stages modified the response of adult fish to low water temperature. We targeted the gilthead sea bream, which develops a condition known as winter syndrome when it is exposed to low water temperatures. Eggs and larvae of sea bream were exposed to four different thermal regimes and then the response of the resulting adults to a low temperature challenge was assessed. Sea bream exposed to a high-low thermal regime as eggs and larvae (HLT; 22°C until hatch and then 18°C until larvae-juvenile transition) had increased plasma cortisol and lower sodium and potassium in response to a cold challenge compared with the other thermal history groups. Plasma glucose and osmolality were increased in cold-challenged HLT fish relative to the unchallenged HLT fish. Cold challenge modified bone homeostasis/responsiveness in the low-high thermal regime group (LHT) relative to other groups, and ocn, ogn1/2, igf1, gr and trα/ß transcripts were all downregulated. In the low temperature group (LT) and HLT group challenged with a low temperature, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities were decreased relative to unchallenged groups, and bone calcium content also decreased in the LT group. Overall, the results indicate that thermal imprinting during early development of sea bream causes a change in the physiological response of adults to a cold challenge.

Olive oil bioactive compounds increase body weight, and improve gut health and integrity in gilthead sea bream (Sparus aurata).

An olive oil bioactive extract (OBE) rich in bioactive compounds like polyphenols, triterpenic acids, long-chain fatty alcohols, unsaturated hydrocarbons, tocopherols and sterols was tested (0, 0·08, 0·17, 0·42 and 0·73 % OBE) in diets fed to sea bream (Sparus aurata) (initial weight: 5·4 (sd 1·2) g) during a 90-d trial (four replicates). Fish fed diets containing 0·17 and 0·42 % OBE were 5 % heavier (61·1 (sd 1·6) and 60·3 (sd 1·1) g, respectively) than those of the control group (57·0 (sd 0·7) g), although feed conversion ratio and specific feed intake did not vary. There were no differences in lipid peroxidation (LPO) levels, catalase, glutathione reductase and glutathione S-transferase activities in the intestine and liver, although there was a tendency of lower intestinal and hepatic LPO levels in fish fed OBE diets. No differences in villus size were found among treatments, whereas goblet cell density in the control group was on average14·3 % lower than in fish fed OBE diets. The transcriptomic profiling of intestinal markers, covering different biological functions like (i) cell differentiation and proliferation, (ii) intestinal permeability, (iii) enterocyte mass and epithelial damage, (iv) IL and cytokines, (v) pathogen recognition receptors and (vi) mitochondria function, indicated that among the eighty-eight evaluated genes, twenty-nine were differentially expressed (0·17 % OBE diet), suggesting that the additive has the potential of improving the condition and defensive role of the intestine by enhancing the maturation of enterocytes, reducing oxidative stress, improving the integrity of the intestinal epithelium and enhancing the intestinal innate immune function, as gene expression data indicated.

Histological study of the gastrointestinal tract in longfin yellowtail (Seriola rivoliana) larvae.

This work contributes basic knowledge on larval development of Seriola rivoliana. A histological study describes the development of the digestive tract and accessory glands in S. rivoliana larvae reared under laboratory conditions at 24 °C from hatching to 30 days post-hatching (DPH). At hatching (2.6 ± 0.12 mm), larvae had an undifferentiated digestive tract with a closed straight tube and a large yolk sac with an oil globule. The liver and pancreas were observed at 1 and 2 days, and the mouth and anus opened at day 2. Enriched rotifers were visible in their digestive tract. At the beginning of the pre-flexion stage, a mixed nutritional period was observed. At day 3, exogenous feeding began; the digestive tract became differentiated into the buccopharynx, esophagus, an undifferentiated stomach, and the intestines. Zymogen granules were visible in the exocrine pancreas. At day 4, supranuclear vacuoles were present in the posterior intestine, indicating the beginning of intracellular digestion. At day 5, goblet cells were present in the esophagus and became functional at day 7 in the esophagus and intestine. The buccopharynx goblet cells developed at day 15. The presence of gastric glands and differentiation of the stomach in the fundic, cardiac, and pyloric regions during the post-flexion stage occurred at day 20. This was the onset of the juvenile period and the beginning of weaning; however, a long co-feeding phase is recommended. Pyloric caeca were observed at day 30 (13.6 ± 1.6 mm). These results provide valuable information on S. rivoliana larvae biology and digestive physiology, which should be useful to improve cultivation techniques and identify ecological features involved in ontogeny.

Dietary fatty acid composition affects food intake and gut-brain satiety signaling in Senegalese sole (Solea senegalensis, Kaup 1858) larvae and post-larvae.

Little is known how dietary lipids affect food intake during larval development of fish, especially with regard to fatty acid (FA) composition. In fact, very little work has been done on appetite regulation and food intake in fish larvae in general, due to biological and technical difficulties associated with this type of studies. A new method using fluorescent microspheres as markers was developed in this study to evaluate food intake and prey selectivity of Senegalese sole larvae and post-larvae. Food intake was quantified in fish fed Artemia metanauplii enriched with oils differing in FA profile: cod liver oil (CLO), linseed oil (LSO), soybean oil (SBO) or olive oil (OO). The fish did not preferentially ingest a specific diet when presented with a choice. However, pre-metamorphic larvae from the CLO treatment ingested more metanauplii per g body weight, while differences in post-larvae were not significant. These findings were developed further by analyzing mRNA levels of a range of putative anorexigenic (pyya, pyyb, glp1, cckl, cart1a, cart1b, cart2a, cart4, pomca, pomcb, crf) and orexigenic (gal, npy, agrp2) genes, to identify those which are significantly affected by feeding and/or dietary FA composition. The variety of expression patterns observed highlighted the complexity of appetite regulatory mechanisms. In general, fish fed the CLO diet tended to show gene expression patterns most dissimilar to the remaining treatments. Expression in pre-metamorphic larvae was generally less in accordance with the putative function of the genes than in post-larvae, which could suggest a yet underdeveloped regulatory system.

Influence of time, storage temperature and freeze/thaw cycles on the activity of digestive enzymes from gilthead sea bream (Sparus aurata).

In this study, we tested the effects of long-term storage (2 years) at -20 °C and short-term storage (several hours) in ice and freeze/thaw cycles on the activities of pancreatic, gastric and intestinal (brush border and cytosolic) digestive enzymes in a teleost fish species. The results revealed a significant lose in activity of pancreatic (trypsin, chymotrypsin, total alkaline proteases and α-amylase) and intestinal cytosolic (leucine-alanine peptidase) enzymes between 140 and 270 days of storage at -20 °C, whereas in contrast, the activity of all the assayed brush border enzymes remained constant during the first 2 years of storage at -20 °C. During short-term storage conditions, the most stable enzymes assayed were those of the enterocytes of the brush border, which did not show any change in activity after being held for 5 h in ice. Five freezing and thawing cycles did not affect the activity of the intestinal brush border enzymes and the cytosolic ones, whereas the activity of trypsin, α-amylase and bile-salt-activated lipase was significantly affected by the number of freezing and thawing cycles. No changes in pepsin activity were found in samples exposed to 1 and 2 freezing and thawing cycles.

Vitamin A supplementation enhances Senegalese sole (Solea senegalensis) early juvenile's immunocompetence: New insights on potential underlying pathways.

Senegalese sole (Solea senegalensis) has been considered since the 1990's to be a promising flatfish species for diversifying European marine aquaculture. However, pathogen outbreaks leading to high mortality rates can impair Senegalese sole commercial production at the weaning phase. Different approaches have been shown to improve fish immunocompetence; with this in mind the objective of the work described herein was to determine whether increased levels of dietary vitamin A (VA) improve the immune response in early juveniles of Senegalese sole. For this purpose, Senegalese sole were reared and fed with Artemia metanauplii containing increased levels of VA (37,000; 44,666; 82,666 and 203,000 total VA IU Kg(-1)) from 6 to 60 days post-hatch (early juvenile stage). After an induced bacterial infection with a 50% lethal dose of Photobacterium damselae subsp. damselae, survival rate, as well as underlying gene expression of specific immune markers (C1inh, C3, C9, Lgals1, Hamp, LysC, Prdx1, Steap4 and Transf) were evaluated. Results showed that fish fed higher doses of dietary VA were more resistant to the bacterial challenge. The lower mortality was found to be related with differential expression of genes involved in the complement system and iron availability. We suggest that feeding metamorphosed Senegalese sole with 203,000 total VA IU Kg(-1) might be an effective, inexpensive and environmentally friendly method to improve Senegalese sole immunocompetence, thereby improving survival of juveniles and reducing economic losses.

Digestive system development and study of acid and alkaline protease digestive capacities using biochemical and molecular approaches in totoaba (Totoaba macdonaldi) larvae.

The present study aimed to describe and understand the development of the digestive system in totoaba (Totoaba macdonaldi) larvae from hatching to 40 days post-hatch (dph) from morphological and functional perspectives. At hatch, the digestive system of totoaba was undifferentiated. The anus and the mouth opened at 4 and 5 dph, respectively. During exogenous feeding, development of the esophagus, pancreas, liver and intestine was observed with a complete differentiation of all digestive organs. Expression and activity of trypsin and chymotrypsin were observed as early as at 1 dph, and increments in their expression and activity coincided with changes in food items (live and compound diets) and morpho-physiological development of the accessory digestive glands. In contrast, pepsin was detected later during development, which includes the appearance of the gastric glands between 24 and 28 dph. One peak in gene expression was detected at 16 dph, few days before the initial development of the stomach at 20 dph. A second peak of pepsin expression was detected at day 35, followed by a peak of activity at day 40, coinciding with the change from live to artificial food. Totoaba larvae showed a fully morphologically developed digestive system between 24 and 28 dph, as demonstrated by histological observations. However, gene expression and activity of alkaline and acid proteases were detected earlier, indicating the functionality of the exocrine pancreas and stomach before the complete morphological development of the digestive organs. These results showed that integrative studies are needed to fully understand the development of the digestive system from a morphological and functional point of views, since the histological organization of digestive structures does not reflect their real functionality. These results indicate that the digestive system of totoaba develops rapidly during the first days post-hatch, especially for alkaline proteases, and the stomach becomes functional between 20 and 24 dph allowing the weaning process to begin at this age.

Dietary administration of ß-1,3/1,6-glucan and probiotic strain Shewanella putrefaciens, single or combined, on gilthead seabream growth, immune responses and gene expression.

It is widely known that ß-glucans and probiotic bacteria are good immunostimulants for fish. In the present work we have evaluated the dietary effect of ß-1,3/1,6-glucan (isolated from Laminarina digitata) and Pdp 11 (Shewanella putrefaciens, probiotic isolated from gilthead seabream skin), single or combined, on growth, humoural (seric level of total IgM antibodies and peroxidase and antiprotease activities) and cellular innate immune response (peroxidase and phagocytic activities of head-kidney leucocytes), as well as the expression of immune-related genes in gilthead seabream (Sparus aurata). Four treatment groups were established: control (non-supplemented diet), Pdp 11 (10(9) cfu g(-1)), ß-1,3/1,6-glucan (0.1%) and ß-1,3/1,6-glucan + Pdp 11 (0.1% and 10(9) cfu g(-1), respectively). Fish were sampled after 1, 2 and 4 weeks of feeding. Interestingly, all supplemented diets produced increments in the seabream growth rates, mainly the Pdp 11-suplemented diet. Overall, Pdp 11 dietary administration resulted in decreased serum IgM levels and peroxidase activity. However, the seric antiprotease activity was increased in fish fed with both supplements together. Furthermore, ß-1,3/1,6-glucan and combined diet increased phagocytic activity after 2 or 4 weeks. At gene level, IL-1ß and INFγ transcripts were always up-regulated in HK but only the interleukin reached significance after 4 weeks in the group fed with ß-glucan. On the contrary, IgM gene expression tended to be down-regulated being significant after 1 week in seabream specimens fed with ß-glucan or ß-glucan plus Pdp 11. These results suggest that ß-1,3/1,6-glucan and Pdp 11 modulate the immune response and stimulates growth of the gilthead seabream, one of the species with the highest rate of production in Mediterranean aquaculture.

Impact of the diet in the gut microbiota after an inter-species microbial transplantation in fish.

Inter-species microbial transplantations offer the possibility of transferring species-specific microbes and their associated functionality. As a conceptual approach, an intestinal microbiota transplant (IMT) between two marine carnivorous fish species that thrive in different environmental conditions was conducted: from donor Atlantic salmon (Salmo salar) to recipient gilthead seabream (Sparus aurata), after obliterating its basal microbiota with an antibiotic treatment. To confirm that the gut microbiota was able to recover after antibiotics without the influence of the diet, a group of gilthead seabream not submitted to the IMT was kept fasted as an internal control. To assess the effect of the diet after the IMT, two groups of gilthead seabream were respectively fed with their typical diet and with Atlantic salmon diet. At 36 days post-IMT, the gut of the individuals fed with their typical diet was dominated by the feed-associated bacteria, while those fed with the salmon diet had developed a unique microbiota from the convergence of the diet, donor, and recipient microbiota. These results suggested that an intestinal microbiota transplantation may be effective if the basal microbiota from the gut is first cleared and a targeted dietary modification is provided to maintain and enrich the novel bacteria species over time.