The potential of a combination of pungent spices as a novel supplement in gilthead seabream (Sparus aurata) diets to aid in the strategic use of fish oil in aquafeeds: a holistic perspective.

This work studied the potential of a combination of pungent spices (capsicum, black pepper, ginger, and cinnamaldehyde) to be used as a supplement in diets of gilthead seabream (Sparus aurata; 44.1 ± 4.2 g). During 90 days, fish were fed three experimental diets with low inclusion of fish oil and containing poultry fat as the main source of lipids, supplemented with graded levels of the tested supplement: 0 (control), 0.1 (SPICY0.1%), and 0.15% (SPICY0.15%). As a result, the pungent spices enhanced the growth performance, the activity of the bile-salt-activated lipase in the intestine, and decreased fat deposit levels within enterocytes. The SPICY0.1% diet reduced the feed conversion ratio and the perivisceral fat index and lipid deposits in the liver. Moreover, the ratio of docosahexaenoic acid/eicosapentaenoic acid in fillet increased in fish fed the SPICY0.1% diet, while the hepatic levels of docosahexaenoic acid and total n-3 polyunsaturated fatty acids increased in fish fed the SPICY0.15% diet. Furthermore, there was an effect on the expression of some biomarkers related to lipid metabolism in 2-h postprandial fish (fasn, elovl6, scd1b, cyp7a1, lpl, and pparß), and in 48 h fasted-fish fed with the SPICY0.1% diet, a regulation of the intestinal immune response was indicated. However, no significant differences were found in lipid apparent digestibility and proximate macronutrient composition. The spices did not affect biomarkers of hepatic or oxidative stress. No differences in microbial diversity were found, except for an increase in Simpson's Index in the posterior intestine of fish fed the SPICY0.1% diet, reflected in the increased relative abundance of the phylum Chloroflexi and lower relative abundances of the genera Campylobacter, Corynebacterium, and Peptoniphilus. In conclusion, the supplementation of gilthead seabream diets with pungent spices at an inclusion of 0.1% was beneficial to enhance growth performance and feed utilization; reduce fat accumulation in the visceral cavity, liver, and intestine; and improve the fish health status and condition. Results suggest that the tested supplement can be used as part of a nutritional strategy to promote a more judicious use of fish oil in fish diets due to its decreasing availability and rising costs.

Dietary Debaryomyces hansenii promotes skin and skin mucus defensive capacities in a marine fish model.

The present study explores the effects of two supplementation levels of Debaryomyces hansenii (1.1% and 2.2%) as a probiotic in a reference low fish meal-based diet on the skin mucosal tissue in Sparus aurata. This study includes the evaluation of fish performance coupled with a holistic study of the skin mucosa: i) a transcriptomic study of the skin tissue, and ii) the evaluation of its secreted mucus both in terms of skin mucosal-associated biomarkers and its defensive capacity by means of co-culture analysis with two pathogenic bacteria. Results showed that after 70 days of diet administration, fish fed the diet supplemented with D. hansenii at 1.1% presented increased somatic growth and a better feed conversion ratio, compared to fish fed the control diet. In contrast, fish fed the diet including 2.2% of the probiotic presented intermediate values. Regarding gene regulation, the probiotic administration at 1.1% resulted in 712 differentially expressed genes (DEGs), among which 53.4% and 46.6% were up- and down-regulated, respectively. In particular, D. hansenii modulated some skin biological processes related to immunity and metabolism. Specifically, D. hansenii administration induced a strong modulation of some immune biological-related processes (61 DEGs), mainly involved in B- and T-cell regulatory pathways. Furthermore, dietary D. hansenii promoted the skin barrier function by the upregulation of anchoring junction genes (23 DEGs), which reinforces the physical defense against potential skin damage. In contrast, the skin showed modulated genes related to extracellular exosome and membrane organization (50 DEGs). This modulated functioning is of great interest, particularly in relation to the increased skin mucus defensive capacity observed in the bacterial co-culture in vitro trials, which could be related to the increased modulation and exudation of the innate immune components from the skin cells into the mucus. In summary, the modulation of innate immune parameters coupled with increased skin barrier function and cell trafficking potentiates the skin's physical barrier and mucus defensive capacity, while maintaining the skin mucosa's homeostatic immune and metabolic status. These findings confirmed the advantages of D. hansenii supplementation in low fish meal-based diets, demonstrating the probiotic benefits on cultured marine species.

A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis.

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.

Phytogenic Bioactive Compounds Shape Fish Mucosal Immunity.

Aquaculture growth will unavoidably involve the implementation of innovative and sustainable production strategies, being functional feeds among the most promising ones. A wide spectrum of phytogenics, particularly those containing terpenes and organosulfur compounds, are increasingly studied in aquafeeds, due to their growth promoting, antimicrobial, immunostimulant, antioxidant, anti-inflammatory and sedative properties. This trend relies on the importance of the mucosal barrier in the fish defense. Establishing the phytogenics' mode of action in mucosal tissues is of importance for further use and safe administration. Although the impact of phytogenics upon fish mucosal immunity has been extensively approached, most of the studies fail in addressing the mechanisms underlying their pharmacological effects. Unstandardized testing as an extended practice also questions the reproducibility and safety of such studies, limiting the use of phytogenics at commercial scale. The information presented herein provides insight on the fish mucosal immune responses to phytogenics, suggesting their mode of action, and ultimately encouraging the practice of reliable and reproducible research for novel feed additives for aquafeeds. For proper screening, characterization and optimization of their mode of action, we encourage the evaluation of purified compounds using in vitro systems before moving forward to in vivo trials. The formulation of additives with combinations of compounds previously characterized is recommended to avoid bacterial resistance. To improve the delivery of phytogenics and overcome limitations associated to compounds volatility and susceptibility to degradation, the use of encapsulation is advisable. Besides, newer approaches and dedicated methodologies are needed to elucidate the phytogenics pharmacokinetics and mode of action in depth.

Medicinal Plant Leaf Extract From Sage and Lemon Verbena Promotes Intestinal Immunity and Barrier Function in Gilthead Seabream (Sparus aurata).

The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins' glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.

Diet, Immunity, and Microbiota Interactions: An Integrative Analysis of the Intestine Transcriptional Response and Microbiota Modulation in Gilthead Seabream (Sparus aurata) Fed an Essential Oils-Based Functional Diet.

Essential oils (EOs) are promising alternatives to chemotherapeutics in animal production due to their immunostimulant, antimicrobial, and antioxidant properties, without associated environmental or hazardous side effects. In the present study, the modulation of the transcriptional immune response (microarray analysis) and microbiota [16S Ribosomal RNA (rRNA) sequencing] in the intestine of the euryhaline fish gilthead seabream (Sparus aurata) fed a dietary supplementation of garlic, carvacrol, and thymol EOs was evaluated. The transcriptomic functional analysis showed the regulation of genes related to processes of proteolysis and inflammatory modulation, immunity, transport and secretion, response to cyclic compounds, symbiosis, and RNA metabolism in fish fed the EOs-supplemented diet. Particularly, the activation of leukocytes, such as acidophilic granulocytes, was suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the gut. Fish growth performance and gut microbiota alpha diversity indices were not affected, while dietary EOs promoted alterations in bacterial abundances in terms of phylum, class, and genus. Subtle, but significant alterations in microbiota composition, such as the decrease in Bacteroidia and Clostridia classes, were suggested to participate in the modulation of the intestine transcriptional immune profile observed in fish fed the EOs diet. Moreover, regarding microbiota functionality, increased bacterial sequences associated with glutathione and lipid metabolisms, among others, detected in fish fed the EOs supported the metabolic alterations suggested to potentially affect the observed immune-related transcriptional response. The overall results indicated that the tested dietary EOs may promote intestinal local immunity through the impact of the EOs on the host-microbial co-metabolism and consequent regulation of significant biological processes, evidencing the crosstalk between gut and microbiota in the inflammatory regulation upon administration of immunostimulant feed additives.

Carvacrol, Thymol, and Garlic Essential Oil Promote Skin Innate Immunity in Gilthead Seabream (Sparus aurata) Through the Multifactorial Modulation of the Secretory Pathway and Enhancement of Mucus Protective Capacity.

One of the main targets for the use of phytogenics in aquafeeds is the mucosal tissues as they constitute a physical and biochemical shield against environmental and pathogenic threats, comprising elements from both the innate and acquired immunity. In the present study, the modulation of the skin transcriptional immune response, the bacterial growth capacity in skin mucus, and the overall health condition of gilthead seabream (Sparus aurata) juveniles fed a dietary supplementation of garlic essential oil, carvacrol, and thymol were assessed. The enrichment analysis of the skin transcriptional profile of fish fed the phytogenic-supplemented diet revealed the regulation of genes associated to cellular components involved in the secretory pathway, suggesting the stimulation, and recruitment of phagocytic cells. Genes recognized by their involvement in non-specific immune response were also identified in the analysis. The promotion of the secretion of non-specific immune molecules into the skin mucus was proposed to be involved in the in vitro decreased growth capacity of pathogenic bacteria in the mucus of fish fed the phytogenic-supplemented diet. Although the mucus antioxidant capacity was not affected by the phytogenics supplementation, the regulation of genes coding for oxidative stress enzymes suggested the reduction of the skin oxidative stress. Additionally, the decreased levels of cortisol in mucus indicated a reduction in the fish allostatic load due to the properties of the tested additive. Altogether, the dietary garlic, carvacrol, and thymol appear to promote the gilthead seabream skin innate immunity and the mucus protective capacity, decreasing its susceptibility to be colonized by pathogenic bacteria.

Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii.

A microencapsulated feed additive composed by garlic, carvacrol and thymol essential oils (EOs) was evaluated regarding its protective effect in gills parasitized by Sparicotyle chrysophrii in Sparus aurata. A nutritional trial (65 days) followed by a cohabitation challenge with parasitized fish (39 days) were performed. Transcriptomic analysis by microarrays of gills of fish fed the EOs diet showed an up-regulation of genes related to biogenesis, vesicular transport and exocytosis, leukocyte-mediated immunity, oxidation-reduction and overall metabolism processes. The functional network obtained indicates a tissue-specific pro-inflammatory immune response arbitrated by degranulating acidophilic granulocytes, sustained by antioxidant and anti-inflammatory responses. The histochemical study of gills also showed an increase of carboxylate glycoproteins containing sialic acid in mucous and epithelial cells of fish fed the EOs diet, suggesting a mucosal defence mechanism through the modulation of mucin secretions. The outcomes of the in vivo challenge supported the transcriptomic results obtained from the nutritional trial, where a significant reduction of 78% in the abundance of S. chrysophrii total parasitation and a decrease in the prevalence of most parasitic developmental stages evaluated were observed in fish fed the EOs diet. These results suggest that the microencapsulation of garlic, carvacrol and thymol EOs could be considered an effective natural dietary strategy with antiparasitic properties against the ectoparasite S. chrysophrii.

Evaluation of a Bacterial Single-Cell Protein in Compound Diets for Rainbow Trout (Oncorhynchus mykiss) Fry as an Alternative Protein Source.

A 60-day trial was conducted in rainbow trout (Oncorhynchus mykiss) fry (initial weight = 2.5 ± 0.6 g) to evaluate the potential use of a bacterial single-cell protein (SCP) as an alternative protein source. Five experimental diets with different levels of fishmeal replacement (0, 25, 50, 75 and 100%) and no amino acid supplementation were tested. At the end of the trial, we found that fry fed diets, replacing 25 and 50% of fishmeal with bacterial SCP, were 9.1 and 21.8% heavier, respectively, than those fed the control diet (p < 0.05), while Feed Conversion Ratio (FCR) values were also lower in comparison to the reference group. These results were also supported by Protein Efficiency Ratio (PER) and Lipid Efficiency Ratio (LER) values that improved in fish fed diets replacing 50% fishmeal by bacterial SCP. The inclusion of SCP enhanced Feed intake (FI) (p < 0.05), although FI was reduced at higher inclusion levels (>50%), which was associated to feed palatability. High levels of bacterial SCP (>50%) affected the muscular amino acid and fatty acid profiles, imbalances that were associated to their dietary content. The broken-line regression analysis using muscle DHA content and weight gain data showed that the maximum levels of fishmeal replacement by bacterial SCP were 46.9 and 52%, respectively.

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.

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.

Dietary Fatty Acid Metabolism is Affected More by Lipid Level than Source in Senegalese Sole Juveniles: Interactions for Optimal Dietary Formulation.

This study analyses the effects of dietary lipid level and source on lipid absorption and metabolism in Senegalese sole (Solea senegalensis). Juvenile fish were fed 4 experimental diets containing either 100 % fish oil (FO) or 25 % FO and 75 % vegetable oil (VO; rapeseed, linseed and soybean oils) at two lipid levels (~8 or ~18 %). Effects were assessed on fish performance, body proximate composition and lipid accumulation, activity of hepatic lipogenic and fatty acid oxidative enzymes and, finally, on the expression of genes related to lipid metabolism in liver and intestine, and to intestinal absorption, both pre- and postprandially. Increased dietary lipid level had no major effects on growth and feeding performance (FCR), although fish fed FO had marginally better growth. Nevertheless, diets induced significant changes in lipid accumulation and metabolism. Hepatic lipid deposits were higher in fish fed VO, associated to increased hepatic ATP citrate lyase activity and up-regulated carnitine palmitoyltransferase 1 (cpt1) mRNA levels post-prandially. However, lipid level had a larger effect on gene expression of metabolic (lipogenesis and ß-oxidation) genes than lipid source, mostly at fasting. High dietary lipid level down-regulated fatty acid synthase expression in liver and intestine, and increased cpt1 mRNA in liver. Large lipid accumulations were observed in the enterocytes of fish fed high lipid diets. This was possibly a result of a poor capacity to adapt to high dietary lipid level, as most genes involved in intestinal absorption were not regulated in response to the diet.

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.

Retinoic acid differentially affects in vitro proliferation, differentiation and mineralization of two fish bone-derived cell lines: different gene expression of nuclear receptors and ECM proteins.

Retinoic acid (RA), the main active metabolite of vitamin A, regulates vertebrate morphogenesis through signaling pathways not yet fully understood. Such process involves the specific activation of retinoic acid and retinoid X receptors (RARs and RXRs), which are nuclear receptors of the steroid/thyroid hormone receptor superfamily. Teleost fish are suitable models to study vertebrate development, such as skeletogenesis. Cell systems capable of in vitro mineralization have been developed for several fish species and may provide new insights into the specific cellular and molecular events related to vitamin A activity in bone, complementary to in vivo studies. This work aims at investigating the in vitro effects of RA (0.5 and 12.5 µM) on proliferation, differentiation and extracellular matrix (ECM) mineralization of two gilthead seabream bone-derived cell lines (VSa13 and VSa16), and at identifying molecular targets of its action through gene expression analysis. RA induced phenotypic changes and cellular proliferation was inhibited in both cell lines in a cell type-dependent manner (36-59% in VSa13 and 17-46% in VSa16 cells). While RA stimulated mineral deposition in VSa13 cell cultures (50-62% stimulation), it inhibited the mineralization of extracellular matrix in VSa16 cells (11-57% inhibition). Expression of hormone receptor genes (rars and rxrs), and extracellular matrix-related genes such as matrix and bone Gla proteins (mgp and bglap), osteopontin (spp1) and type I collagen (col1a1) were differentially regulated upon exposure to RA in proliferating, differentiating and mineralizing cultures of VSa13 and VSa16 cells. Altogether, our results show: (i) RA affects proliferative and mineralogenic activities in two fish skeletal cell types and (ii) that during phenotype transitions, specific RA nuclear receptors and bone-related genes are differentially expressed in a cell type-dependent manner.

Genomic characterization, phylogeny and gene regulation of g-type lysozyme in sole (Solea senegalensis).

The g-type lysozyme is a key protein of the innate immune system to fight bacterial infections. In this study we cloned and characterized the gene encoding for g-type lysozyme in Senegalese sole (Solea senegalensis). The deduced amino acid sequence comprised 195 residues containing the three conserved catalytic residues and two cysteines. A BAC analysis revealed that the gene is structured in 5 exons and 4 introns. Also, two polyadenylation signals that generate two cDNAs differing in 3'-UTR length were detected. Promoter analysis showed the presence of the main cis-acting elements involved in the transcriptional regulation of the gene. At genomic level, the g-type lysozyme was associated with mucolipin 1 and the peptidoglycan recognition protein 2 conforming a cluster of antidefensive genes with a well-conserved synteny across Percomorpha. FISH analysis using the BAC clone revealed a single hybridization signal located in an acrocentric chromosome pair. The phylogenetic analysis confirmed that the g-type lysozyme represents a complex group in fish that has been shaped by gene duplications and diversification with several positions under Darwinian selection. Expression analysis in juvenile tissues indicated that transcript levels were higher in gills, spleen and heart. During development, gene expression activated just at the beginning of metamorphosis, increasing progressively until climax. Hormonal treatments demonstrated that this gene was regulated positively by thyroid hormones during development and negatively by dexamethasone. In contrast, no response was observed after all-trans retinoic acid or 4-diethylaminobenzaldehyde treatments. Finally, treatments using lipopolysaccharide, lipoteichoic acid, peptidoglycan, zymosan and poly(I:C) activated gene expression in a time- and tissue-specific manner. Taken together, data indicate that g-type lysozyme is a high evolutionary conserved gene that diversified to adapt to changing environment and pathogen conditions. Gene expression can be activated by diverse pathogen stimuli and modulated by physiological factors with important consequences for the aquaculture of this species.

Effect of nature of dietary lipids on European sea bass morphogenesis: implication of retinoid receptors.

The effect of the nature and form of supply of dietary lipids on larval development was investigated in European sea bass larvae, by considering the expression of several genes involved in morphogenesis. Fish were fed from 7 to 37 d post-hatch with five isoproteic and isolipidic compound diets incorporating different levels of EPA and DHA provided by phospholipid or neutral lipid. Phospholipid fraction containing 1.1 % (PL1 diet) to 2.3 % (PL3 diet) of EPA and DHA sustained good larval growth and survival, with low vertebral and cephalic deformities. Similar levels of EPA and DHA provided by the neutral lipid fraction were teratogenic and lethal. Nevertheless, dietary phospholipids containing high levels of DHA and EPA (PL5 diet) induced cephalic (8.5 %) and vertebral column deformities (35.3 %) adversely affecting fish growth and survival; moreover, a down-regulation of retinoid X receptor alpha (RXRalpha), retinoic acid receptor alpha, retinoic acid receptor gamma and bone morphogenetic protein-4 genes was also noted in PL5 dietary group at day 16. High levels of dietary PUFA in neutral lipid (NL3 diet) first up-regulated the expression of RXRalpha at day 16 and then down-regulated most of the studied genes at day 23, leading to skeletal abnormalities and death of the larvae. A moderate level of PUFA in neutral lipids up-regulated genes only at day 16, inducing a lesser negative effect on growth, survival and malformation rate than the NL3 group. These results showed that retinoid pathways can be influenced by dietary lipids leading to skeletal malformation during sea bass larvae development.