Unravelling the main immune repertoire of Paracentrotus lividus following Vibrio anguillarum bath challenge.

Paracentrotus lividus is the most abundant echinoid species in the North East Atlantic Ocean and Mediterranean Sea. Although there is abundant genomic information of the species, there is no deep characterisation of the genes involved in the immune response. Here, a reference transcriptome of male and female coelomocytes was produced. The generated P. lividus transcriptome assembly has 203,511 transcripts, N50 transcript length of 1079 bp, and more than 90% estimated gene completeness in Eukaryota and Metazoa BUSCO databases, respectively. Differential gene expression analyses showed 54 and 55 up-regulated genes in P. lividus female and male coelomocyte tissues, respectively. These results suggest a similar immune gene repertoire between sexes. To examine the immune response, P. lividus was challenged with Vibrio anguillarum, one of the candidate pathogens for bald disease. Immune parameters were evaluated at cell and humoral levels, as well as the expression analysis of immune related genes at an early response stage. No differences were found at cellular and humoral levels with the exception of the increase of nitric oxide in perivisceral fluid of challenged animals. At the gene expression level, a total of 2721 genes were upregulated in challenged animals, 13.6 times higher expression than control group. Our analysis revealed that four major KEGG pathways were enriched in challenged animals: Autophagy (KEGG:04140), Endocytosis (KEGG:04144), Phagosome (KEGG:04145) and Protein processing in endoplasmic reticulum (KEGG:04141). Several toll-like receptors (TLR), scavenger receptors cysteine-rich (SRCR) or nucleotide-binding oligomerisation domain like receptors (NLR) were identified as major family genes for pathogen recognition and immune defence. This study provides a valuable transcriptomic resource and unfolds the molecular basis of immune response to V. anguillarum exposure. Overall, our findings contribute to the conservation effort of the P. lividus populations, as well as its sustainable exploitation in an aquaculture context.

Gut physiology of rainbow trout (Oncorhynchus mykiss) is influenced more by short-term fasting followed by refeeding than by feeding fishmeal-free diets.

Supplementing a fishmeal-free diet with yeast extract improves rainbow trout (Oncorhynchus mykiss) growth performance and modulates the hepatic and intestinal transcriptomic response. These effects are often observed in the long term but are not well documented after short periods of fasting. Fasting for a few days is a common practice in fish farming, especially before handling the fish, such as for short sorting, tank transfers, and vaccinations. In the present study, rainbow trout were subjected to a 4-day fast and then refed, for 8 days, a conventional diet containing fishmeal (control diet) or alternative diets composed of terrestrial animal by-products supplemented or not with a yeast extract. During the refeeding period alone, most of the parameters considered did not differ significantly in response to the different feeds. Only the expression of claudin-15 was upregulated in fish fed the yeast-supplemented diet compared to the control diet. Conversely, fasting followed by refeeding significantly influenced most of the parameters analyzed. In the proximal intestine, the surface area of villi significantly increased, and the density of goblet cell tended to decrease during refeeding. Although no distinct plasma immune response or major signs of gut inflammation were observed, some genes involved in the structure, complement pathway, antiviral functions, coagulation, and endoplasmic reticulum stress response of the liver and intestine were significantly regulated by refeeding after fasting. These results indicate that short-term fasting, as commonly practiced in fish farming, significantly alters the physiology of the liver and intestine regardless of the composition of the diet.

Search for new biomarkers of tolerance to Perkinsus olseni parasite infection in Ruditapes decussatus clams.

The grooved carpet shell (Ruditapes decussatus) is a clam species with high economic and social importance in several European and Mediterranean countries. Production of this species suffered a decline caused by biotic (parasite infection) and abiotic factors (environmental factors, stress, poor management methods and intensive culture of the introduced species Ruditapes philippinarum). The protozoan parasite Perkinsus olseni is also responsible for the decline of production, being nowadays one of the major issues for clam culture. Molecular biomarkers that might represent tolerance of R. decussatus to P. olseni have already been uncovered, shedding light in a possible production improvement by selecting those clams with a strongest immune response. In the present study, new tolerance biomarkers to P. olseni infection in R. decussatus were identified. The haemolymph proteomic profiles of naturally non/low-infected (tolerant) and highly-infected (susceptible) clams by the parasite across several heavy affected areas of Europe were characterized through a shotgun proteomics approach. Also, the mechanisms that might be involved in the responses against the disease in chronic infections were explored. Proteins related to energy restoration and balance, metabolic regulation, energy accumulation, ROS production, lysosomal activity, amino acid synthesis, proteolytic activity, iron regulation, iron withholding, and immune response modulation were significantly regulated in susceptible clams. In the tolerant group, proteins related to phagocytosis regulation, control of cell growth and proliferation, gonadal maturation, regulation of apoptosis, growth modulation, response to oxidative stress, iron regulation, shell development and metabolic regulation were significantly expressed. In summary, the protein expression profile of tolerant individuals suggests that an efficient pathogen elimination mechanism coupled to a better metabolic regulation leads to a tolerance to the parasite infection by limiting the spread through the tissues.

Effect of the alveolate parasite Perkinsus olseni infection on sexual maturation and spawning efficiency of the clam Ruditapes decussatus.

The effect of Perkinsus olseni infection on the reproduction ability of clams has been underestimated so far. Although some studies found evidence of reduction of egg production and delay in gonad maturation after infection, the total effect of the infection is still unclear. In this study, Ruditapes decussatus clams from a naïve population were injected with two different doses of P. olseni parasites, a low dose leading to a light infection and a high dose leading to a heavy infection. Clams were maintained during 2 months for maturation, and at the end of the experiment, the spawning was induced, the number of larvae release and mortality were evaluated. During the maturation period, infection level, gonadal stage, condition index, gross biochemical composition and oxidative status of progenitors were evaluated at days 0, 30 and 60 post-injection. The effects of P. olseni infection on clams showed alterations on biochemical parameters, namely lipid peroxidation, a significant mortality and a delayed gonad maturation, with a greater effect in the highly infected individuals. The reproductive capacity of the clams was impaired in both infected groups showing a lower production and a higher mortality rate of larvae. Finally, this study indicates that the production of natural beds with a high prevalence of P. olseni could be compromised by a deregulation of the natural reproduction cycle and a decrease in larvae production by infected animals, probably due to a combination of lower egg production and lower lipid reserves in larvae from infected clams.

Early Molecular Immune Responses of Turbot (Scophthalmus maximus L.) Following Infection with Aeromonas salmonicida subsp. salmonicida.

Turbot aquaculture production is an important economic activity in several countries around the world; nonetheless, the incidence of diseases, such furunculosis, caused by the etiological agent A. salmonicida subsp. salmonicida, is responsible for important losses to this industry worldwide. Given this perspective, this study aimed to evaluate early immune responses in turbot (S. maximus L.) following infection with A. salmonicida subsp. salmonicida. For this, 72 fish were individually weighed and randomly distributed into 6 tanks in a circulating seawater system. For the bacterial challenge, half of the individuals (3 tanks with 36 individuals) were infected using a peritoneal injection with the bacterial suspension, while the other half of individuals were injected with PBS and kept as a control group. Several factors linked to the innate immune response were studied, including not only haematological (white blood cells, red blood cells, haematocrit, haemoglobin, mean corpuscular volume, mean cell haemoglobin, mean corpuscular haemoglobin concentration, neutrophils, monocytes, lymphocytes, thrombocytes) and oxidative stress parameters, but also the analyses of the expression of 13 key immune-related genes (tnf-α, il-1ß, il-8, pparα-1, acox1, tgf-ß1, nf-kB p65, srebp-1, il-10, c3, cpt1a, pcna, il-22). No significant differences were recorded in blood or innate humoral parameters (lysozyme, anti-protease, peroxidase) at the selected sampling points. There was neither any evidence of significant changes in the activity levels of the oxidative stress indicators (catalase, glutathione S-transferase, lipid peroxidation, superoxide dismutase). In contrast, pro-inflammatory (tnf-α, il-1ß), anti-inflammatory (il-10), and innate immune-related genes (c3) were up-regulated, while another gene linked with the lipid metabolism (acox1) was down-regulated. The results showed new insights about early responses of turbot following infection with A. salmonicida subsp. salmonicida.

Dietary supplementation with Gracilaria gracilis by-products modulates the immune status and oxidative stress response of gilthead seabream (Sparus aurata) stimulated with Photobacterium damselae subsp. piscicida.

This study evaluated the effects of agar waste (AW) dietary supplementation, obtained from the seaweed Gracilaria gracilis cultivated under two different spectral lights, neutral (NT) and blue (BL), on haematological parameters, inflammatory response, and antioxidant biomarkers of gilthead seabream (Sparus aurata). Three diets were prepared: i) a basal diet (CTR), ii) a diet supplemented with 2.5% NT, and iii) a diet supplemented with 2.5% BL. After 15 days of feeding, fish were injected with PBS (placebo) or inactivated Photobacterium damselae subsp. piscicida (stimulated) and sampled at 4 h and 24 h post-stimulus. Results indicated that fish fed NT and BL supplemented diets had lower Ht value and mean corpuscular volume (MCV) than fish fed the CTR diet, regardless of the stimulus and the sampling time. No differences in mean corpuscular haemoglobin (MCH) were found between fish fed the different diets, while the mean corpuscular haemoglobin concentration (MCHC) increased in fish fed AW supplemented diets compared to fish fed the CTR diet, regardless of the stimulus and the sampling time. In response to inflammation, fish fed the NT diet displayed higher neutrophils count in blood when compared to the CTR group, regardless of the stimulus and sampling time. Thrombocyte count was higher in fish fed NT and BL diets than in the CTR group, especially in the stimulated fish (Diet*injection (D*I), P = 0.004). An increase in plasma protease activity was detected in fish fed NT or BL diets in both placebo and stimulated fish regardless of the sampling time. Hepatic catalase activity was higher in fish fed the NT and BL than in the CTR group, particularly in the stimulated fish (D*I, P < 0.001). In addition, both stimulated and placebo fish that received the BL diet showed an increase in hepatic GR activity compared to the CTR group, regardless of the sampling time. Dietary supplementation with AW by-products obtained from G. gracilis cultured under NT and BL conditions showed to improve the inflammatory and antioxidant mechanisms in gilthead seabream in response to a UV-killed bacterial stimulus, having valuable applications for the sustainable use of seaweed toward improving the health and welfare of cultured fish.

Evaluation of the Ruditapes decussatus immune response after differential injected doses of Perkinsus olseni.

Ruditapes decussatus is a highly appreciated clam species, with a high demand, especially in European countries. However, natural populations of this species have been affected by over-exploitation, abiotic factors and also by diseases mainly caused by bacteria, virus and protozoans. Within the latter group of pathogens, Perkinsus spp. has been attracting attention for causing relevant mortality rates on bivalves around the world. From all species of this genus, Perkinsus olseni is one of the most impactful for R. decussatus, as it may cause chronic infection with fatal consequences under adverse conditions. Therefore, in this study, naïve clams were injected with two doses of P. olseni in order to better understand the response of R. decussatus against this parasite. So, one of the main goals of this experiment was to evaluate the differential response of the clams at gene expression level after injection and also to monitor the possible mortalities related to parasite. Results indicated that the methods applied for injection and detection of parasite load are effective. Furthermore, altogether, R. decussatus gene expression studies indicate differences on expression of some of the addressed genes among distinct levels of infection over time, which require further studies to confirm if these genes could be used as molecular markers of P. olseni infection. According to the results obtained, Adiponectin-C1q seems to be a promising gene to be used as molecular marker of chronic infection in clams haemolymph.

Chlorella vulgaris Extracts as Modulators of the Health Status and the Inflammatory Response of Gilthead Seabream Juveniles (Sparus aurata).

This study aimed to evaluate the effects of short-term supplementation, with 2% Chlorella vulgaris (C. vulgaris) biomass and two 0.1% C. vulgaris extracts, on the health status (experiment one) and on the inflammatory response (experiment two) of gilthead seabream (Sparus aurata). The trial comprised four isoproteic (50% crude protein) and isolipidic (17% crude fat) diets. A fishmeal-based (FM), practical diet was used as a control (CTR), whereas three experimental diets based on CTR were further supplemented with a 2% inclusion of C. vulgaris biomass (Diet D1); 0.1% inclusion of C. vulgaris peptide-enriched extract (Diet D2) and finally a 0.1% inclusion of C. vulgaris insoluble fraction (Diet D3). Diets were randomly assigned to quadruplicate groups of 97 fish/tank (IBW: 33.4 ± 4.1 g), fed to satiation three times a day in a recirculation seawater system. In experiment one, seabream juveniles were fed for 2 weeks and sampled for tissues at 1 week and at the end of the feeding period. Afterwards, randomly selected fish from each group were subjected to an inflammatory insult (experiment two) by intraperitoneal injection of inactivated gram-negative bacteria, following 24 and 48 h fish were sampled for tissues. Blood was withdrawn for haematological procedures, whereas plasma and gut tissue were sampled for immune and oxidative stress parameters. The anterior gut was also collected for gene expression measurements. After 1 and 2 weeks of feeding, fish fed D2 showed higher circulating neutrophils than seabream fed CTR. In contrast, dietary treatments induced mild effects on the innate immune and antioxidant functions of gilthead seabream juveniles fed for 2 weeks. In the inflammatory response following the inflammatory insult, mild effects could be attributed to C. vulgaris supplementation either in biomass form or extract. However, the C. vulgaris soluble peptide-enriched extract seems to confer a protective, anti-stress effect in the gut at the molecular level, which should be further explored in future studies.

Tryptophan Modulatory Role in European Seabass (Dicentrarchus labrax) Immune Response to Acute Inflammation under Stressful Conditions.

The present work aimed to study the role of dietary tryptophan supplementation in modulating the European seabass (Dicentrarchus labrax) immune condition during stressful rearing conditions (i.e., 15 days exposure to high density), as well as the immune response to acute inflammation after intraperitoneal injection of a bacterial pathogen. Stress alone did not compromise seabass health indicators. In contrast, a clear peripheral and local inflammatory response was observed in response to the inoculated bacteria. Moreover, exposure to a high stocking density seemed to exacerbate the inflammatory response at early sampling points, compared to fish stocked at a lower density. In contrast, stressed fish presented some immune-suppressing effects on the T-cell surface glycoprotein receptor expressions at a late sampling point following inflammation. Regarding the effects of dietary tryptophan, no changes were observed on seabass immune indicators prior to inflammation, while a small number of immunosuppressive effects were observed in response to inflammation, supporting tryptophan's role in the promotion of immune-tolerance signals during inflammation. Nonetheless, tryptophan dietary supplementation improved the inflammatory response against a bacterial pathogen during stressful conditions, supported by a reduction of plasma cortisol levels, an up-regulation of several immune-related genes at 48 h, and an inversion of the previously observed, stress-induced T-cell suppression. Finally, the involvement of tryptophan catabolism in macrophages was confirmed by the up-regulation of genes involved in the kynurenine pathway. The present study brings new insights regarding the immune modulatory role of tryptophan during stressful conditions in fish, thus allowing for the development of novel prophylactic protocols during vaccination by intraperitoneal injection in the European seabass.

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida.

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Functional and Molecular Immune Response of Rainbow Trout (Oncorhynchus mykiss) Following Challenge with Yersinia ruckeri.

Currently, aquaculture production of rainbow trout (Oncorhynchus mykiss) is a multibillion dollar industry; nevertheless, the development of this sector has not been exempt from pitfalls related to the recurrent presence of pathogens of bacterial origin. This is the case of Yersinia ruckeri, the etiologic agent of the infectious pathology known as Enteric Red Mouth Disease (ERM), causing serious economic losses that can be as high as 30-70% of production. Although several studies have been performed regarding pathogen features and virulence factors, more information is needed about the host defense mechanism activation after infection. Given this perspective, this study aimed to evaluate rainbow trout's short-term innate immune response against infection with Y. ruckeri. A series of factors linked to the innate immune response were evaluated, including determination of hematological parameters, oxidative stress biomarkers, and analysis of the expression of immune-related genes. Results showed a significant decrease in several hematological parameters (white blood cell count, hematocrit, neutrophils, monocytes, lymphocytes, and thrombocytes) and oxidative stress indicators (SOD) between the control and infected groups. In addition, there were significant differences in the level of gene expression between infected individuals and the control group. Most of these genes (il-1ß, il-8, il-10, tnf-α1, tnf-α2, socs3, mmp-9, cath, hsp-70, saa, fer, pcb) were upregulated within the first 24 h following infection. Results from this study showed more insights into the short-term immune response of rainbow trout to infection with Y. ruckeri, which may be useful for the establishment of biomarkers that may be used for the early detection of ERM.

Dietary methionine as a strategy to improve innate immunity in rainbow trout (Oncorhynchus mykiss) juveniles.

Methionine ability to enhance fish immune status and inflammatory response by the modulation of methionine-related pathways has been verified in several fish species. However, no attention has been given to the role of methionine as an immune-modulatory additive in rainbow trout (Oncorhynchus mykiss). Hence, this study was designed to evaluate the effect of short-term feeding a diet supplemented with dl-methionine on the rainbow trout immune status. For this purpose, two diets were formulated: a control (CTRL) diet including the AA profile required to meet the ideal pattern estimated for rainbow trout; and an experimental diet (MET) identical to the CTRL but supplemented with dl-methionine two fold above its requirement level. After 2 and 4 weeks, fish haematological profile, peripheral cell populations, plasma and skin mucus humoral immune parameters as well as head-kidney gene expression were analysed. Results showed that methionine was able to improve peripheral neutrophil numbers after 4 weeks of feeding while reducing the expression of pro-inflammatory genes (i.e. IL1ß and IL8). Also, indications of fish physiological ability to regulate polyamine biosynthesis were found by the reduced expression of the spermine synthase enzyme (SMS). Together, these results point to some level of enhancement of the cellular-related innate immune status by dietary dl-methionine supplementation after a short-term feeding period, which could be used as a prophylactic strategy for rainbow trout health management.

Immune Status and Hepatic Antioxidant Capacity of Gilthead Seabream Sparus aurata Juveniles Fed Yeast and Microalga Derived ß-glucans.

This work aimed to evaluate the effects of dietary supplementation with ß-glucans extracted from yeast (Saccharomyces cerevisiae) and microalga (Phaeodactylum tricornutum) on gene expression, oxidative stress biomarkers and plasma immune parameters in gilthead seabream (Sparus aurata) juveniles. A practical commercial diet was used as the control (CTRL), and three others based on CTRL were further supplemented with different ß-glucan extracts. One was derived from S. cerevisiae (diet MG) and two different extracts of 21% and 37% P. tricornutum-derived ß-glucans (defined as Phaeo21 and Phaeo37), to give a final 0.06% ß-glucan dietary concentration. Quadruplicate groups of 95 gilthead seabream (initial body weight: 4.1 ± 0.1 g) were fed to satiation three times a day for 8 weeks in a pulse-feeding regimen, with experimental diets intercalated with the CTRL dietary treatment every 2 weeks. After 8 weeks of feeding, all groups showed equal growth performance and no changes were found in plasma innate immune status. Nonetheless, fish groups fed ß-glucans supplemented diets showed an improved anti-oxidant status compared to those fed CTRL at both sampling points (i.e., 2 and 8 weeks). The intestinal gene expression analysis highlighted the immunomodulatory role of Phaeo37 diet after 8 weeks, inducing an immune tolerance effect in gilthead seabream intestine, and a general down-regulation of immune-related gene expression. In conclusion, the results suggest that the dietary pulse administration of a P. tricornutum 37% enriched-ß-glucans extract might be used as a counter-measure in a context of gut inflammation, due to its immune-tolerant and anti-oxidative effects.

Dietary methionine supplementation improves the European seabass (Dicentrarchus labrax) immune status following long-term feeding on fishmeal-free diets.

Methionine is a limiting amino acid (AA) in fish diets, particularly in those containing high levels of plant protein (PP), and is key in the immune system. Accordingly, outcome on the fish immune mechanisms of methionine-deficient and methionine-supplemented diets within the context of 0 % fishmeal formulation, after a short and prolonged feeding period, was studied in European seabass (Dicentrarchus labrax). For this, seabass juveniles were fed a (i) fishmeal-free diet, meeting AA requirements, but deficient in methionine (MET0·65); (ii) as control, the MET0·65 supplemented with l-methionine at 0·22 % of feed weight (CTRL); (iii) two diets, identical to MET0·65 but supplemented at 0·63 and 0·88 % of feed weight of l-methionine (MET1·25 and MET1·5, respectively); and (iv) a fishmeal-based diet (FM), as positive control. After 2 and 12 weeks of feeding, blood and plasma were sampled for leucocyte counting and humoral parameter assays and head-kidney collected for gene expression. After 2 weeks of feeding, a fishmeal-free diet supplemented with methionine led to changes in the expression of methionine- and leucocyte-related genes. A methionine immune-enhancer role was more evident after 12 weeks with an increased neutrophil percentage and a decreased expression of apoptotic genes, possibly indicating an enhancement of fish immunity by methionine dietary supplementation. Furthermore, even though CTRL and FM present similar methionine content, CTRL presented a reduced expression of several immune-related genes indicating that in a practical PP-based diet scenario, the requirement level of methionine for an optimal immune status could be higher.

Dietary arginine and citrulline supplementation modulates the immune condition and inflammatory response of European seabass.

The present study was designed to determine the modulatory effects of arginine and citrulline dietary supplementation on the immune condition and inflammatory response of European seabass, Dicentrarchus labrax. Four diets were manufactured: a control diet (CTRL) was formulated to meet the indispensable amino acids profile established for seabass. Based on this formulation, three other diets were supplemented with l-arginine at two different levels (0.5% and 1%, ARG1 and ARG2, respectively) and l-citrulline at 0.5% (CIT). Fish were fed these diets for 2 or 4 weeks under controlled conditions. At the end of 4 weeks, fish from all dietary treatments were intraperitoneally-injected with Photobacterium damselae piscicida and sampled after 4, 24 our 48 h. Immune status was characterized by a lymphocyte time-dependent decrease regardless of dietary treatment, whereas peroxidase values dropped in time in fish fed ARG1 and ARG2 and was lower at 4 weeks in fish fed ARG1 than in fish fed CTRL. Up-regulation of several genes was more evident in ARG1-and CIT-fed fish, though pro-inflammatory cytokines were down-regulated by CIT dietary treatment. Following immune stimulation, seabass fed ARG1 showed a decrease in neutrophils and monocytes circulating numbers. On the other hand, expression of 17 selected immune and inflammatory responses genes was barely affected by dietary treatments. Based on the analyzed parameters, results suggest an active role of dietary arginine/citrulline supplementation in modulating immune defences that seem to translate into a suppressed immune repertoire, mostly at the cell response level. The observed changes due to citrulline dietary supplementation were in part similar to those caused by arginine, suggesting that citrulline might have been used by macrophages as an arginine precursor and then engaged in similar immune-impairment leading mechanisms.

New Perspectives Related to the Bioluminescent System in Dinoflagellates: Pyrocystis lunula, a Case Study.

Pyrocystis lunula is considered a model organism due to its bioluminescence capacity linked to circadian rhythms. The mechanisms underlying the bioluminescent phenomenon have been well characterized in dinoflagellates; however, there are still some aspects that remain an enigma. Such is the case of the presence and diversity of the luciferin-binding protein (LBP), as well as the synthesis process of luciferin. Here we carry out a review of the literature in relation to the molecular players responsible for bioluminescence in dinoflagellates, with particular interest in P. lunula. We also carried out a phylogenetic analysis of the conservation of protein sequence, structure and evolutionary pattern of these key players. The basic structure of the luciferase (LCF) is quite conserved among the sequences reported to date for dinoflagellate species, but not in the case of the LBP, which has proven to be more variable in terms of sequence and structure. In the case of luciferin, its synthesis has been shown to be complex process with more than one metabolic pathway involved. The glutathione S-transferase (GST) and the P630 or blue compound, seem to be involved in this process. In the same way, various hypotheses regarding the role of bioluminescence in dinoflagellates are exposed.

Dietary tryptophan supplementation induces a transient immune enhancement of gilthead seabream (Sparus aurata) juveniles fed fishmeal-free diets.

European aquaculture is an industry with a high sustainability profile contributing to the supply of safe seafood. However, several diseases can affect farmed fish and it is imperative to find alternatives for chemotherapeutic treatments when disease outbreaks occur. Maintenance of health through nutrition is well-establish in modern animal farming, and amino acids (AA) are promising candidates as functional additives to improve fish health. Therefore, the goal of this research is to provide a better understanding of the influence of tryptophan supplementation on nutritional condition and immune mechanisms in fish. Triplicate groups of fish (13.3 ±â€¯0.3g) previously fed with a fishmeal-based diet were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the SUP diet, formulated as the CTRL with an increase in tryptophan (TRP) content. After 2 and 13 weeks of feeding, head-kidney (HK), liver (L) and white skeletal muscle (WSM) were collected for gene expression, whereas plasma was suited for humoral immune parameters. A holistic approach using transcriptomic, humoral and zootechnical parameters was undertaken. The expression of 29-31 genes for WSM, L or HK confirms an effect due to the treatment across time. A two-way ANOVA analysis revealed that 15-24 genes varied significantly depending on the tissue, and the multivariate analysis by means of PLS-DA explained (R2) and predicted (Q2) with four components up to 93% and 78% of total variance, respectively. Component 1 (R2 = 50.06%) represented the time effects, whereas components 2 (24.36%) and 3 (13.89%) grouped fish on the basis of dietary treatment, at early sampling. The HK results in particular suggest that fish fed SUP diet displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, despite an increase in antiprotease and peroxidase activities after 13 weeks regardless of dietary treatment. These results suggest that tryptophan supplementation may improve the seabream immune status after 2 weeks. Hence, the use of functional feeds is especially relevant during a short-term feeding period before a predictable stressful event or disease outbreak, considering that these putative advantageous effects seem to disappear after a 13 weeks feeding period.

Isolation and characterization of native probiotics for fish farming.

BACKGROUND: Innovations in fish nutrition act as drivers for the sustainable development of the rapidly expanding aquaculture sector. Probiotic dietary supplements are able to improve health and nutrition of livestock, but respective bacteria have mainly been isolated from terrestrial, warm-blooded hosts, limiting an efficient application in fish. Native probiotics adapted to the gastrointestinal tract of the respective fish species will establish within the original host more efficiently. RESULTS: Here, 248 autochthonous isolates were cultured from the digestive system of three temperate flatfish species. Upon 16S rRNA gene sequencing of 195 isolates, 89.7% (n = 175) Gram-negatives belonging to the Alpha- (1.0%), Beta- (4.1%) and Gammaproteobacteria (84.6%) were identified. Candidate probiotics were further characterized using in vitro assays addressing 1) inhibition of pathogens, 2) degradation of plant derived anti-nutrient (saponin) and 3) the content of essential fatty acids (FA) and their precursors. Twelve isolates revealed an inhibition towards the common fish pathogen Tenacibaculum maritimum, seven were able to metabolize saponin as sole carbon and energy source and two isolates 012 Psychrobacter sp. and 047 Paracoccus sp. revealed remarkably high contents of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Furthermore, a rapid and cost-effective method to coat feed pellets revealed high viability of the supplemented probiotics over 54 d of storage at 4°C. CONCLUSIONS: Here, a strategy for the isolation and characterization of native probiotic candidates is presented that can easily be adapted to other farmed fish species. The simple coating procedure assures viability of probiotics and can thus be applied for the evaluation of probiotic candidates in the future.

Humoral and mucosal immune responses in meagre (Argyrosomus regius) juveniles fed diets with varying inclusion levels of carob seed germ meal.

Many studies have assessed the effects of incorporation of plant feedstuffs in fish diets on growth performance, whereas few studies have addressed the effects of fish meal replacement by plant protein sources on fish immune parameters. Thus, the aim of this study was to evaluate the effects on immune response of different inclusion levels of carob seed germ meal (CSGM) as partial replacement for fish meal in diets for meagre (Argyrosomus regius) juveniles. Fish were fed four experimental diets with increased CSGM inclusion levels [0% (control), 7.5% (CSGM7.5), 15% (CSGM15) and 22.5% (CSGM22.5)]. After 1, 2, and 8 weeks of feeding fish were sampled to determine haematological profile and several humoral parameters in plasma and intestine. Results showed that dietary inclusion of CSGM did not negatively affect the immune parameters of meagre. In addition, total numbers of red and white blood cells, as well as thrombocytes, lymphocytes, monocytes, and neutrophils counts were not affected by dietary treatments. All parameters evaluated in plasma were unaffected by dietary CSGM inclusion after 1 and 2 weeks of feeding, with only the haemolytic complement activity showing an increase in fish fed diets with CSGM after 1 week and in fish fed CSGM22.5 diet after 2 weeks. Regarding the innate immune parameters analysed in the intestine, it could be highlighted the increase in alkaline phosphatase and antiprotease activities in fish fed the diet with the higher inclusion of CSGM at 8 weeks. Overall, results suggest that high dietary CSGM inclusion do not compromise immune status or induce an inflammatory response in meagre juveniles.

Acute hyperoxia induces systemic responses with no major changes in peripheral tissues in the Senegalese sole (Solea senegalensis Kaup, 1858).

Senegalese sole Solea senegalensis is currently farmed in recirculation aquaculture systems that often involve water re-oxygenation, which in turn may cause acute or prolonged hyperoxia exposures. In order to understand the impact of acute hyperoxia on the fish immune system and peripheral tissues such as gills and gut, Senegalese sole juveniles (30.05 ±â€¯1.72 g) were exposed to normoxia (100% O2sat) as control and two hyperoxic conditions (150 and 200% O2sat) and sampled at 4 and 24 h. Fish haematological profile, total and differential blood cell counts and plasma immune parameters were analysed. Histomorphology and immunofluorescence analyses of gills and intestine were performed, respectively, whereas head-kidney samples were used for assessing the expression of immune-related genes. Results indicate that acute hyperoxia exposure may reduce fish erythrocyte and haemoglobin levels. Moreover, decreases in total leucocytes numbers, circulating lymphocytes, monocytes, alternative complement pathway activity and expression of cyclooxygenase-2 were observed in fish exposed to hyperoxia. In contrast, hyperoxia did not induce major effects on gill histomorphology nor in the protein content of ion and glucose cotransporters as well as a macrophage marker (V-ATPase) in the intestine. Although the activation of humoral mechanisms and immune-related genes were not dramatically affected by acute hyperoxia, the compromised immune cell status and the reduction of some inflammatory indicators are issues to consider under acute hyperoxia conditions.