Aquaculture is an essential economic activity worldwide, supplying the requirements of various nations. The larval stage is one of the most critical periods during the production cycle in aquaculture. To contribute to amplifying the knowledge related to viable methods to enhance this activity, the present study investigated the effects of prebiotic fructooligosaccharide (FOS) and the synthetic peptide GHRP-6 in the growth of fish, as well as its repercussions on a series of digestive enzyme activities and immune response parameters. Tilapias (Oreochromis niloticus) larvae were divided into three groups: group 1, fed with pre-formulated feed; group 2, fed with feed + FOS; group 3, fed with feed + GHRP-6. We measured the gain in size and weight at 72 and 87 days after starting the diets. At the end of treatment, we continue feeding the animals with a standard diet using the pre-formulated feed. We measured immune system parameters and digestive enzyme activities on larvae homogenate, focusing on lysozyme and antiprotease activity and α-amylase, trypsin and esterase activity, respectively. Results show a significant increment in size and weight in those fish fed with feed + FOS. Meanwhile, the primary enzyme activity was detected in those fish fed with feed + GHRP-6. On rough terms, both food combinations stimulated the immune system. Our study suggests that FOS and GHRP-6 could be desirable additives to incorporate into the tilapia larvae feeding and increase production efficiency. Keywords: aquaculture, digestive enzymes activity, FOS, GHRP-6, immune system, larvae

Teleost IgT/Z plays a principal role in the defense mechanisms against infectious agents in the mucosal compartments and in systemic immunity. Previously, Nile tilapia (Oreochromis niloticus) IgT was discovered and characterized at transcription level. In this work, we generated a monoclonal antibody (mAb) that specifically recognized the Nile tilapia IgT. BALB/c mice were immunized with three synthetic peptides conjugated to KLH. The sequences of these peptides derived from the constant region of the Nile tilapia IgT heavy chain. ELISA and Western blotting confirmed the specificity of the polyclonal sera and the culture supernatant from a positive hybridoma clone. We observed immunoreactivity against a recombinant IgT fragment and native IgT in skin mucus. The anti-IgT mAb did not cross-react with purified tilapia IgM. Direct ELISA analysis allowed the quantification of skin mucus IgM and IgT concentrations. Flow cytometry analysis revealed differences in the percentage of IgT+ B cell populations between juveniles and adults in peripheral blood, head kidney and spleen lymphocytes and among the tissues analyzed. For further validation of the anti-IgT mAb utility, a recombinant vaccine candidate against sea lice (TT-P0 Ls) was injected into juvenile tilapia. Direct ELISA results revealed a differential secretion of skin mucus IgT and IgM after immunostimulation. In addition, the percentages of IgT+ B cells were determined at 7 days after booster and ex-vivo stimulation by flow cytometry. This mAb constitutes an important immunological tool to study the biological function and structural characteristics of tilapia IgT.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide belonging to the glucagon/secretin superfamily. Its role in fish growth and immunity has been previously demonstrated using different administration routes such as injection or immersion bath. The present study evaluated the physiological effects of a new feed formulation containing Clarias gariepinus synthetic PACAP-38 administered to rainbow trout (Oncorhynchus mykiss) fingerlings. With this aim, a new oiled formulation containing the peptide was developed and two diets with different PACAP concentrations were prepared. After two months of administration, dietary supplementation with PACAP markedly improved the PUFA content in muscle, increased the total number of caliciform cells within the intestinal villi and modulated the transcription of some of the studied cytokines in head kidney. This study suggests new potential outcomes in further advancing of the use of small peptides as feed additives and formulations to improve the quality of culture fish fingerlings.

Growth Hormone-Releasing Peptide 6 (GHRP-6) (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2) is an agonist of the growth hormone secretagogue receptor. GHRP-6 mimics the effect of ghrelin. The present study focuses on the immunomodulatory effects of GHRP-6 in tilapia with and without the presence of Pseudomonas aeruginosa infection. GHRP-6 up-regulated the transcription levels of three piscidin-like antimicrobial peptides (Oreochromicins I, II, and III) and granzyme in a tissue-dependent manner. Antimicrobial activity stimulation in serum (lysozyme and anti-protease activity) was also confirmed. Besides, GHRP-6 enhanced the in vitro antimicrobial activity against P. aeruginosa in tilapia gills mucus and serum samples and decreased the bacterial load in vivo after infection with this Gram-negative bacterium. Our results evidenced, for the first time, a direct link between a growth hormone secretagogue ghrelin mimetic in fish and the enhancement of antimicrobial peptides transcription, which suggests that this secretagogue is capable to lead the activation of microbicidal activity in tilapia. Thus, these results open new possibilities for GHRP-6 application in aquaculture to stimulate the teleost immune system as an alternative treatment against opportunistic bacteria.

Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide based strategy. The peptide sequence was selected from the surface-exposed region between CH3-CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.

Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebrates, by protecting the organism from a wide variety of pathogens. Nile tilapia (Oreochromis niloticus) is extensively cultivated worldwide, with a strong established market demand. It constitutes one of the model species for the study of fish immunology and its genome is currently fully sequenced. The presence of the immunoglobulin M gene in this species is well documented, as well as its major role in systemic immunity. To date, the IgT gene from O. niloticus has not been identified and, therefore, no information is available on the role of this immunoglobulin isotype in the immune response in tilapia. In the present work, novel secreted and membrane immunoglobulin T isotypes and a fragment of IgM were isolated from tilapia head kidney lymphocytes. Their transcriptional profiles were analyzed by quantitative PCR in larval development and in different tissues of healthy or lipopolysaccharide/Edwardsiella tarda-challenged tilapia adults. The presence of IgT and IgM were detected in early stages of larval development. Additionally, these genes exhibited differential expression profiles in basal conditions and after E. tarda infection in adult tilapia, in accord with the proposed effector functions of these immunoglobulins in the systemic and mucosal compartments. Our results suggest the potential involvement of this new Ig in mucosal immunity in tilapia.

Growth hormone (GH) release is a process that is well regulated by several factors, including GH secretagogues. GH can mediate the regulation of the fatty acid level and composition. The aim of this study was to determine the effect of a synthetic GH secretagogue peptide (A233) on the growth and fatty acid composition in tilapia (Oreochromis niloticus). To address this objective, we administrated a diet supplemented with A233 to juvenile tilapia for 60 days. The group fed with a diet supplemented with 600 μg of A233 per kg of feed increased in weight (4.81 ± 0.09 g) and specific growth rate (2.49 ± 0.03%/day) compared to the control diet group (3.63 ± 0.08 g, 2.07 ± 0.04%/day; respectively) (p < 0.001). In the muscle, the total lipids for the control diet group were higher than that in the group fed with 600 μg of A233 per kg feed; however, no differences were detected in the liver. In both tissues, the patterns of fatty acid composition and content were generally similar, with some exceptions. Tilapia fed with 600 μg of A233 per kg of feed showed, in liver and muscle, a significantly higher composition and content of n-3 polyunsaturated fatty acids (such as 20:5n-3, 22:5n-3, 22:6n-3) and n-3/n-6 PUFA than animals fed with the control diet. To our knowledge, this is the first report on the the effects of natural or synthetic GH secretagogues (GHS) on fatty acid composition, implying an increase in the nutritional quality of the tilapia.

Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. This cytokine plays a very important role in defining Th1 immune response in all vertebrates. In the present study, we identified and isolated for the first time the gene coding for Nile tilapia (Oreochromis niloticus) IFNγ from spleen lymphocytes. The isolated tilapia IFNγ has between 24 and 62% of amino acid identity as compared to reported sequences for other teleost fishes. It has close phylogenetic relationships with IFNγ molecules belonging to the group of Perciforms and presents the typical structural characteristics of gamma interferon molecules. The tissue expression analysis showed that IFNγ is expressed constitutively in head kidney, skin, intestine, muscle and brain. Its expression was not detected in gills by conventional RT-PCR. However, under conditions of stimulation with Poly I:C and LPS, IFNγ expression was up-regulated in gills after 24 h post-stimulation. IFNγ expression was also induced in gills 24 h after Edwardsiella tarda infection suggesting its important role in immunity against intracellular bacteria. The recombinant protein produced in Escherichia coli induced Mx gene transcription in head kidney primary culture cells. These results are the first steps to characterize the role of tilapia IFNγ in the defense against pathogens in tilapia. Furthermore, the isolation of this molecule provides a new tool to characterize the cellular immune response to various stimuli in this organism.

Modern subunit vaccines have excellent safety profiles and improved tolerability, but do not elicit strong immune responses without the addition of adjuvants. Developing a safe and affective adjuvant remains a challenge for peptide-based vaccine design. Growth Hormone Releasing Peptide-6 (GHRP-6) is one of the earliest-developed, synthetic, peptidyl growth hormone secretagogue receptor agonists. These compounds mimic the effect of the endogenous ligand, ghrelin. In the present study, we evaluated the ability of GHRP-6 to enhance the humoral immune response against co-injected antigens in mice, tilapia and African catfish. This peptide was able to increase the antigen-specific antibody response using heterologous proteins and peptides as antigens, which were also formulated in "water in oil" emulsions (Freund and Montanide). As long as we know there is no previous report describing any ghrelin analogous as molecular immunomodulator stimulating a humoral immune response. Further studies will be conducted to evaluate the functionality of this humoral immune response in challenge trials.

Aquaculture is part of the solution for meeting the growing world demand for food, mainly as an animalprotein source. However, current yields are insufficient for aquaculture to play this crucial role. Growth-rateenhancement is one of the approaches that have been exploited in this regard. Also, losses caused by bacteria,viruses and parasites remain a significant problem. It has been demonstrated that growth stimulants contributenot only to growth enhancement but also to fish health improvement. Growth Hormone Releasing Peptide-6(GHRP-6) is one of the earliest-developed, synthetic, peptidyl growth hormone secretagogue receptoragonists. These compounds mimic the effect of the endogenous ligand, ghrelin. This peptide has shown itsbenefits for both growth enhancement and immune system stimulation in fish and crustaceans. In the presentstudy, we demonstrate that intraperitoneal administration of GHRP-6 induces liver insulin growth factor-Imessenger RNA, and increases growth hormone levels in juvenile tilapia (Oreochromis sp.), in a time-courseexperiment. In addition, administration of GHRP-6 in formulated feed to tilapia larvae was assessed. Growthand immune parameters such as lectin titers and intestinal intraepithelial lymphocyte numbers were increasedin treated larvae. We also evaluated the effect of GHRP-6 injection over feed intake in shrimp and its effectson shrimp growth when the peptide was administrated by successive immersion baths. GHRP-6 increasedfeed intake, body weight and size, the number of rostral spines and gill branches, protein concentration andhaemocyte number in treated shrimps.

Aquaculture is part of the solution for meeting the growing world demand for food, mainly as an animal protein source. However, current yields are insufficient for aquaculture to play this crucial role. Growth-rate enhancement is one of the approaches that have been exploited in this regard. Also, losses caused by bacteria, viruses and parasites remain a significant problem. It has been demonstrated that growth stimulants contribute not only to growth enhancement but also to fish health improvement. Growth Hormone Releasing Peptide-6 (GHRP-6) is one of the earliest-developed, synthetic, peptidyl growth hormone secretagogue receptor agonists. These compounds mimic the effect of the endogenous ligand, ghrelin. This peptide has shown its benefits for both growth enhancement and immune system stimulation in fish. In the present study, we demonstrate that intraperitoneal administration of GHRP-6 induces liver insulin growth factor-I messenger RNA, and increases growth hormone levels in juvenile tilapia (Oreochromis sp.), in a time-course experiment. GHRP-6 also stimulates growth using non-encapsulated and encapsulated peptide administered by a plastic tube to the pharyngeal cavity to juveniles. In addition, administration of GHRP-6 in formulated feed to tilapia larvae was assessed. Growth and immune parameters such as lectin titers and intestinal intraepithelial lymphocyte numbers were increased in treated larvae. Based on these results, GHRP-6 could be an interesting candidate for development into a new biotechnology product for improving fish farming.

In teleost fish, secretion of the growth hormone (GH) is regulated by several hypothalamic factors that are influenced by the physiological state of the animal. GH in fish is involved in many physiological processes that are not overtly growth-related such as: saltwater osmoregulation, antifreeze protein synthesis, and the regulation of sexual maturation and immune functions. This study was conducted to characterize a decapeptide A233 (GKFDLSPEHQ) designed by molecular modeling to evaluate its function as a GH secretagogue (GHS). In pituitary cell culture, the peptide A233 induces GH secretion and it is also able to increase superoxide production in tilapia head-kidney leukocyte cultures. This effect is blocked by preincubation with the GHS receptor antagonist [D-Lys3]-GHRP6. Immunoneutralization of GH by addition of anti-tilapia GH monoclonal antibody blocked the stimulatory effect of A233 on superoxide production. These experiments suggest a GH-mediated mechanism for the action of A233. The in vivo biological action of the decapeptide was also demonstrated for growth stimulation in goldfish and tilapia larvae (p < 0.001). Superoxide dismutase levels, antiprotease activity, and lectin titer were enhanced in tilapia larvae treated with this novel molecule. The decapeptide A233 is able to function as GHS in teleosts and enhance parameters of the innate immune system in the fish larvae. This study won the Annual Award of the Academy of Sciences of Cuba in 2012.

In teleost fi sh, secretion of the growth hormone (GH) is regulated by several hypothalamic factors that are infl uenced by the physiological state of the animal. GH in fi sh is involved in many physiological processes that are not overtly growth-related such as: saltwater osmoregulation, antifreeze protein synthesis, and the regulation of sexual maturation and immune functions. This study was conducted to characterize a decapeptide A233 (GKFDLSPEHQ) designed by molecular modeling to evaluate its function as a GH secretagogue (GHS). In pituitary cell culture, the peptide A233 induces GH secretion and it is also able to increase superoxide production in tilapia head-kidney leukocyte cultures. This effect is blocked by preincubation with the GHS receptor antagonist [D-Lys3]-GHRP6. Immunoneutralization of GH by addition of anti-tilapia GH monoclonal antibody blocked the stimulatory effect of A233 on superoxide production. These experiments suggest a GH-mediated mechanism for the action of A233. The in vivo biological action of the decapeptide was also demonstrated for growth stimulation in goldfi sh and tilapia larvae (p < 0.001). Superoxide dismutase levels, antiprotease activity, and lectin titer were enhanced in tilapia larvae treated with this novel molecule. The decapeptide A233 is able to function as GHS in teleosts and enhance parameters of the innate immune system in the fi sh larvae. This study won the Annual Award of the Academy of Sciences of Cuba in 2012.

In teleosts fish, secretion of GH is regulated by several hypothalamic factors that are influenced by the physiological state of the animal. There is an interaction between immune and endocrine systems through hormones and cytokines. GH in fish is involved in many physiological processes that are not overtly growth related, such as saltwater osmoregulation, antifreeze synthesis, and the regulation of sexual maturation and immune functions. This study was conducted to characterize a decapeptide compound A233 (GKFDLSPEHQ) designed by molecular modeling to evaluate its function as a GH secretagogue (GHS). In pituitary cell culture, the peptide A233 induces GH secretion and it is also able to increase superoxide production in tilapia head-kidney leukocyte cultures. This effect is blocked by preincubation with the GHS receptor antagonist [d-Lys(3)]-GHRP6. Immunoneutralization of GH by addition of anti-tilapia GH monoclonal antibody blocked the stimulatory effect of A233 on superoxide production. These experiments propose a GH-mediated mechanism for the action of A233. The in vivo biological action of the decapeptide was also demonstrated for growth stimulation in goldfish and tilapia larvae (P<0.001). Superoxide dismutase levels, antiprotease activity, and lectin titer were enhanced in tilapia larvae treated with this novel molecule. The decapeptide A233 designed by molecular modeling is able to function as a GHS in teleosts and enhance parameters of the innate immune system in the fish larvae.

To date, many technologies have been developed to increase efficiency in aquaculture, but very few successful biotechnology molecules have arrived on the market. In this context, marine biotechnology has an opportunity to develop products to improve the output of fish in aquaculture. Published in vivo studies on the action of the pituitary adenylate cyclase-activating polypeptide (PACAP) in fish are scarce. Recently, our group, for the first time, demonstrated the biological role of this neuropeptide administrated by immersion baths in the growth and development of larval fish. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on growth performance and feeding behavior in juvenile fish. Our results showed the physiological role of this peptide for growth control in fish, including the juvenile stage, and confirm that its biological functions are well conserved in fish, since C. gariepinus PACAP stimulated growth in juvenile tilapia Oreochromis niloticus. In addition, we have observed that the growth-promoting effect of PACAP in juvenile tilapia was correlated with higher GH concentration in serum. With regard to the neuroendocrine regulation of growth control by PACAP, it was demonstrated that PACAP stimulates food intake in juvenile tilapia. In general, PACAP appears to act in the regulation of the growth control in juvenile fish. These findings propose that PACAP is a prominent target with the potential to stimulate fish growth in aquaculture.

In a previous study, we unexpectedly found that tilapia growth hormone (tiGH) secreted to the culture media by transformed cells of the yeast Pichia pastoris lacks 46 amino acids from the C-terminal end. In the present study, we cloned the exact fragment that code for this truncated variant and demonstrated its growth promoting activity in goldfish when it's administered by immersion bath. Furthermore, a better characterization of this polypeptide was performed. Administration of the polypeptide derived from tiGH increased superoxide anion production and has a mitogenic effect on peripheral blood leukocytes. This molecule binds to liver membranes proteins in vitro in a saturable manner. Beside, we cloned and expressed tiGH and its truncated variant in mammalian cells using the signal peptide of this hormone and we observed that the secretion was drastically reduced in the truncated tiGH as compared to the intact molecule. Truncated tilapia growth hormone lacking the helix 4 and two disulfide loops is still a bioactive hormone, suggesting that the disulfide bonds and the helix 4 are not essential for the biological activities examined in this work. However, the growth hormone C-terminal portion seems to be essential for this hormone to be secreted by cultured cells in vitro.

The manipulation of the growth characteristics of aquatic organisms constitutes a high priority for the biotechnological industry, given the expected worldwide increase in food demand and the current food shortages, especially in animal protein, for significant segments of the population. The stimulation of growth, aimed at reducing the extension of harvest cycles for aquaculture and at decreasing the high mortality typical of larval stages in fish; as well as the use of immunostimulants to allow aquacultural populations to cope with the conditions of intensive culture, are two of the main targets of modern aquacultural biotechnology. Recent data about the immuneendocrine connection in fish have shown that growth hormone has a stimulatory effect on several parameters of the innate immune system. Therefore, the use of biotechnological means for the stimulation of growth hormone and, consequently, the innate immune system, is a promising mean for fulfilling this goal. The present work summarizes our study of the influence of the pituitary adenylate cyclase-activating peptide (PACAP) and the PACAPrelated peptide (PRP) from Clarias gariepinus, as well as neuropeptide Y from Oreochromis sp. on the growth characteristics and innate immune system of fish. The results show that these neuropeptides not only have growthpromoting and development-related effects, but also stimulate several elements of the innate immune response such as lysozyme, lectins, nitric oxide and anti-oxidative defenses (catalase, superoxide dismutase and reduced glutathione levels).

Growth hormone (GH) is a single chain polypeptide of approximately 22 kDa, produced by the pituitary gland and with pleiotropic functions among vertebrates. It mainly regulates body growth, being also involved in reproduction, immunity and osmoregulation in teleost fish. In order to obtain the tilapia (Oreochromis hornorum) growth hormone (tiGH) in Pichia pastoris cells, its gene was cloned into expression vectors in both with and without a heterologous secretion signal. The tiGH, obtained either intracellularly or extracellularly in P. pastoris cells, was characterized showing its production as associated to the cellular rupture precipitate with an approximate molecular weight of 22 kDa; or being secreted with an approximate molecular weight of 18 kDa, respectively. The mass spectrometry analysis of the recombinant protein obtained in the culture supernatant corroborated the identity of the protein as tiGH but lacking 46 aminoacids of its carboxyl terminal sequence. The tiGH biological activity of P. pastoris intact cells producing this protein was carried out in tilapia larvae (Oreochromis sp.), showing, for the first time, that it is possible to stimulate fish growth by immersion baths with recombinant tiGH-producing yeast. On the contrary what was previously postulated for mammals, the evaluation of P. pastoris cells expressing the truncated variant of tiGH demonstrated that this protein is also able to stimulate growth and immune system in fish. This is the first report of a biologically active, truncated GH variant in fish.

La manipulacion del crecimiento en organismos acuaticos es una tarea fundamental de la biotecnologia actual, teniendo en cuenta que uno de los problemas cruciales que afronta la humanidad es el hambre y en especifico, la carencia de proteina animal. La estimulacion del crecimiento dirigida a disminuir los tiempos de cosecha y las grandes mortalidades en las etapas larvales de los peces, asi como el uso de inmunoestimulantes que los preparen para enfrentar cultivos intensivos, son objetivos esenciales de la biotecnologia agropecuaria moderna. Recientemente se han obtenido evidencias que demuestran la relacion existente entre el sistema inmune y el sistema endocrino en los peces por la accion estimuladora de la hormona de crecimiento sobre varios parametros de la inmunidad innata, que constituye su respuesta defensiva mas importante. Lograr con la utilizacion de productos biotecnologicos estimular la hormona de crecimiento y a la vez, el sistema inmune innato de los peces constituye una de las principales promesas de exito que tiene la biotecnologia para la acuicultura. En el presente trabajo se estudia la influencia del peptido activador de la adenilato ciclasa de pituitaria (PACAP), del peptido relacionado a PACAP (PRP) de Clarias gariepinus y del neuropeptido Y de Oreochromis sp sobre el crecimiento y el sistema inmune innato en peces. Los resultados obtenidos muestran que estos neuropeptidos, ademas de tener actividad promotora del crecimiento y del desarrollo estimulan varios elementos de la inmunidad innata como lisozima, lectinas y oxido nitrico, y de las defensas antioxidantes (actividad de la catalasa, de la superoxido/dismutasa y concentracion de glutation reducido)