Fidel Herrera

Mendelian transmission, transgene dosage and growth phenotype in transgenic tilapia (Oreochromis hornorum) showing ectopic expression of homologous growth hormone

Abstract Gene transfer has offered a new tool for the development of improved fish strains for aquaculture. However, characterization is required before these strains can be introduced into national aquaculture programs. Transgenic tilapia ( O. hornorum urolepis ) were produced by the microinjection into early embryos of a transgene containing the tilapia growth hormone (tiGH) cDNA under the regulatory sequences derived from the human cytomegolovirus (CMV). A male containing 1 copy/cell of the transgene was selected to establish a transgenic tilapia line. The transgene was transmitted to F 1 –F 4 generations in a Mendelian fashion. Previous studies showed ectopic, low level expression of tiGH in brain, heart, gonad, liver and muscle cells of transgenic tilapia. Biochemical analyses indicated lower levels of cholesterol, free alanine and aspartic acid in the muscle of transgenic animals. Four month old transgenic homozygous (F 2 +/+ ) and heterozygous (F 2 −/+ ) tilapia and non-transgenic siblings ( N TRANSGENICS =14; N CONTROLS =11; N F2 −/+ =8; N F2 +/+ =6) were studied for 3 months grown communally in the same pond. Transgenic (F 2 −/+ +F 2 +/+ ), F 2 −/+ , and F 2 +/+ progeny were larger than non-transgenic siblings at P =0.009, P =0.005 and P =0.07 (Student t -test), respectively, suggesting a transgene-dosage effect. These results indicate stable germ line transformation in this fast-growing transgenic tilapia line.

Cloning, expression and growth promoting action of red tilapia (Oreochromis sp.) neuropeptide Y

Neuropeptide Y, a 36 amino acid peptide abundantly expressed in the brain, is the most potent orexigenic factor known to date in mammals. It has been shown to be one of the most conserved neuropeptides in vertebrate evolution. It seems that neuropeptide Y functions, in addition to sequence conservation, are also well conserved in fish. In the present study, we cloned and reported the cDNA sequence coding for tilapia 36 aminoacid neuropeptide Y. We express the tilapia neuropeptide Y gene in Escherichia coli driven by T7 promoter. The recombinant neuropeptide Y was purified up to 80% by affinity chromatography. We developed both, a food intake and a growth performance experiment to evaluate the effects of neuropeptide Y administration. Juvenile tilapia receiving recombinant neuropeptide Y (1 microg/g of body weight) by intraperitoneal injection increased food intake compared to controls (p < 0.05). Similarly, in the growth performance experiment, we observed an increase in body weight (p < 0.05) of tilapia fry receiving the same dose of the peptide. Neuropeptide Y treatment had no significant effect on hepatosomatic index and muscle moisture content. On the other hand, muscle protein content was increased in treated animals. These results demonstrate that administration of biologically active E. coli-derived neuropeptide Y resulted in a growth promoting action in fish.

A novel GH secretagogue, A233, exhibits enhanced growth activity and innate immune system stimulation in teleosts fish.

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.

The biological role of pituitary adenylate cyclase-activating polypeptide (PACAP) in growth and feeding behavior in juvenile fish.

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. Copyright

Novel IFNγ homologue identified in Nile tilapia (Oreochromis niloticus) links with immune response in gills under different stimuli

Abstract Interferon gamma (IFN‐&ggr;) 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) IFN&ggr; from spleen lymphocytes. The isolated tilapia IFN&ggr; has between 24 and 62% of amino acid identity as compared to reported sequences for other teleost fishes. It has close phylogenetic relationships with IFN&ggr; molecules belonging to the group of Perciforms and presents the typical structural characteristics of gamma interferon molecules. The tissue expression analysis showed that IFN&ggr; 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, IFN&ggr; expression was up‐regulated in gills after 24 h post‐stimulation. IFN&ggr; 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 IFN&ggr; 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. HighlightsA novel Nile tilapia IFN&ggr; was cloned from spleen lymphocytes stimulated with LPS.The isolated cDNA sequence has the typical features of IFN&ggr;.IFN&ggr; expression was induced in gills 24 h after stimulation with poly I:C and LPS.E. tarda infection induces IFN expression levels in gills 24 h after infection.Recombinant IFN&ggr; activates Mx gene transcription in head kidney primary culture cells.

New insights into the biological activity and secretion properties of a polypeptide derived from tilapia somatotropin

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 its 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.

Growth Hormone Secretagogue (A233) Improves Growth and Changes the Tissue Fatty Acid Profile in Juvenile Tilapia (Oreochromis niloticus)

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.

Discovery of immunoglobulin T in Nile tilapia (Oreochromis niloticus): A potential molecular marker to understand mucosal immunity in this species

ABSTRACT 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. HIGHLIGHTSTwo novel isoforms of IgT were cloned from Nile tilapia head kidney lymphocytes.The isolated cDNA sequence has the typical features of IgT.Early expression of IgT and IgM was detected in tilapia larvae.IgT and IgM are differentially expressed in basal conditions and after E. tarda infection.Infection by immersion suggested IgT specialization in mucosal compartments.