Mario Pablo Estrada

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.

Novel gene isolated from Caligus rogercresseyi: A promising target for vaccine development against sea lice

Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry in the last 30 years. Caligus rogercresseyi is the most important species affecting Chiles salmon industry. Vaccines against caligid copepods have the potential to be a cost-effective means of controlling the infestation and avoid many of the disadvantages of medicine treatments. However, research in the development of such vaccines has begun only recently and approaches used thus far have met with little or no success. In the present study, we characterized a novel gene (denoted as my32) from C. rogercresseyi which has the highest identity with the Lepeophtheirus salmonis gene akirin-2. To assess the function of the gene an RNA interference experiment was developed and a reduction in the number of ectoparasites on fish in the my32-dsRNA treated group was observed. The recombinant my32 protein was used in a vaccination-challenge trial to evaluate its ability to protect against sea lice infestations. A significant reduction in the number of parasites per fish was observed at 24 days post-challenge. These results, together with the delay observed in the development of parasites from the vaccinated group suggest that the major effect of immunization was on the second parasite generation. The results of these experiments suggest that the my32 protein may be a promising target for vaccine development to control sea lice infestations in fish.

Safety Evaluation of Transgenic Tilapia with Accelerated Growth.

Abstract. Recent advances in modern marine biotechnology have permitted the generation of new strains of economically important fish species through the transfer of growth hormone genes. These transgenic fish strains show improved growth performance and therefore constitute a better alternative for aquaculture programs. Recently, we have obtained a transgenic tilapia line with accelerated growth. However, before introducing this line into Cuban aquaculture, environmental and food safety assessment was required by national authorities. Experiments were performed to evaluate the behavior of transgenic tilapia in comparison to wild tilapia as a way to assess the environmental impact of introducing transgenic tilapia into Cuban aquaculture. Studies were also conducted to evaluate, according to the principle of substantial equivalence, the safety of consuming transgenic tilapia as food. Behavior studies showed that transgenic tilapia had a lower feeding motivation and dominance status than controls. Food safety assessment indicated that tilapia growth hormone has no biological activity when administered to nonhuman primates. Furthermore, no effects were detected in human healthy volunteers after the consumption of transgenic tilapia. These results showed, at least under the conditions found in Cuba, no environmental implications for the introduction of this transgenic tilapia line and the safety in the consumption of tiGH-transgenic tilapia as an alternative feeding source for humans. These results support the culture and consumption of these transgenic tilapia.

Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus schmitti. Functional analysis by double-stranded RNA interference technique.

The crustacean hyperglycemic hormone (CHH) plays an important role in the regulation of hemolymph glucose levels, but it is also involved in other functions such as growth, molting and reproduction. In the present study we describe the first CHH family gene isolated from the Atlantic Ocean shrimp Litopenaeus schmitti. Sequence analysis of the amplified cDNA fragment revealed a high nucleotide sequence identity with other CHHs. Northern blot analysis showed that the isolated CHH mRNA from L. schmitti is present in the eyestalk but not in muscle or stomach. We also investigated the ability of dsRNA to inhibit the CHH function in shrimps in vivo. Injection of CHH dsRNA into the abdominal hemolymh sinuses resulted in undetectable CHH mRNA levels within 24 h and a corresponding decrease in hemolymph glucose levels, suggesting that functional gene silencing had occurred. These findings are the first evidence that dsRNA technique is operative in adult shrimps in vivo.

Regulation of body mass growth through activin type IIB receptor in teleost fish

Myostatin is a TGF-beta family member that plays a key role in regulating skeletal muscle growth. Previous studies in mammals have demonstrated that myostatin is capable of binding the two activin type II receptors. Additionally, activin type II receptors have been shown to be capable of binding a number of other TGF-beta family members besides myostatin. An injection of a soluble form of activin type IIB receptor obtained from CHO cells into wild-type mice generated up to a 60% increase in muscle mass in 2 weeks. The knowledge on the role of activin receptors in fish is limited. In the present study, we examined the growth effect of administering a recombinant, soluble form of goldfish activin type IIB receptor extracellular domain to juvenile and larval goldfish (Carassius auratus), African catfish (Clarias gariepinus) larvae and tilapia (Oreochromis aureus) larvae. We have expressed the goldfish activin type IIB receptor extracellular domain in the yeast Pichia pastoris and we have demonstrated for the first time that this recombinant molecule stimulates growth in teleost fish in a dose-dependent manner. We provide evidence that this body weight increase is achieved by an increase in muscle mass and protein content. Histological analysis of the goldfish muscle revealed that treated fish exhibited hyperplasia as compared to controls. These findings contribute to the understanding of the mechanisms that regulate growth in non-mammalian vertebrates and suggest a powerful biotechnology approach to improving fish growth in aquaculture.

An inducible nitric oxide synthase (NOS) is expressed in hemocytes of the spiny lobster Panulirus argus: cloning, characterization and expression analysis.

Nitric oxide (NO) is a free radical gas involved in a variety of physiological processes in invertebrates, such as neuromodulation, muscle contraction and host defense. Surprisingly, little is known about the involvement of NO synthase (NOS) in the immune system of crustaceans. This work is focused on the study of the NOS gene of the spiny lobster Panulirus argus, a crustacean with commercial interest, and its relationship with the immune response to a microbial elicitor. A NOS full-length DNA was isolated from hemocytes by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers. The open reading frame (ORF) encodes a protein of 1200 amino acids, with an estimated molecular mass of 135.9 kDa, which contains the conserved domains and binding motifs of NOS found in a variety of organisms. NOS gene expression in lobster gills, heart, stomach, digestive gland, abdominal muscle, gut and hemocytes was studied by Real Time quantitative PCR (Real Time qPCR). The expression was higher in hemocytes, heart and gills. In addition, when lobster hemocytes were exposed in vitro to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase in the NOS activity and also in the NOS gene expression evaluated by Real Time qPCR was observed, thus demonstrating the presence of an inducible crustacean NOS by a microbial elicitor of the immune response. The information is relevant in providing basic knowledge for further studies of crustacean defense mechanisms.

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 tick antigen shows high vaccine efficacy against the dog tick, Rhipicephalus sanguineus

Ticks are acaridae ectoparasites that, while taking a blood meal, can transmit viruses, bacteria, protozoa and filarial nematodes, which cause a variety of human and animal illnesses. The use of chemical pesticides constitutes the primary measure for control of these ectoparasites. However, the intensive use of these chemicals has drawbacks such as the contamination of food, environmental pollution and development of resistance by ectoparasites. Vaccination is considered a promising alternative for controlling infestations by ectoparasites. Although emerging tick proteins have been identified recently, and have been proposed as potential targets for generating protective molecules, only a limited number of them have been evaluated in vaccine trials. More than 80 proteins are found in eukaryotic ribosomes. The protein P0 is essential for the assembly of the 60S ribosomal subunit. We have identified an immunogenic region of the ribosomal protein P0 from Rhipicephalus sp. ticks that is not very conserved compared to host P0. The efficacy of a 20 amino acid synthetic peptide from this sequence was assayed as a vaccine antigen against Rhipicephalus sanguineus infestations in an immunization and challenge experiment on rabbits. A remarkable diminution in the viability of newly molted nymphs from larvae fed on vaccinated rabbits was observed. The number of adults and the number of eggs hatching were significantly reduced, with an overall efficacy of 90%. Our results demonstrated that immunization with an immunogenic peptide of tick protein P0 greatly reduced survival of ticks, suggesting that it has promise as an effective tick control agent.

Novel function of recombinant pituitary adenylate cyclase-activating polypeptide as stimulator of innate immunity in African catfish (Clarias gariepinus) fry

There are several studies that clearly indicate a close bidirectional communication between the neuroendocrine and immune systems. In this sense, hypothalamic releasing hormones, besides their neuroendocrine role, have been shown to influence immune functions. Despite studies developed in mammals, there is, as yet, no information available about the role of the pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-related peptide (PRP) in the fish innate immune system. The present study has evaluated the effect of PACAP and PRP administered by bath immersion, on important parameters of innate immunity and antioxidant defenses in African catfish (Clarias gariepinus) fry. We have shown, for the first time, that administration of recombinant C. gariepinus PACAP not only promotes growth but also increases lysozyme, nitric oxide synthase-derived metabolites and antioxidant defenses in treated fry. From our results, PACAP appears to act as a regulator of the teleostean immune system, in addition to its physiological role in controlling growth of fish.

Recombinant truncated tilapia growth hormone enhances growth and innate immunity in tilapia fry (Oreochromis sp.).

Pichia pastoris cells transformed with a plasmid engineered for the expression of tilapia growth hormone as a secreted product produced a proteolytically cleaved form of the recombinant protein. The sequence of this truncated variant was obtained by mass spectrometry analysis. The cleavage site was determined to be between residues Tyr 158 and Tyr 159. The resulting truncated tilapia growth hormone was a single chain protein lacking 46 amino acids of the C-terminal portion. In this study, we showed that the truncated growth hormone produced in the P. pastoris culture supernatant has growth promoting effects and stimulates innate immune parameters (lysozyme and lectins) in tilapia larvae. These results suggest that the C-terminal portion of growth hormone is not required for its growth promoting activity and the innate immune functions studied herein in fish. In addition, we found that the culture supernatant containing truncated tilapia growth hormone has a stronger effect over growth and immune system than cells lysate containing intact tilapia growth hormone expressed in P. pastoris.