8th National Seminary and first Central-American and Caribbean Congress of Sciences and Marine Technologies.

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COMPARATIVE MORPHOLOGY OF THE NURSE SHARK BLOOD CELLS, Ginglymostoma cirratum (Bonnaterre, 1788), WITH OTHER FISH.

This work is a comparation of the blood cells morphology between 9 nurse sharks, a southern stingray and 9 bone fishes, moreover, it was maked a differential leucocytes count, red and white cells counts by blood mm3 for any fish.

BIOLOGICAL ASPECTS OF ANY SPECIES OF ELASMOBRANCH FISHES.

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Effects of the Haematopoietic Organs Extracts of Nurse Shark, Ginglymostoma cirratum (Bonnaterre, 1788) in a tumoral cells in vitro culture.

A preliminary study of the effects of the aquoses extracts of the nurse shark (Ginglymostoma cirratum) haematopoietic organs (liver, epigonadal organ, kidney, cartilage and plasma), on the culture of the tumorals cells producer of specific antibodies with the A blood group (hibridomas) was carried out. The experiments were complemented with a control of specifity using the liver and cartilage extracts of the skate Rhinobatos percellens. The monitoring of the growth and metabolism showed that the epigonal organ extract caused apoptosis in the hibridomas, the liver extract and the plasma provoked cell necrosis and the other extracts as the liver and cartilage of the skate caused inhibition on the growth and cell metabolism.

Effects of the Haematopoietic Organs Extracts of Nurse Shark, Ginglymostoma cirratum (Bonnaterre, 1788) in a tumoral cells in vitro culture.

A preliminary study of the effects of the aquoses extracts of the nurse shark (Ginglymostoma cirratum) haematopoietic organs (liver, epigonadal organ, kidney, cartilage and plasma), on the culture of the tumorals cells producer of specific antibodies with the A blood group (hibridomas) was carried out. The experiments were complemented with a control of specifity using the liver and cartilage extracts of the skate Rhinobatos percellens. The monitoring of the growth and metabolism showed that the epigonal organ extract caused apoptosis in the hibridomas, the liver extract and the plasma provoked cell necrosis and the other extracts as the liver and cartilage of the skate caused inhibition on the growth and cell metabolism.

National Seminary “The Aquaculture: Alternative beef production for 21st century”

Comparative Morphology of Blood Cells of Nurse Shark, Ginglymostoma cirratum (Bonnaterre, 1788), with others Fish.

This work is a comparison of the blood cell morphology between 9 Nurse Sharks, Southern Stingray and 9 teleost fishes. A differential count of red and white cells by mm3 blood, was made.

Position of Corals at the reef.

Talks about the principal coral reefs present in differents seas and distribution of coral inside.

Desarrollo de anticuerpos monoclonales para el estudio de la respuesta inmune en el rodaballo (Scophthalmus maximus Linnaeus, 1758).

Monoclonal antibodies (mAbs) can be used in several applications, v.g in the aquaculture field. In attempt to study the immune system of the turbot fish, we wanted to develop mAbs directed against turbot’s leucocytes. BALB/c mice were immunized with turbot thymocytes, the precursors cells of the T lymphocytes. Splenocytes from immunized mice were fused with mouse myeloma cells to generate hybridomas. The screening of secretory hybridomas was first performed by ELISA. Then, the specific recogition was analyzed by immunochemistry on turbot lymphoid tissues and by flow cytometry using turbot thymocytes, splenocytes, and pronephros. We have generated several mAbs, and we selected the RoD4 mAb that it is able to identify a population of thymocytes, and works in both flow cytometry and immunochemistry. The mAb recognizes a membrane marker that could be useful in the identification of thymic populations.

CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO TURBOT (Scophthalmus maximus) THYMIC CELLS.

Monoclonal antibodies (mAbs) can be used in several applications, v.g in the aquaculture field. In attempt to study the immune system of the turbot fish, we wanted to develop mAbs directed against turbot’s lymphocytes, mainly T cells. BALB/c mice were immunized with turbot thymic cells and mouse splenocytes were fused with mouse myeloma cells to generate hybridomas. The screening of secretory hybridomas was first performed by ELISA. Then, specificity of different monoclonal antibodies was determined by using immunohistochemical and immunofluorescence methods and, on the basis on their reactivity with cells from lymphoid tissues of turbot, three antibodies called RoA3, RoA4 and RoD4 were selected. RoA3 positively reacted with the membrane components of cells resembling to thymocytes, RoA4 stained cells with extended cytoplasmic processes, compatible with reticular cells. In the case of RoD4 mAb, it stains large cells mainly in the medullar zone of the thymus, compatible with macrophages and reticular cells. Flow cytometry studies showed its specificity for thymic cells (no reaction with spleen or pronephros cells), further confirmed by western blot, indicating that RoD4 mAb recognizes a specific protein of 130-135 kD only on thymic tissue.

Inmunodetección en Biología Marina.

El perfeccionamiento de herramientas inmunológicas ha promovido un desarrollo en la biología molecular y celular en diferentes campos, no solo en biomedicina humana o animal, sino también en el campo marino. Con la aparición de los anticuerpos monoclonales como herramienta inmunológica se ha hecho posible la detección de larvas marinas, líneas celulares sanguíneas en peces, toxinas, proteínas específicas y seguimiento de las mismas dentro de sistemas celulares y tejidos, etc. Además de poder complementar su uso con anticuerpos policlonales y fagos recombinantes, empleando técnicas como Inmunofluorescencia, Citometría de flujo, Western Blot o Microscopia Confocal, para su consecuente caracterización.

Advances in Immunotherapy.

Marine resources exploitation. PharmAtlantic project (Knowledge Transfer Network for Prevention of Mental Diseases and Cancer in the Atlantic Area).

Phage Display Techniques to obtain antibodies against turbot (Scophthalmus maximus) blood cells.

Chemistry and Cancer: From Causes to Diagnosis and Therapy.

Fourth Scientific Biomedical Capacities Support Program (BIOCAPS) Seminar 2013. Instituto de Investigación Biomédica de Vigo. University of Vigo.

The Epigenetic Mechanisms in Human Diseases.

Fifth Scientific BIOCAPS Seminar. Instituto de Investigación Biomédica de Vigo (IBI). University of Vigo.

Use of ethanol as an anesthetic in Octopus vulgaris paralarvae: A contribution to the well-being of cephalopods in culture.

Trials were performer with ethanol in order to test their effect as anaesthetic in Octopus vulgaris paralarvae. Ethanol was an effective anaesthetic with limited toxic or stressing effect only at high concentrations (4%). Anesthetized specimens show relaxed musculature, non-active chromatophores and normal respiratory processes. Specimens’ recovery after anesthesia is good and no adverse effects were observed after.

Is alkali labile phosphate (ALP) a suitable biomarker for endocrine disruption in marine mussels?

Alkali labile phosphate (ALP), a method that detects inorganic phosphate liberated from phosphorylated proteins, is usually measured as a proxy for vitellogenin in fish and molluscs. In mussels, it has been measured either in gonad, hemolymph or digestive gland and used in both laboratory and field experiments as a biomarker of endocrine disruption. However, recent studies have shown that vitellogenin is only produced in the female gonad of bivalves and that the phosphorilated proteins measured by ALP in hemolymph correspond to extrapallial protein precursor and not to vitellogenin, that is absent in this fluid. This work aims to demonstrate if ALP can still be used as a proxy of vitellogenin in the marine mussel Mytilus galloprovincialis when measured in the gonad, the primary site of vitellogenin production and accumulation.

Comparison of Proteomic and Spectrophotometric Methods for Vitellogein measurement in Mytilus galloprovincialis

Vitellogenin (Vtg), the egg-yolk precursor in female oviparous animals, is used as a biomarker of estrogenic endocrine disruption due to its increase after exposure to xenoestrogenic pollutants, and specially to its anomalous appearance in males. A common indirect technique for Vtg analysis is the measure of alkali labile phosphates (ALP), a method which detects inorganic phosphate released from phosphorylated proteins. Recent studies have shown that the ALP method detects other phosphorylated proteins isn`t that are not Vtg. Therefore, it is necessary to develop a more accurate measurement of Vtg in mussels.

Morphometric of the ocular surface in the Small-Spotted Catshark Scyliorhinus canicula (Linnaeus, 1758)

The ocular surface (OS) values was obtained of 38 Small-spotted Catsharks and were analysed by sex. The intraspecific analysis shows a similar value by sex. The OS values are valid and could be useful to ecological or physiological analysis.

Application of label-free shotgun proteomics to identify and quantify vitellogenin in marine mussels

Vitellogenin (vtg), the egg-yolk precursor in female oviparous animals, is used as a biomarker of estrogenic endocrine disruption due to its induction by xenoestrogenic pollutants, and specially to its anomalous appearance in males. A common indirect technique for quantification of vtg is the measure of alkali labile phosphates (ALP), but the main disadvantage of this method is that it measures all the phosphorylated proteins present in the sample, not only vtg. Therefore, it is necessary to develop a more accurate technique to measure vtg in marine mussels. In this study, we have applied shotgun proteomics using high resolution LC-MS/MS to identify and quantify vtg in mussel gonads. The aim of this work is to standardize a technique for the measurement of vtg in the gonads of the marine mussel Mytilus galloprovincialis, and to check whether vtg synthesis is induced after exposing individuals to a synthetic estrogen: 17α-etynil estradiol (EE2).

Pixelar index: a new quantitative approach for the assessment of reproductive condition in the purple sea urchin, Paracentrotus lividus, by image analysis

The assessment of the gametogenic cycle and gonadal stage in commercial species relevant for shellfisheries and aquaculture is essential for the optimal management of the resource. However, currently this assessment frequently relies on the subjective classification of the specimens into discrete stages of development, with a strong dependence on the observer. The present study developed a quantitative, continuous, and observer-independent method for the analysis of the gonadal stage of the edible sea urchin, Paracentrotus lividus. This new approach takes advantage of image analysis to assess in histological preparations of the gonads the cellular growth of germ cells and calculate a Pixelar index (PI). The PI can be calculated taking into account just the proportion of the area covered by germinal vs somatic cells in the gonad follicle (PI1) or computing also the empty space created in the follicle lumen after spawning (PI2), which reduces the variability of the index. In both cases, PI showed a bell-shaped dependence on the traditional gametogenic stage that could be fitted to a Gaussian model, providing a tool that can be used for the quantitative evaluation of effects on sea urchin gonads of any kind of environmental factors, from aquaculture rearing conditions to ecotoxicological effects of pollutants on wild stocks. Further assessment of intraindividual—both among gonads and within gonads—variability in this index will be necessary for refinement of this quantitative tool.

Shotgun Proteomics Analysis Discards Alkali Labile Phosphate as a Reliable Method To Assess Vitellogenin Levels in Mytilus galloprovincialis

Vitellogenin, the egg yolk precursor, is a well known biomarker of endocrine disruption in oviparous vertebrates. In invertebrates, such as bivalves, it has been used in the last 10 years for the same purpose, despite the limited knowledge of invertebrate endocrinology. In bivalves, vitellogenin levels are usually estimated using an indirect technique, alkali labile phosphate (ALP), that assumes that vitellogenin is the most abundant phosphorylated protein in the analyzed tissue. In this study, we applied shotgun proteomics for the identification and quantification of vitellogenin in marine mussel gonads and compared the results with those obtained with the ALP method. The proteomic analysis revealed that vitellogenin is only detected in female gonads with expression levels that are rather variable among female mussels at different stages of gonad development. ALP analysis, on the contrary, detected similar amounts of phosphorylated proteins regardless sex or gonad development stage. These results show evidence that the ALP method is not providing reliable information about Vtg levels, at least in marine mussel gonads. ALP is not a good proxy to assess Vtg levels in marine mussels and careful verification of the adequacy of the procedure should be done before ALP is further assumed as a proxy of Vtg in other bivalve mollusks.

Phage Display Techniques to obtain antibodies against turbot (Scophthalmus maximus) blood cells.

Since the development of the monoclonal antibody technique, these proteins have become an essential tool for diagnosis and therapy on the biomedical field, but also on many other applications such as in the marine field. However, this technique presents several disadvantages such as the hibridoma instability, the necessity of animal facilities and frequent low response against some antigens. The display of single chain variable fragment (scFv) antibodies on the surface of filamentous phages can be a very nice alternative to overcome all these problems. In this work, we present for the first time, the use of a human phage library to obtain scFv against turbot blood cells. After four rounds of panning and selection against turbot mononuclear peripheral blood cells, several individual clones were isolated. Their specificity was analyzed using several immunological techniques including flow cytometry on cells from several tissues. We selected the monoclonal phage-scFv P2B2 that specifically recognizes turbot erythrocytes. Our results suggest that phage display libraries provide a valuable method for an easier and faster generation of specific antibody fragments against cellular antigens.

Development of the olfactory system in turbot (Psetta maxima L.).

We have examined the histogenesis of the olfactory system during turbot development using histological and immunohistochemical methods. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was used to detect dividing cells, whereas calretinin (CR) immunohistochemistry was used to distinguish some neuronal components of the olfactory system. Around hatching, the olfactory placode of embryos transforms into an olfactory pit, which enlarges progressively during development. In metamorphic turbots, the right olfactory organ moves to the tip of the head. Each olfactory chamber opens to the external medium by two nostrils and accessory nasal sacs develop during metamorphosis. The order of birth of olfactory receptor cells in the sensory epithelium follows the pattern of most teleosts: ciliated cells differentiate prior to microvillous cells in turbot larvae, and crypt cells are generated during metamorphosis. Axons of olfactory sensory neurons reach the rostral forebrain by hatching, and calretinin-immunoreactive (CR-ir) glomerular fields were apparent during the subsequent larval development. During metamorphosis olfactory bulbs become strongly distorted by head torsion and glomeruli acquire asymmetric organization. The spatio-temporal course of proliferation in the olfactory system reveals changes in the distribution of dividing cells in the sensory epithelium throughout the developmental period investigated. In the olfactory bulb, proliferative activity becomes restricted to the ventral periventricular zone in turbot larvae, as well as in metamorphic specimens.

EL CULTIVO DEL PULPO. UNA ALTERNATIVA DE FUTURO?

El pulpo es un recurso pesquero tradicionalmente capturado en las costas españolas. Este molusco tiene gran importancia, sobre todo en las pesquerías artesanales, donde sus capturas representan una importante fracción de las ganancias de los pescadores. Sin embargo, y sobre todo este último año, el pulpo ha sido escaso y en algunas comunidades como Galicia, se teme que pueda peligrar como recurso. En este contexto, el cultivo industrial del pulpo facilitaría un medio eficiente y controlado para suplir la demanda de este recurso sin sobreexplotar las poblaciones naturales e, incluso, daría la posibilidad de regenerar aquéllas muy explotadas.

Lista de especies Elasmobranquias de Colombia.

This work is a bibliography of species of elasmobranchs fish reported for Colombia (South America), and the data refer only to sharks, skates and rays.

Morfología comparativa de las células sanguíneas del tiburón nodriza Ginglymostoma cirratum (Bonnaterre, 1788) con diversos peces.

This work is a comparation of the blood cells morphology between 9 nurse sharks, a southern stingray and 9 bone fishes, moreover, it was maked a differential leucocytes count, red and white cells counts by blood mm3 for any fish.

Desarrollo de herramientas inmunológicas y estudio de la organogénesis del sistema olfatorio del rodaballo (Scophthalmus maximus, L.).

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EFECTO DE LOS EXTRACTOS DE ORGANOS HEMATOPOYETICOS DEL TIBURON NODRIZA, Ginglymostoma cirratum (BONNATERRE, 1788) EN UN CULTIVO in vitro DE CELULAS TUMORALES

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Desarrollo de herramientas para inmunodetección: Generación de Anticuerpos Policlonales, Monoclonales y Bibliotecas de Fagos

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Bioassays stress the ecotoxicological differences between polymers and plastics additives in the marine environment.

PLASTICS IN THE OCEAN: MICROPLASTICS The global ocean is the final sink of land-based plastic pollution. Until recently plastic objects were thought to persist for hundreds of years in the environment, but according to an emerging paradigm they undergo fragmentation as a result of leaching of plasticisers, exposure to UV light, chemical oxidation and the high energy hydrodynamics of some coastal areas. As a result, plastic particles of micron size, called secondary microplastics, are increasingly abundant in marine ecosystems. CONCLUSIONS -Conventional polymers microplastics currently do not pose a risk to planktonic invertebrates; in contrast some plastic additives show acute toxicity and evidence of estrogenic effects. -Development of practical tools to assess endocrine disruption is urgentlyneeded since many plastic additives are suspected EDCs and we lack standard methods to detect EDCs with marine organisms.

Estrogenic effects of an Organophosphorous Flame Retardant (TCPP) on Edible Sea Urchin Paracentrotus lividus L.

Tris (2-Chloro-1-Methlethyl) Phosphate (TCPP) Organophosphorus Flame Retardant used in plastics, mainly in polyurethane foam, are one of the most common OPFRs detected in aquatic systems. This new problem in water contamination and coastal waters, are due to the incomplete elimination in the wastewater treatment plants. Possible toxic effect of this chemical has not been deeply evaluated yet. This study aims to explore the possible effect of TCPP as an endocrine disruptor on edible sea urchin, Paracentrotus lividus, (Lamarck, 1816).

Possible estrogenic effects of ethinyl estradiol on gonadal maturation in Edible Sea Urchin Paracentrotus lividus L.

17α-Ethinylestradiol (EE2) is a synthetic estrogen widely used in combination with other steroid hormones in contraceptives. EE2 has been detected in sewage treatment plant effluents in the low ng/L range and occasionally in surface waters. The main objective of this study was to test the possible effects of exposure to 10 ng/L EE2 on edible sea urchins, since echinoderms are considered valuable test species in marine ecotoxicology. 110 adult individuals of Paracentrotus lividus, with a diameter ranging between 4 and 6 cm were used under experimental conditions. These organisms were maintained in aquaria by controlled conditions in Toralla Marine Science Station (ECIMAT) at University of Vigo. The sea urchins were distributed in three groups: Seawater Control, Solvent Control (Acetone), and EE2. Finally, gonad samples were collected and analized by histology and Gonad Index (GI) after 7 and 28 days of exposure. EE2 diluted in water did not induce tissue damage in gonads, but it seems to produce an increase in gonad maturation and Pixel Index in histological preparations of gonad, after 28 days of exposure in both sexes. Gonad Index (GI) increased significantly compared to solvent control at 28 days, suggesting estrogenic effects in females. Preliminary proteomics results showed differences in proteins of ~170kDa weight, that increase in females treated with EE2, and decrease in males. Further studies are necessary to identify proteins by mass spectrometry. Finally, the use of acetone as a solvent is not recommended, since it caused a decrease of gonad index and therefore it could affect also protein synthesis.