1

Sponsors

GENERALITAT VALENCIANA (GV)

PROGRAMA PROMETEO

(Prometeo II 2015/085 y II 2015/051)

CONSEJO SUPERIOR DE INVESTIGACIONES

CIENTÍFICAS (CSIC)

SOCIEDAD IBÉRICA DE ENDOCRINOLOGÍA

COMPARADA (AIEC)

2

Local Organizing

Committee Members

(in alphabetical order)

Josep Calduch-Giner

José Miguel Cerdá-Reverter

Alicia Felip Edo

Ana Gómez Peris

Félix Hernández

Evaristo Mañanós Sánchez

Jaime Nácher-Mestre

Jaume Pérez-Sánchez (Chair)

Society Council Members

(in alphabetical order)

Pedro Guerreiro

Esther Isorna

Juan Miguel Mancera

José Antonio Muñoz-Cueto

Isabel Navarro (Vicepresident)

Jaume Pérez-Sánchez

Deborah Power (President)

José Luis Soengas

3

Scientific Program

4

5

WEDNESDAY (23rd

September 2015)

OPENING LECTURE

OL - ON THE SHOULDERS OF GH-IANTS: A SOMATOTROPE JOURNEY

FROM COMPARATIVE TO TRANSLATIONAL ENDOCRINOLOGY. J.P.

Castaño, F. Gracia-Navarro, S. Pedraza-Agüera, A. Sarmento-Cabral, M.C.

Vázquez-Borrego, F. López-López, D. Hormaechea-Agulla, A. Villa-Osaba, E.

Rivero-Cortés, A.J. Martínez-Fuentes, A. Ibáñez-Costa, M.D. Gahete, R.M.

Luque.

SESSION 1: REPRODUCTION

Chairperson: Dr. Fransesc Piferrer

O1 - SPECIFIC RECOMBINANT GONADOTROPINS INDUCE

SPERMATOGENESIS AND SPERMIATION BY FIRST TIME IN A TELEOST

FISH, THE EUROPEAN EEL. David S. Peñaranda, V. Gallego, M.C. Vílchez,

L. Pérez, A. Gómez, I. Giménez, J.F. Asturiano

O2 - CHARACTERIZATION OF NUCLEAR AND MEMBRANE

PROGESTIN RECEPTORS IN EUROPEAN EEL, AND THEIR

EXPRESSION IN VIVO THROUGHOUT SPERMATOGENESIS. Marina

Morini, D.S. Peñaranda, M.C. Vílchez, R. Nourizadeh-Lillabadi, J.F.

Asturiano, F.A. Weltzien, L. Pérez

O3 - ASSESSMENT OF THE RA-SIGNALLING PATHWAY DURING

GONADAL DEVELOPMENT AND MATURATION IN THE EUROPEAN

SEA BASS (Dicentrarchus labrax). Paula Medina, A. Gómez, S. Zanuy, M.

Blázquez

O4 - DIFFERENTIAL ACTIVATION OF KISS RECEPTORS BY Kiss1 and

Kiss2 PEPTIDES IN THE SEA BASS. Alicia Felip, F. Espigares, S. Zanuy, A.

Gómez

O5 – EFFECT OF HIGH REARING DENSITY ON SEX DIFFERENTIATION

IN ZEBRAFISH (Danio rerio): SEX RATIO AND GENE EXPRESSION

ANALYSIS. Alejandro Valdivieso, L. Ribas, F. Piferrer

6

THURSDAY (24th

September 2015)

JOSEP PLANAS' CONFERENCE

JP'C - FROM INSULIN TO IGFS IN FISH: A SCIENTIFIC JOURNEY.

Gutiérrez, J., Navarro, I., Fernández-Borràs, J., Blasco, J., Planas, J.V., Vélez,

E.J., Lutfi, E. and Capilla, E. E-mail: jgutierrez@ub.edu

SESSION 2: ENERGY METABOLISM

Chairperson: Dr. José Luis Soengas

O6 - DAILY VARIATIONS OF CARBOHYDRATE AND LIPID

METABOLISM-RELATED PARAMETERS IN LIVER OF RAINBOW

TROUT. INFLUENCE OF LIGHT AND FOOD AVAILABILITY. Juan

Hernández-Pérez, J.M. Míguez, J.L. Muñoz, F. Naderi, M. Librán-Pérez, R.

Ceinos, M.A. López-Patiño

O7 - EFFECTS OF GHRELIN ON THE RESPONSE TO OLEATE OF

HYPOTHALAMIC FATTY ACID SENSORS IN RAINBOW TROUT

(Oncorhynchus mykiss): INVOLVEMENT IN THE CONTROL OF FOOD

INTAKE. Cristina Velasco, M. Librán-Pérez, C. Otero-Rodiño, F. Naderi, R.

Álvarez-Otero, M.A. López-Patiño, J.L. Soengas

O8 - FEEDING-INDUCED CHANGES IN THE GHRELINERGIC SYSTEM

IN THE PLASMA, BRAIN AND INTESTINAL BULB OF GOLDFISH

(Carassius auratus). Ayelén M. Blanco, M. Gómez-Boronat, I. Redondo, A.I. ,

M.J. Delgado

O9 - SERUM METABOLOME OF FASTED GILTHEAD SEA BREAM. A

NON-TARGETED APPROACH FOR THE IDENTIFICATION OF ROBUST

BIOMARKERS OF MALNUTRITION IN FISH. Rubén Gil Solsona, J.

Nácher-Mestre, L. Lacalle, J.A. Calduch-Giner, J.V. Sancho, F. Hernández, J.

Pérez-Sánchez

O10 - ENERGY SENSING IN GILTHEAD SEA BREAM. MOLECULAR

AND FUNCTIONAL CHARACTERIZATION OF SIRTUINS. Paula Simó,

J.A. Calduch-Giner, J. Pérez-Sánchez

O11 - METFORMIN EXERTS DIRECT ANTI-PROLIFERATIVE EFFECTS

7

IN PROSTATE CANCER CELLS IN VITRO AND INHIBITS PROSTATE

TUMOR GROWTH IN VIVO UNDER NORMAL-FED AND, SPECIALLY,

UNDER HIGH FAT FED CONDITIONS. André Sarmento-Cabral, F. López-

López, M.D. Gahete, J.P. Castaño, R.M. Luque

SESSION 3: WIDE FUNCTIONAL ANALYSIS

Chairperson: Dr. Deborah Power

O12 - WIDE-TRANSCRIPTOMIC ANALYSIS OF INTESTINE IN

MEDITERRANEAN FISH. Jaume Pérez-Sánchez, J.A. Calduch-Giner, A.

Sitjà-Bobadilla

O13 - TRANSCRIPTOMICS PROVIDE INSIGHT INTO MUSSEL (Mytilus

galloprovincialis) MANTLE FUNCTION. Nadège Zaghdoudi-Allan, T. Yarra,

A. Churcher, R. Felix, M. Clark, D.M. Power

O14 - THE SKIN AS AN ENDOCRINE ORGAN: INSIGHTS FROM THE

SEA BASS SKIN TRANSCRIPTOME. Patrícia I. S. Pinto, M.A.S. Thorne, D.

M. Power

O15 - A MICROARRAY STUDY OF SKIN AND SCALE REGENERATION

IN THE SEA BREAM (Sparus aurata). Rita Alves Costa, F. Vieira, D.M.

Power

O16 - MULTI-CLASS SCREENING OF FEEDSTUFF CONTAMINANTS

AND POTENTIAL ENDOCRINE DISRUPTORS IN FARMED FISH. Jaime

Nácher-Mestre, R. Serrano, M. Ibáñez, T, Portolés, M.H.G. Berntssen, F.

Hernández, J. Pérez-Sánchez

SESSION 4: STRESS AND IMMUNE RESPONSE

Chairperson: Dr. Juan Miguel Mancera

O17 - ACUTE STRESS REGULATES VASOTOCINERGIC AND

ISOTOCINERGIC SYSTEM IN THE GILTHEAD SEA BREAM (Sparus

aurata). Arleta Krystyna Skrzyńska, E. Maiorano, M.Bastaroli, G. Martínez-

Rodríguez, J.A. Martos-Sitcha, J.M. Mancera

O18 - BRAIN MONOAMINERGIC NEUROTRANSMITTERS DURING

CHRONIC STRESS IN RAINBOW TROUT (Oncorhynchus mykiss). Fatemeh

Naderi, M.A. López-Patiño, M. Gesto, J.M. Cerdá-Reverter, C. Otero-Rodiño,

8

C. Velasco, J.M. Míguez

O19 - PLASTIC PHYSIOLOGY, SOCIAL BEHAVIOUR AND ENDOCRINE

PROFILES OF AN INVASIVE CICHLID IN SOUTHERN PORTUGAL. Flávia

B. Silva, J.L. Saraiva, M. Vargas, M. Silva, J. Soares, A.V.M. Canário, P.M.

Guerreiro

O20 - TEMPERATURE IMPRINTING DURING EARLY DEVELOPMENT

AND THE ADULT STRESS AND IMMUNE RESPONSE. Ana Patrícia

Mateus, R. Costa, J.R. Cardoso, E. Gisbert, D.M. Power

FRIDAY (25th

September 2015)

SESSION 5: GROWTH FACTORS AND MOLECULAR EVOLUTION

Chairperson: Dr. Isabel Navarro

O21 - ACTIVATION OF SOMATOSTATIN RECEPTOR 3 REDUCES CELL

VIABILITY AND HORMONAL SECRETION IN NON-FUNCTIONING

PITUITARY ADENOMAS THROUGH MAPK SIGNALING. Mª Carmen

Vázquez-Borrego, A. Ibáñez-Costa, E. Venegas-Moreno, A. Toledano-Delgado,

A. Soto-Moreno, M.A. Gálvez, MD. Gahete, J.P. Castaño, R.M. Luque

O22 - LACK OF CORTISTATIN OR SOMATOSTATIN PLAYS

DIFFERENTIAL ROLES IN THE CONTROL OF MAMMARY GLAND

TUMORIGENESIS IN LEAN AND OBESE MICE. Alicia Villa-Osaba, M.D.

Gahete, F López-López, A.I. Pozo-Salas, R. Sánchez-Sánchez, R. Ortega-Salas,

M. Álvarez-Benito, J. López-Miranda, R.M. Luque, J.P. Castaño

O23 – THE TRUNCATED SOMATOSTATIN RECEPTOR sst5TMD4 IS

OVEREXPRESSED IN PROSTATE CANCER AND INCREASES

AGGRESSIVENESS FEATURES THROUGH REGULATION OF

ANGIOGENIC FACTORS AND Wnt/B-CATENIN PATHWAY. Daniel

Hormaechea-Agulla, M.D. Gahete, A. Ibáñez-Costa, E. Gómez-Gómez, J.A.

Ramos-Fernández, J. Valero-Rosa, J. Carrasco-Valiente, M.M. Moreno, M.D.

Culler, M.J. Requena, J.P. Castaño, R.M. Luque

O24 - IN1-GHRELIN INCREASES MALIGNANCY FEATURES OF BREAST

CANCER CELL LINES. David Rincón-Fernández, M.D. Gahete, V. Ruiz-

Murillo, R. Santamaría, R.M. Luque, J.P. Castaño

9

O25 - EXPLORING ALLATOSTATIN-A, KISS AND GALANIN GPCRS IN

LOPHOTROCOZOANS. João C.R. Cardoso, N. Zaghdoudi-Allan, R.C. Félix,

D.M. Power

SESSION 6: MINERALIZATION AND PIGMENTATION

Chairperson: Dr. Pedro Guerreiro

O26 - RESPONSIVENESS OF SEA BASS SCALES TO ESTRADIOL AND

GENISTEIN. M. Dulce Estêvão, P. I. S. Pinto, A. Andrade, S. Santos, D. M.

Power O27 - AN APPROACH TO STANNIOCALCIN SECRETION IN GILTHEAD

SEABREAM (Sparus aurata). Ignacio Ruiz-Jarabo, S.F. Gregório, J. Fuentes

O28 - TARGETED PTH4 CELL ABLATION LEADS TO AN UNCOUPLING

OF BONE FORMATION AND MINERALIZATION. Paula Suarez-Bregua, A.

Saxena, M.E. Bronner, P. Moran, J. Rotllant

O29 - THE GENE REGULATORY NETWORK UNDERPINNING DORSAL–

VENTRAL PIGMENTATION PATTERNING IN FISH. ANALYSIS OF asip1

CIS-REGULATORY LANDSCAPE. Laura Cal, I. Braasch, J.L. Gómez-

Skarmeta, R. Kelsh, J.M. Cerdá-Reverter, J. Rotllant

CLOSING LECTURE

CL - 40 YEARS OF RESEARCH IN COMPARATIVE ENDOCRINOLOGY.

Olivier Kah

10

Plenary Lectures

11

Opening Lecture (OL)

ON THE SHOULDERS OF GH-IANTS: A SOMATOTROPE JOURNEY

FROM COMPARATIVE TO TRANSLATIONAL ENDOCRINOLOGY

J.P. Castaño, F. Gracia-Navarro, S. Pedraza-Agüera, A. Sarmento-Cabral,

M.C. Vázquez-Borrego, F. López-López, D. Hormaechea-Agulla, A. Villa-

Osaba, E. Rivero-Cortés, A.J. Martínez-Fuentes, A. Ibáñez-Costa, M.D. Gahete,

R.M. Luque. Email: justo@uco.es

1Department of Cell Biology, Physiology and Immunology, University of

Córdoba, Hospital Universitario Reina Sofía (HURS), Instituto Maimónides de

Investigación Biomédica de Córdoba (IMIBIC), CIBERobn and, ceiA3;

Córdoba, Spain.

Pituitary somatotropes provide a unique cell model to investigate the regulation

of hormone secretion from an integrative viewpoint. Over the last thirty years,

our group has investigated multiple aspects of the biology of somatotropes, in a

journey encompassing different animal species, organizational levels, and

functional research targets. A retrospective view of the steps covered reveals the

genuine value of comparative endocrinology, and its potential to develop novel

scientific knowledge. Our initial research, focused on frog pituitary, aimed at

understanding the role of hypothalamic hormones on the regulation of

somatotrope function. Combining in vivo and in vitro studies, EM-

immunocytochemistry and quantitative image analysis, we demonstrated for the

first time that TRH is a stimulatory factor for frog somatotropes. This discovery

was later extended by studies on cells from porcine and rat pituitary, and human

pituitary tumors, which unveiled a multifactorial regulation of somatotropes

across species by multiple neuropeptides, including GHRH, ghrelin, PACAP,

TRH, somatostatin, cortistatin, kisspeptin, etc, and helped to define the common

and unique signaling pathways and receptors mediating such regulation.

Ultrastructural analysis of somatotropes led us to the notion of cell

heterogeneity, and prompted us to investigate its biological meaning by

characterizing the different subpopulations of somatotropes composing the

pituitary during lifespan. Morphological and functional in vitro studies in pig

and rat somatotropes unveiled a unique dynamic secretory cell cycle that

includes not only hormone storage and secretion, but also the distinct ability of

somatotropes to respond to regulatory cues through an integrated control of

their molecular and cellular identity. The knowledge gathered in this travel led

us to explore the pathological side of somatotropes and GH. First, by studying

the cell biology of somatotropes from human pituitary tumors, we discovered

and characterized the presence and pathological role of aberrantly spliced

receptors for somatostatin (sst5TMD4) and of the In1-ghrelin variant in

12

acromegaly patients, and examined their potential as therapeutic targets. A

second area of study aims at understanding the pathophysiological importance,

beyond somatic growth, of GH and somatotropes, which function as a true

endo-metabolic hub integrating, in a balanced manner, multiple components of

the homeostatic regulatory machinery of the glucose/insulin/IGF-I axis, whose

deregulation critically influences pathologies like diabetes, obesity and cancer.

Thus, departing from a comparative endocrinology onset, our journey provided

us a unique opportunity to achieve scientific knowledge, enjoy discoveries,

traverse fascinating translational research avenues, and treasure the privilege of

receiving the wisdom from GH-iant mentors.

13

Josep Planas' Conference (JP'C)

FROM INSULIN TO IGFS IN FISH: A SCIENTIFIC JOURNEY

Gutiérrez, J., Navarro, I., Fernández-Borràs, J., Blasco, J., Planas, J.V., Vélez,

E.J., Lutfi, E. and Capilla, E. E-mail: jgutierrez@ub.edu

Department of Physiology and Immunology, School of Biology, University of

Barcelona, Barcelona, Spain

A lecture in honor of Professor José Planas has been created in the 10th

congress of AIEC and we believe that it is fair to dedicate it to who can be

considered one of the first comparative endocrinologists in Spain. Dr. Planas

presented his thesis at the University of Barcelona (UB) in 1955, directed by

Prof. F. Ponz entitled “Insulin extraction from tuna (Thunnus thynnus, L.)” that

demonstrated his early interest in fish hormones. Later he also studied tuna

glucagon showing its effects and all these findings were reported in

international publications.

After a period at the University of Valladolid Dr. Planas returned to UB and

recovered the line of research in fish, directing the thesis of S. Zanuy and M.

Carrillo (1975) and later those of J. Fernández-Borràs and J. Gutiérrez (1985).

In 1984, in collaboration with A. Thorpe and IATS-CSIC, the first paper on sea

bass insulin was published, which was followed by a series of papers on insulin

function in different fish species. In 1986 the first international report on fish

plasma glucagon levels was published. Insulin receptor research was initiated

with E. Plisetskaya (1988/89) and was followed by the first characterization of

IGF-I receptors in fish (1993). During these years J. Planas and the group was

very active publishing an interesting series of papers on phylogenetic aspects of

insulin and IGF-I receptors and their role in fish glucose intolerance. After Dr.

Planas dead the group followed this research and E. Capilla and J.V. Planas

published the first report of a fish Glut4 (2000) and in 2001 the group published

for the first time the existence of IGF-II receptors in ectotherms.

In the last years, I. Navarro and J. Gutiérrez have dedicated a big effort to study

the regulation, mainly by IGFs and GH, of adipose tissue and muscle

development and physiology, generating numerous thesis and publications.

However, all this research has the origin in the pioneer work of Dr. Planas, who

was able to start it in a very difficult time for science in Spain, being the

predecessor of different teams that have contributed importantly to develop this

scientific area in our country.

(Supported by funds from MINECO, EU-FPs and SGR).

14

Closing Lecture (CL)

40 YEARS OF RESEARCH IN COMPARATIVE ENDOCRINOLOGY

Olivier Kah. E-mail: olivier.kah@univ-rennes1.fr

Research Institute in Health, Environment and Occupation (INSERM U1085),

University of Rennes 1, Rennes, France

Comparative endocrinology played an important role in understanding how our

hormones and their receptors are inherited from our ancestors, diversify through

genome duplications and gain new functions. By taking the example of GnRH,

this lecture intends to cover about 40 years of research in comparative

neuroendocrinology. The primary sequence of GnRH was published in 1971

and through the evolution of the field, we shall see how ideas evolved, dogma

collapsed and new concepts appear that, in some cases, totally changed the way

we have to consider our past and future research.

15

Oral

Communications

16

O1

SPECIFIC RECOMBINANT GONADOTROPINS INDUCE

SPERMATOGENESIS AND SPERMIATION BY FIRST TIME IN A

TELEOST FISH, THE EUROPEAN EEL

David S. Peñaranda1, V. Gallego

1, M.C. Vílchez

1, L. Pérez

1, A. Gómez

2, I.

Giménez3, J.F. Asturiano

1. E-mail: dasncpea@upvnet.upv.es

1Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología

Animal. Universitat Politècnica de València. Spain. 2Instituto de Acuicultura de

Torre la Sal (CSIC). Castellón, Spain. 3Rara Avis Biotec S.L., Valencia, Spain.

Eel recombinant gonadotropins have induced spermatogenesis in vitro, but had

limited effect in vivo. New specific single-chain recombinant gonadotropins

(Rara Avis Biotec S.L.) were tested treating European eel males with rFSH and

rLH. Males received rFSH in three doses (2.8, 1.4 and 0.7 µg/fish; high,

medium and low treatments) during 3 weeks. Later, an increasing rLH dose

(every 3 weeks; 1, 2, 6 µg/fish) was combined with rFSH. Three

males/treatment were sacrificed every 3 weeks. Blood and testis samples were

taken for steroids and histological analyses and GSI was calculated. Sperm

quality was determined by CASA software.

rFSH by itself was able to induce the spermatogenesis (week 3), causing higher

androgen levels and development until the spermatogonia 2 stage in the high

rFSH group. The rLH (week 6) promoted further maturation to spermatocyte 2

stage in the medium and high groups, but no gonadal progression was observed

in the low group. Higher T levels were reached in all the groups, and the biggest

11KT values in the rFSH high group. A second increase of rLH (week 9)

induced further maturation to spermatid-spermatozoa 1 and spermatocyte 1

stages in the high and low groups, respectively. The medium group showed no

further maturation (spermatocyte 1-2 stages). The highest dose of rLH (week

12) induced the most advanced stage (spermatozoa 2), and significant GSI

increase in all the groups. A progressive 11KT (high group) and T (all

treatments) decrease was observed from week 9 to 12. All treatments induced

spermiating males, however, the best sperm quality with ?50% motile cells and

volumes ~0.4 ml was observed in males treated with the highest rFSH dose and

a progressive increase of rLH treatment. On the other hand, a 20% of non-

responders were observed and the sperm quality was variable. So, further

experiments are required to improve these treatments.

Funded by IMPRESS (Marie Sklodowska-Curie Actions; Grant agreement nº:

642893) and MINECO (REPRO-TEMP; AGL2013-41646-R).

17

O2

CHARACTERIZATION OF NUCLEAR AND MEMBRANE

PROGESTIN RECEPTORS IN EUROPEAN EEL, AND THEIR

EXPRESSION IN VIVO THROUGHOUT SPERMATOGENESIS

Marina Morini1, D.S. Peñaranda

1, M.C. Vílchez

1, R. Nourizadeh-Lillabadi

2,

J.F. Asturiano1, F.A. Weltzien

2, L. Pérez*

1. E-mail: mlpereig@dca.upv.es

1Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología

Animal. Universitat Politècnica de València, Valencia, Spain. 2Department of

Basic Sciences and Aquatic Medicine, Norwegian University of Life Sciences.

Campus Adamstuen, Oslo, Norway.

In male teleost, the role of progestins as 17α,20β-dihydroxy-4-pregnen-3-one

(DHP) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20βS), have been reported

in the regulation of spermatogenesis. Two mechanisms of action can mediate

the progestins action: the classic genomic mechanism of steroid action relatively

slow involving nuclear receptors (nPRs or Pgrs), members of the nuclear steroid

receptor superfamily; and the membrane progestin receptors (mPRs), members

of the progestin and adipoQ receptor (PAQR) family, which exert an often non

genomic, rapid activation of intracellular signal transduction pathways.

We characterized and studied the mRNA expression of two nuclear progestin

receptors (pgr1 and pgr2) and for the first time in a teleost of five membrane

progestin receptors (mPRα, mPRγ, mPRδ, mPRAL1, mPRAL2) in brain,

pituitary and gonads, in the European eel. mPR phylogeny placed three eel

mPRs together with vertebrate mPRα, and were called mPRα, mPRAL1 (alpha-

like1) and mPRAL2 (alpha-like2). The two other eel mPRs (i.e.: mPRγ, mPRδ)

clustered together with their respective mPR types amongst vertebrate

representatives.

Eel tissue distributions reveal that nPR and mPR transcripts were expressed in

neuroendocrine and non-reproductive tissues, which suggest the involvement of

these receptors not only in reproduction, but also in other physiological

functions. Furthermore, in vivo studies of mRNA expression throughout

spermatogenesis suggest that progestins exert their actions in the eel brain and

pituitary by both nPRs and mPRs. In the testis, the nuclear pgr2 and membrane

mPRγ and mPRδ seem to be involved on the induction of meiosis, whereas

mPRa, mPRAL1 and mPRAL2 seems to be involved on the process of final

sperm maturation.

The emergence of both mPRs and nPRs in early vertebrates and the

colocalisation of both mPRs and nPRs mRNA suggest the possibility that they

may act in concert to regulate the progestin-mediated functions.

18

03

ASSESSMENT OF THE RA-SIGNALLING PATHWAY DURING

GONADAL DEVELOPMENT AND MATURATION IN THE EUROPEAN

SEA BASS (Dicentrarchus labrax)

Paula Medinaac

, A. Gómezb, S. Zanuy

b, M. Blázquez

a. E-mail:

blazquez@icm.csic.es

aInstitut de Ciències del Mar (ICM-CSIC), Barcelona, Spain.

bInstituto de Acuicultura de Torre la Sal (IATS-CSIC), Castellón, Spain.

cUniversidad de Antofagasta, Antofagasta, Chile.

In higher vertebrates, the retinoic acid (RA) signalling pathway stimulates

meiosis by regulating the transcription of several genes that result in a spatio-

temporal balance between the synthesis and degradation of RA. The goal of this

research is to gain a better understanding of the role of the RA-signalling

pathway in fish meiosis using the European sea bass as a model. The study is

focussed on the expression of nine RA-related genes by the time of sex

differentiation and the onset of meiosis in males and females. Moreover, in

females it was also extended to the first vitellogenesis and final maturation. The

selected genes include a retinol receptor (stra6), oxidizing proteins that

synthesize RA (aldh1a2, aldh1a3), binding proteins (crabp, crbp4), nuclear

receptors (rarα, rxrα, pparγ), and the RA catabolizing enzyme (cyp26a1). The

results show that in males, stra6, crbp4, rarα, rxrα, cyp26a1, and crabp reached

lowest levels by the onset of puberty. The highest expression of aldh1a2,

aldh1a3, and pparγ was attained after sex differentiation, decreasing to their

lowest levels by the time of puberty. In females, a steady increase of stra6, rarα,

rxrα, and cyp26a1 up to the start of vitellogenesis, was followed by a

progressive decrease during maturation. A similar scenario applied for aldh1a2,

aldh1a3, pparγ and crabp, although in this case, a final increase occurred by the

time of maturation. The decrease in the expression of cyp26a1 in both sexes

marked the onset of meiosis, indicating that an increase in the availability of RA

is required to trigger the process. In addition, in females, an increase in the

levels of both aldhs and thus of RA was also observed by the time of final

oocyte maturation, coinciding with the resumption of meiosis, further

supporting the need of high RA levels for the process. Regardless of these

evidences, more studies are required to elucidate the influence of RA in fish

meiosis and the network of genes that might be involved.

Funded by projects Reprosex (AGL2011-28890) and Reprobass

(PROMETEOII/2014/051). PM was sponsored by a grant MECE2 (ANT0806).

19

04

DIFFERENTIAL ACTIVATION OF KISS RECEPTORS BY KISS1 AND

KISS2 PEPTIDES IN THE SEA BASS

Alicia Felip, F. Espigares, S. Zanuy, A. Gómez. E-mail: afelip@iats.csic.es

Instituto de acuicultura de Torre de la Sal (CSIC), Ribera de Cabanes,12595

Castellón, Spain

Two forms of kiss gene (kiss1and kiss2) have been described in the teleost sea

bass. This study assesses the cloning and characterization of two Kiss receptor

genes, namely kissr2 and kissr3 (known as gpr54-1b and gpr54-2b,

respectively), and their signal transduction pathways in response to Kiss1 and

Kiss2 peptides. Phylogenetic and synteny analyses indicate that these paralogs

originated by duplication of an ancestral gene before teleost specific

duplication. The kissr2 and kissr3 mRNAs encode proteins of 368 and 378

amino acids, respectively, and share 53.1% similarity in amino acid sequences.

In silico analysis of the putative promoter regions of the sea bass Kiss receptor

genes revealed conserved flanking regulatory sequences among teleosts.

Both kissr2 and kissr3 are predominantly expressed in brain and gonads of sea

bass, medaka and zebrafish. In the testis, the expression levels of sea bass

kisspeptins and Kiss receptors point to a significant variation during the

reproductive cycle.

In vitro functional analyses revealed that sea bass Kiss receptor signals are

transduced both via the protein kinase C (PKC) and protein kinase A (PKA)

pathway. Synthetic sea bass Kiss1-15 and Kiss2-12 peptides activated Kiss

receptors with different potencies, indicating differential ligand selectivity. Our

data suggest that Kissr2 and Kissr3 have a preference for Kiss1 and Kiss2

peptides, respectively, thus providing the basis for future studies aimed at

establishing their physiologic roles in sea bass.

Supported by GV (GV06/268) and (ACOMP2010), Programa PROMETEO

(Prometeo/2010/003; PrometeoII/2014/051) and EU grant 655 FP7222719

(LIFECYCLE). A.F was supported by a Ramón y Cajal and F.E by a JAE-

Predoc CSIC.

20

05

EFFECT OF HIGH REARING DENSITY ON SEX DIFFERENTIATION

IN ZEBRAFISH (Danio rerio): SEX RATIO AND GENE EXPRESSION

ANALYSIS

Alejandro Valdivieso, L. Ribas, F. Piferrer. E-mail:valdivieso@icm.csic.es

Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas.

Passeig Marítim, 37–49. 08003 Barcelona

Fish manifest all kind of reproduction systems known in vertebrates in which

sex determination and sex differentiation establish the final gender. Rearing

density is one of the most studied environmental factors in fish and it has been

shown that some species placed at high density can exhibit altered sex ratios,

usually in favor of males. In addition, it has been shown that some fish

subjected to high density exhibit increased cortisol plasma levels, which can

have masculinizing effects. Here, we used a domesticated zebrafish strain,

which behaves as having a polygenic system of sex determination, being more

susceptible to the environmental factors than the wild type strains. To

understand the effect of density and cortisol on sex determination and

differentiation in zebrafish we examined population sex ratios and gene

expression in ovaries and testes of juveniles by RT-PCR. We were interested in

canonical reproduction-related genes, genes involved in the glucocorticoid

pathway and genes related to epigenetic regulatory mechanisms. To better

determine the role of density and final sex of the individual we set up two

experiments. In experiment one fish were subjected to different densities during

6–90 days post fertilization (dpf) while in experiment two fish were fed using

different cortisol treatments between 10–45 dpf. Elevated densities resulted in

an increase in the number of males while cortisol treatment resulted in complete

masculinization of the population. Moreover, elevated density downregulated

nr3c1, cy19a1a and dnmt1 in both ovaries and testes. However, despite the

observed masculinization, cortisol treatment did not result in significant

differences in gene expression. Taken together, these results show that density

affects sex ratios in zebrafish, inducing masculinization. Results also show that

high density downregulated the expression of gene related to the female

differentiation pathway. Nevertheless, the underlying mechanisms related to the

masculinizing effects of cortisol still remain unclear.

Supported by MINECO grant AGL2013-41047-R “Epifarm” to FP.

21

06

DAILY VARIATIONS OF CARBOHYDRATE AND LIPID

METABOLISM-RELATED PARAMETERS IN LIVER OF RAINBOW

TROUT. INFLUENCE OF LIGHT AND FOOD AVAILABILITY.

Juan Hernández-Pérez*, Jesús M. Míguez, J.L. Muñoz

#, Fatemeh Naderi, Marta

Librán-Pérez, Rosa Ceinos, Marcos A. López-Patiño. E-mail:

juan.hernandez.perez@uvigo.es

Laboratorio de Fisioloxía Animal. Dept. Bioloxía Funcional e Ciencias da

Saúde, Facultade de Bioloxía, Universidade de Vigo; and ECIMAT. #Current

address: Centro I-Mar. Universidad de Los Lagos (Chile).

Most physiological processes, such as feeding behaviour and energy

metabolism, exhibit circadian rhythmicity, thus playing a main role in the

maintenance of the functional homeostasis. Light-dark and feeding-time cycles

are mainly entraining such processes in order to adjust them to the

chronological arrangement of the external world. The aim of the current study

was to investigate in rainbow trout the existence of daily changes in activity and

mRNA abundance of proteins involved in major pathways of carbohydrate and

lipid metabolism in liver. The influence exerted by both the light-dark cycle and

food availability in synchronizing such rhythms was also evaluated. For this

purpose, four cohorts of animals previously adapted to 12L:12D (lights on at

ZT0; feeding time at ZT2) were subjected to: normal conditions (LD); 48-h

light isolation (DD); 96-h food deprivation (LD + Fasting); or food deprivation

and light isolation (DD + Fasting) respectively. After such time periods, fish

were sacrificed and sampled every 4-h on the following 24-h period (ZT/CT0,

4, 8, 12, 16, 20 and 0´). Our results reveal gk, pepck, g6pase, pk, glut2, hoad

and fas rhythms of mRNA abundance to persist in the absence of light and/or

food as synchronizers. Enzyme activities did not display significant daily

variations although these parameters mostly fluctuated in parallel to the

respective gene expression. Rhythms of mRNA abundance did mostly depend

on the presence of food, but the influence of the light/dark cycle could not be

discarded. The present results indicate that liver metabolism in trout is linked to

a circadian system which may play a main role in generating rhythms of

enzymes expression, but also influences enzyme activities. Further research

must be carried out in order to understand such interaction.

Acknowledgements: Funded by Ministerio de Economía y Competitividad and

European Fund for Regional Development (AGL2013-46448-3-1-R and

FEDER) and AEO-ECIMAT.

22

O7

EFFECTS OF GHRELIN ON THE RESPONSE TO OLEATE OF

HYPOTHALAMIC FATTY ACID SENSORS IN RAINBOW TROUT

(Oncorhynchus mykiss): INVOLVEMENT IN THE CONTROL OF FOOD

INTAKE

Cristina Velasco, M. Librán-Pérez, C. Otero-Rodiño, F. Naderi, R. Álvarez-

Otero, M.A. López-Patiño, J.L. Soengas. E-mail: cvrubial@uvigo.es

Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e

Ciencias da Saúde. Facultade de Bioloxía, Universidade de Vigo and ECIMAT.

We have previously demonstrated the existence in hypothalamus of rainbow

trout of fatty acid sensing systems responding to changes in the levels of LCFA

like oleate or MCFA like octanoate. The activation of these systems induces an

anorectic effect through changes in the expression of hypothalamic

neuropeptides like NPY/AgRP and POMC/CART. The hypothalamic neurons

possessing these sensors are known in mammals to integrate information from

levels of circulating hormones like insulin, leptin or ghrelin. Ghrelin is a

gastrointestinal hormone that plays an important role in the regulation of

hormone release, energy balance, and control of food intake. We hypothesize

that ghrelin counteracts the response to increased fatty acid levels of

hypothalamic fatty acid sensing systems. Thus, we administered ICV to 100g

rainbow trout 1µl of saline alone (control) or containing 2 ng ghrelin, 1 µmol

oleate or both ghrelin+oleate. After oleate treatment we observed an activation

of fatty acid sensor systems, increase of anorectic potential and inhibition of

food intake in agreement with our previous studies thus validating the

experimental design. After ghrelin treatment, we observed a significant

increased production of orexigenic factors (NPY, AgRP) and a significant

decrease in the production of anorexigenic factors (POMC and CART) whose

overall balance would be an increased orexigenic potential also in agreement

with the finding of a raised food intake. The administration of oleate+ghrelin

resulted in a response of fatty acid sensing systems, expression of

neuropeptides, and food intake similar to control group, thus suggesting that

ghrelin is effectively counteracting the effects induced by raised oleate levels.

We therefore demonstrate, for the first time in fish, the modulatory action of

ghrelin of the metabolic hypothalamic integration related to the control of food

intake.

Acknowledgements: Funded by Ministerio de Economía y Competitividad and

European Fund for Regional Development (AGL2013-46448-3-1-R and

FEDER) and AEO-ECIMAT.

23

O8

FEEDING-INDUCED CHANGES IN THE GHRELINERGIC SYSTEM

IN THE PLASMA, BRAIN AND INTESTINAL BULB OF GOLDFISH

(Carassius auratus)

Ayelén M. Blanco, M. Gómez-Boronat, I. Redondo, A.I. Valenciano, M.J.

Delgado. E-mail: ayelenmelisablanco@ucm.es

Department of Physiology (Animal Physiology II), Faculty of Biology,

University Complutense of Madrid, Spain

Ghrelin is a gut-derived peptide hormone with a key role in food intake

regulation and energy balance. In mammals, ghrelin secretion and expression is

known to increase prior to a meal, suggesting a role for this hormone in hunger

and meal initiation. However, results regarding this aspect in fish are

controversial. Therefore, the aim of this study was to characterize the pre- and

postprandial pattern of plasma acyl-ghrelin (AG), desacyl-ghrelin (DAG) and

ghrelin O-acyl transferase (GOAT) in goldfish (Carassius auratus) that were

either fed or unfed the day of the experiment. Moreover, the expression of

preproghrelin, goat and growth hormone secretagogue receptors (ghs-r) was

quantified in brain and peripheral tissues. Circulating hormone levels were

measured by immunoassays, and gene expression was determined by real-time

RT-PCR. Results show a significant preprandial rise in circulating levels of AG,

but not DAG, and GOAT, supporting the role of AG as a meal initiator in this

teleost. Consistently, preproghrelin and ghs-r1a1 expression increases 1-h

before scheduled feeding time in intestinal bulb, suggesting that this receptor

subtype might be involved in the preprandial action of ghrelin in this tissue.

Neither preproghrelin nor ghs-r1a1 are observed to be modified postprandially

in intestinal bulb; by contrary, goat and ghs-r1a2 mRNA levels are significantly

higher in unfed fish after subjective feeding time. In brain, none of the studied

genes are modified preprandially, except for goat in the hypothalamus, whose

expression is reduced 1-h before scheduled feeding time. Postprandially, a

significant increase in mRNA levels of unfed fish is detected for preproghrelin

in telencephalon, goat in both telencephalon and hypothalamus, ghs-r1a1 in

vagal lobe, ghs-r1a2 and ghs-r2a1 in hypothalamus, and ghs-r2a2 in

telencephalon and vagal lobe. Collectively, the periprandial variations observed

in this study support the role of ghrelin as an orexigenic factor in fish, and show

for the first time a tissue-dependent implication of GOAT and the four subtypes

of GHS-R in the responses of the ghrelinergic system to feeding.

Acknowledgements. Supported by the Spanish MINECO (AGL 2013-46448-C3-

2-R). A.M. Blanco and M. Gómez-Boronat are predoctoral fellows from the

Spanish MEC and MINECO, respectively.

24

O9

SERUM METABOLOME OF FASTED GILTHEAD SEA BREAM. A

NON-TARGETED APPROACH FOR THE IDENTIFICATION OF

ROBUST BIOMARKERS OF MALNUTRITION IN FISH

Rubén Gil Solsona1, J. Nácher-Mestre

1, L. Lacalle

1, J.A. Calduch-Giner

2, J.V.

Sancho1, F. Hernández

1, J. Pérez-Sánchez

2. E-mail: ruben.gil@uji.es

1Research Institute for Pesticides and Water (IUPA), University Jaume I,

Castellón, Spain.

2Nutrigenomics and Fish Growth Endocrinology Group, Institute of

Aquaculture Torre de la Sal (CSIC), Castellón, Spain.

Up-stream regulators of the endocrine cascade are largely altered by

malnutrition due to feed deprivation or unbalanced diet composition. This

includes many changes in circulating catabolic and growth-promoting factors,

also related to plasma metabolite levels. This complex trade-off has been

addressed in both fish and higher vertebrates, although modern omic

approaches are required to identify and validate the most robust biomarkers

with either diagnostic and/or prognostic value. For this goal, a non-targeted

metabolomics approach is developed.

Nuclear magnetic resonance has been often used for routine metabolomic

profiling in a vast array of species due to its high elucidation power and

universality. However, liquid chromatography-high resolution mass

spectrometry (LC-HRMS) has emerged as a reliable alternative technique with

greater sensitivity and selectivity. This methodological advantage has been

exploited to assess the serum metabolome of two-year old gilthead sea bream

(Sparus aurata) in continuously fed fish and 10-days fasted fish.

Feed deprivation resulted in a significant reduction of condition factor (2.91 vs

2.63), hepatosomatic index (1.32 vs 0.90) and mesenteric fat index (1.90 vs

1.64). Deproteinized serum samples were analysed by LC-HRMS for individual

fish (n=10) and data was peak picked, aligned and normalized with XCMS.

Partial Least Squares–Discriminant Analysis (PLS-DA) clearly separated the

two experimental groups, explaining the first component more than 90% of

total variance. Orthogonal PLS-DA highlighted more than 850 differentiation

ions (P>0.80), which primarily yielded 46 compounds that were classified into

different physiological pathways (phospholipid metabolism, lysophosphatidic

acid metabolism, fatty acid oxidation, bile salt metabolism, methionine-cysteine

metabolism, amino acid catabolism, biotin metabolism). A targeted refined

search in combination with transcriptional validation procedures were

conducted to go further into fish biomarker research.

The study was partially funded by the ARRAINA EU Project.

25

O10

ENERGY SENSING IN GILTHEAD SEA BREAM. MOLECULAR AND

FUNCTIONAL CHARACTERIZATION OF SIRTUINS

Paula Simó, J.A Calduch-Giner, J. Pérez-Sánchez. E-mail: paula.simo@csic.es

Nutrigenomics and Fish Growth Endocrinology, Institute of Aquaculture Torre

de la Sal, CSIC, Spain.

Sirtuins (SIRTs) use nicotinamide adenine dinucleotide (NAD+) as co-substrate

in their acetylated reactions, sensing specific metabolic states that intimately

link the intracellular NAD+/NADH ratio with post-translational modifications

of histones and metabolic enzymes. Several examples in mammals agree with

this, and the increase of the intracellular NAD+/NADH ratio with dietary

restriction and exercise leads to increase SIRT activity and mRNA expression.

Thus, SIRTs are master regulators of energy metabolism and nearly all enzymes

involved in glycolysis, gluconeogenesis, FA oxidation, AA catabolism and

OXPHOS have been identified as acetylated proteins in the proteome of

mammalian cells. The SIRT family is virtually ubiquitous throughout all

kingdoms of life with a number of SIRTs that ranges from one in bacteria to

seven in vertebrates, but few studies have addressed the molecular

characterization and functional analysis of SIRTs in livestock animals and fish

in particular. To solve this gap of knowledge, the molecular characterization and

gene expression regulation of SIRTs was assessed in gilthead sea bream.

Searches in the Nutrigroup transcriptomic database (www.nutrigroup-

iats.org/seabreamdb) yielded seven full nucleotide sequences with a conserved

enzymatic core domain (SIRT1-7) and mitochondrial targeting sequences

(SIRT3-5). Phylogenetic analysis identified three major clades corresponding to

SIRT1-3, SIRT4-5 and SIRT6-7 that reflected the present hierarchy of

vertebrates. Transcriptional studies corroborated a ubiquitous gene expression

that was tissue-specific for each SIRT isoform. This was evidenced by PCA and

hierarchical clustering which identified two main clusters corresponding to

SIRT isoforms with relatively high (SIRT1, 2 and 5) and low (SIRT3, 4, 6 and

7) expression levels. A nutritional regulation was also evidenced in 10-days

fasted fish, and SIRT2, 3 and 4 exhibited an overall down-regulated expression.

In contrast, the overall response of SIRT 1, 5, 6 and 7 was the up-regulation,

which is indicative of the different adaptive responses to cope with the

changing tissue-energy demands.

The study was funded by EU (ARRAINA) and Spanish (Mi2-Fish,

PrometeoII/2014/85) Projects.

26

O11

METFORMIN EXERTS DIRECT ANTI-PROLIFERATIVE EFFECTS IN

PROSTATE CANCER CELLS IN VITRO AND INHIBITS PROSTATE

TUMOR GROWTH IN VIVO UNDER NORMAL-FED AND,

SPECIALLY, UNDER HIGH FAT FED CONDITIONS.

André Sarmento-Cabral, F. López-López, M.D. Gahete, J.P. Castaño, R.M.

Luque. E-mail: amsbcabral@gmail.com

Department of Cell Biology, Physiology and Immunology, University of

Córdoba; Hospital Universitario Reina Sofía, Instituto Maimónides de

Investigación Biomédica de Córdoba (IMIBIC); CIBER Fisiopatología de la

Obesidad y Nutrición (CIBERobn); and, Campus de Excelencia Internacional

Agroalimentario (ceiA3), Córdoba, Spain.

Obesity (Ob) is a chronic endocrine-metabolic disease and one of the most

serious and complex threats for the human health, which is associated with an

increased incidence of some types of cancers such as prostate cancer (PC), the

second most common cancer in men worldwide. Interestingly, metformin (Met),

an antidiabetic drug, might represents a very promising opportunity to treat Ob

and PC as some retrospective clinical studies have shown that the incidence of

PC is lower in patients treated with Met. However, the endocrine-metabolic,

cellular and molecular mechanisms underlying the association between Ob and

higher incidence/aggressiveness of PC and the putative pharmacological

effectiveness of Met in PC are still unknown. We used primary normal prostate

(NP) cell cultures from mice and human PC cell lines (PC3, 22Rv1 and LNCaP)

as well as, immuno-suppressed mice inoculated with PC3 cells, fed a high-fat

diet (HFD) or low-fat diet (LFD), as models to test the beneficial effect of Met

(in vitro: 10µM-10mM; in vivo: 250mg/Kg). Our results indicate that Met

modulates key metabolic, endocrine and pathologic components (e.g.

expression of components of insulin/IGF-I/somatostatin/ghrelin systems) in NP

cell cultures. Interestingly, Met had no evident effect on 22Rv1 cells

proliferation but significantly reduced PC3 and LNCaP cells proliferation

and/or migration. Remarkably, we found that Met also have a significant

beneficial in vivo effect as it reduced tumor volume and weight in mice fed a

LFD and, specially, on those fed a HFD compared to their vehicle-treated

control mice. Altogether, our data suggest that Met modulates NP cell function

and exerts beneficial effects in the inhibition of PC cells growth in vitro and in

vivo, specially, under HF-conditions.

Funding: PI13-00651, BFU2013-43282-R, BIO-0139, CTS-1406, PI-0639-

2012, and CIBERobn.

27

O12

WIDE-TRANSCRIPTOMIC ANALYSIS OF INTESTINE IN

MEDITERRANEAN FISH

J. Pérez-Sánchez1, Josep A. Calduch-Giner

1, A. Sitjà-Bobadilla

2. E-mail:

jaime.perez.sanchez@csic.es

1Nutrigenomics and Fish Growth Endocrinology, Institute of Aquaculture Torre

de la Sal, CSIC, Spain.

2Fish Pathology, Institute of Aquaculture Torre de la Sal, CSIC, Spain.

The intestinal tract of teleosts is involved in water and electrolyte balance,

immunity, and endocrine regulation of nutrient availability and metabolism.

Hence, gastrointestinal syndromes due to nutritional deficiencies and infectious

agents are important causes of low growth performance, poor health or serious

mortalities in farmed fish. We presented the current research conducted in our

laboratory with gilthead sea bream and European sea bass. Attention is mostly

focused on the plasticity at the molecular level of the intestine tract in order to

identify/validate new intestine health biomarkers of diagnostic and/or

prognostic value.

In a first stage, microarray expression profiling of sea bass intestine highlighted

almost a constant gene expression profile of middle (MID) and anterior (ANT)

intestine segments. Conversely, more than 1,800 genes were differently

expressed between posterior (POS) and MID-ANT intestine. Thus, POS

intestine emerged as a highly immune-regulated tissue, with also relevance on

vitamin B12 and bile acid metabolism (entero-hepatic pathway). The

differential expression of different chemosensors along the intestine tract was

also evidenced. This issue was further verified in sea bream and more than

5,000 unique genes were differentially expressed across the intestine, although

this different expression pattern is especially evident in active feeding periods

(summer). Microarray and RNA-seq only revealed minor gene expression

changes in fish fed balanced low FM/FO diets and interestingly the use of feed

additives is able to reverse most of the observed changes towards the wild

phenotype. When season was considered, the number of differentially expressed

genes (summer vs winter) was higher than 6,500 genes. The magnitude of the

change is even higher than that reported after massive destruction of intestine

epithelium in chronically parasitized fish. Likewise, previous studies reflected

that the plasticity of the transcriptome of anaerobic and highly oxidative muscle

tissues is quite lower in comparison to that of the intestine tract.

The study was funded by EU (ARRAINA) and Spanish (Mi2-Fish,

PrometeoII/2014/85) Projects.

28

O13

TRANSCRIPTOMICS PROVIDE INSIGHT INTO MUSSEL (Mytilus

galloprovincialis) MANTLE FUNCTION

Nadège Zaghdoudi-Allan1, T. Yarra

2, A. Churcher

1, R. Felix

1, M. Clark

2 and

D.M. Power1. E-mail: nallan@ualg.pt

1Comparative Endocrinology and Integrative Biology, Centre of Marine

Sciences, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro,

Portugal. 2British Antarctic Survey, Natural Environment Research Council,

High Cross, Madingley Road, Cambridge, CB3 0ET, UK.

With over 90,000 extant species, phylum Mollusca is the most successful group

of animals on Earth. Common to all molluscs, the mantle is a multi-functional

highly muscular tissue that contacts the shell and envelops vital organs. In

bivalves, the epithelial cells of the mantle secrete the external shell by a

complex network of mechanisms that remain poorly understood. We

hypothesize that the mantle edge in Mytilus species is heterogenous in cellular

structure and function and use next generation sequencing to test this

hypothesis. The mantle edge of the Mediterranean mussel (Mytilus

galloprovincialis) was sectioned into three parts and sequenced using Illumina

(HiSeq 1500). The transcriptome sequences generated assembled into 18440

genes with an N50 of 2055. The transcriptome was annotated and GO

(geneontology.org) and String analysis (http://www.string-db.org) used for a

preliminary characterisation of biological processes. To test our hypothesis, we

compared the transcripts from the 3 mantle segments and the expression levels

of 8 candidate genes in the sectioned mantle of 6 individuals using qPCR.

Candidates were chosen based on their regulatory function and potential

involvement in shell formation. Candidates such as Dermatopontin and SMAD

are known to regulate cell proliferation and growth. While the Buccalin-related

neuropeptide mediates neuromuscular transmission in molluscs. Our results

show significant differences in transcript abundance and cellular function

amongst the three mantle sections. Combining our transcriptomic study with

histological studies of the mantle tissue, we present evidence of both molecular

and structural heterogeneity of the mussel mantle and identify several putative

regulatory networks.

Acknowledgements: This study was financed by the European Regional

Development Fund (ERDF) COMPETE and Portuguese funds through

Foundation for Science and Technology (FCT, project UID/Multi/04326/2013)

and CACHE a Marie Curie ITN (PITN-GA-2013-605051).

29

O14

THE SKIN AS AN ENDOCRINE ORGAN: INSIGHTS FROM THE SEA

BASS SKIN TRANSCRIPTOME

Patrícia I. S. Pinto1, M. A. S. Thorne

2 and D. M. Power

1. E-mail:

ppinto@ualg.pt

1 CCMAR - Centre of Marine Sciences, Universidade do Algarve, Faro,

Portugal. 2 British Antarctic Survey (BAS), Cambridge, UK

The skin is an important organ with a wide range of roles including mechanical,

thermal and immune barrier functions and its sensory and communication

functions. Significant attention has been paid to understanding skin homeostasis

and regeneration in mammals but much less is known about this organ in other

vertebrates. The skin is a target for several hormones that regulate its multitude

of functions including pigmentation and appendage growth. Fish skin has a

similar structural organization to mammals with the exception of the calcified

scale and the outer non-keratinized epithelia and it has specialized roles in

osmoregulation and mineral homeostasis. The endocrine regulation of the

function of skin in fish is not very well explored and the emerging concept of

skin as an endocrine organ is unstudied in fish. The present study uses next-

generation sequencing to explore the potential endocrine function of skin, by

generating the transcriptome of sea bass (Dicentrarchus labrax) dorsal skin.

The sea bass skin transcriptome contained 31,856 unique transcripts with

minimum 500 and average 1070 base pairs of length, of which approx. 97%

were assigned to the sea bass genome and 94% to existing proteins or cDNAs in

databases. A focused analysis identified several hormones and receptors from

different systems (including steroid or thyroid hormones and calcemic

hormones) which are expressed in the skin. The results from in silico and

targeted quantitative PCR support the role of fish skin as both a target and a

source of hormones. They will be an important resource for future research on

the impact of the environment on skin functions as well as the recognition of

the skins contribution to whole organism physiology, with potential relevance to

aquaculture production, fish welfare and conservation.

Acknowledgments: Funded by FCT – Portuguese Science and Technology

Foundation through projects UID/Multi/04326/2013 and PTDC/AAG-

GLO/4003/2012 and grant FCT SFRH/BPD/25247/2005 to PISP.

30

O15

A MICROARRAY STUDY OF SKIN AND SCALE REGENERATION IN

THE SEA BREAM (Sparus aurata)

Rita Alves Costa, F. Vieira and D.M. Power. Email: racosta@ualg.pt

Comparative Endocrinology and Integrative Biology, Centre of Marine

Sciences, University of Algarve, Campus of Gambelas, 8005-139 Faro,

Portugal

Wound healing in teleost fish is a multistage process that involves several

biological processes that overlap in time and space and re-establish tissue

integrity. We previously reported using a validated microarray that 3 days after

scale removal in sea bream significantly up-regulated genes were involved in

cell cycle regulation, cell proliferation and adhesion, immune response and

antioxidant activities and that by day 7 only 8 genes were up-regulated (Vieira

et al. 2011). In the present study we targeted down-regulated genes in the

microarray to identify regulatory factors and immune processes that are

suppressed during scale regeneration. The results revealed that 3 days after scale

removal 109 genes were down-regulated. The 72 annotated probes were

analysed using STRING and the most represented biological processes were

vascular endothelial growth factor receptor signalling (3.2e-4

), endothelial cell

proliferation (3.2e-4

), cell migration and motility (8.4e e-4

), hydrogen peroxide

metabolic process (6e-3

) and leukocyte migration (6.7 e-3

). Several genes

associated with innate and acquired immunity and the inflammatory response

(17/72) were identified amongst down-regulated transcripts, although in the

previous study up-regulated immunoendocrine genes were identified (10/53).

To carry out a detailed analysis of the neuroimmunoendocrine response during

regeneration a new experiment was performed in which scales were removed

from the left flank of adult sea bream and regenerating and intact skin and

scales were collected at 0 and 6 hours and 1, 2, 3 and 4 days after scale removal.

The results revealed that genes related with the cellular response of the innate

immunity, such as myeloperoxidase (mpo), cytochrome b-light chain (p22phox)

and macrophage colony stimulating factor 1 receptor (csf1r) were down-

regulated while the expression of galectin-3-binding protein precursor (gal3)

and receptor-transporting protein 2 (rtp2) varied during the time course studied.

This study demonstrated that during four days of skin and scale regeneration in

sea bream there is a balance between different regulatory processes that dictate

the stimulation and impairment of immune related genes.

Acknowledgements: RC is in receipt of FCT scholarship SFRH/BD/81625/2011

grant. Vieira et al. BMC Genomics 2011, 12:490

31

O16

MULTI-CLASS SCREENING OF FEEDSTUFF CONTAMINANTS AND

POTENTIAL ENDOCRINE DISRUPTORS IN FARMED FISH

Jaime Nácher-Mestre1, R. Serrano

1, M. Ibáñez

1, T, Portolés

1, M.H.G.

Berntssen2, F. Hernández

1, J. Pérez-Sánchez

3. E-mail: nacher@uji.es

1Research Institute for Pesticides and Water (IUPA), University Jaume I.

2National Institute of Nutrition and Seafood Research (NIFES), PO Box 2029,

Nordnes, N-5817 Bergen, Norway.

3Nutrigenomics and Fish Growth Endocrinology Group, Institute of

Aquaculture Torre de la Sal (CSIC).

Food and feedstuffs could seat undesirables associated with potential risk for

animal welfare and food safety. Pesticides, polycyclic aromatic hydrocarbons

(PAHs), classical halogenated organic contaminants (HOCs), mycotoxins,

emergent pharmaceuticals and personal care products (PPCPs), marker dyes

among others, may be an unintentionally contamination source. These

contaminants have several potential toxic outcomes by several modes of action,

including endocrine disruption and genotoxicity. To be aware of current and

recurrent disrupting affairs, an analytical strategy based on a generic extraction

followed by ultra-high performance liquid chromatography (LC) and gas

chromatography (GC) both coupled to high resolution mass spectrometry

(HRMS), using a quadrupole time-of-flight mass analyzer (QTOF MS), has

been developed and applied to real world samples.

Up to our knowledge, HOCs and mycotoxines do not represent a problem in

food safety although their study remains relevant. Only residue levels of

pirimiphos-methyl and chlorpyrifos have been mainly found in plant based feed.

These pesticides may disturb steroid production and biosynthesis, and

histopathological changes have been reported in animals. In addition, some light

PAHs have been found. These compounds may be related to steroid level

suppression and steroid synthesis inhibition as well as retinoid signaling

disruption. In some cases, the presence of unintended background levels of

illegal pharmaceutical dyes, unreported antibiotics, or unauthorized anti-

inflammatory agents might give cause to legal actions, making their monitoring

relevant. The potential applicability of this strategy helps to assure possible

emergent risk exposition of food and feed industry to more than 2500

compounds, including previous non-reported undesirables. The proposed

strategy is presented as a useful risk assessment tool in order to widen the

knowledge on novel ingredients and also traditional ingredients under use.

The study was funded by the ARRAINA EU Project.

32

O17

ACUTE STRESS REGULATES VASOTOCINERGIC AND

ISOTOCINERGIC SYSTEM IN THE GILTHEAD SEA BREAM (Sparus

aurata)

Arleta Krystyna Skrzyńska1, E. Maiorano

1, M.Bastaroli

1, G. Martínez-

Rodríguez2, J.A. Martos-Sitcha

2, J.M. Mancera

1. E-mail:

arleta.skrzynska@uca.es

1Department of Biology, Faculty of Marine and Environmental Sciences,

Campus de Excelencia Internacional del Mar (CEI-MAR), University of Cádiz,

11510 Puerto Real, Cádiz, Spain; 2Instituto de Ciencias Marinas de Andalucía,

Consejo Superior de Investigaciones Científicas (ICMAN-CSIC), 11510 Puerto

Real, Cádiz.

The hypothalamus-pituitary-interrenal axis is involved in stress response.

Although corticotrophin releasing hormone (CRH) is considered the main

hormone that activates this axis, other hormones as CRH binding protein (CRH-

BP), arginine vasotocin (AVT) and isotocin (IT) are considered as important

players in stress regulation. This study assessed, using the gilthead sea bream

(Sparus aurata) as a biological model, hypothalamic mRNA expression changes

of different endocrine factors (CRH, CRH-BP), as well as different

neuropeptides (AVT e IT) and their receptors (AVTR V1-type, AVTR V2-type,

and ITR) after an acute stress situation. Specimens were exposed to air for 3

min by lifting the wire-net cage out of the tank and put back in their respective

tanks after that, being sampled in a time course response (15 min, 30 min, 1, 2,

4 and 8 hours post-stress). At hypothalamic level, acute stress affects mRNA

expression of all precursors (pro-VT and pro-IT) and receptors (AVTRs and

ITR), as well as hormonal factors (CRH and CRH-BP), by increasing and

modulating their mRNA expression levels from the first 15 min post-emersion.

Furthermore, our data provide evidence on existence of different variation

pattern in the mRNA expression of both AVT receptors suggesting that each of

them can be involved in different phases of stress response to cope with this

adverse situation. Thus, the AVT V1-type receptor can be responsible for

primary endocrine control while the AVTR V2-type can be involved in the

regulation of several metabolic pathways to support energy demand

enhancement required during stress processes. Our results demonstrate the

activation of both endocrine pathways in the regulation of hypothalamic-

pituitary-interrenal axis of S. aurata, which is stated, at least, through changes in

mRNA expression levels of these genes.

33

O18

BRAIN MONOAMINERGIC NEUROTRANSMITTERS DURING

CHRONIC STRESS IN RAINBOW TROUT (Oncorhynchus mykiss).

Fatemeh Naderi1*

, Marcos A. López-Patiño1, Manuel Gesto

$, José M. Cerdá-

Reverter2, Cristina Otero-Rodiño

1, Cristina Velasco

1, Jesús M. Míguez

1. E-mail:

Fatemeh.Naderi79@uvigo.es

1Lab. Fisioloxía Animal. Dept. Bioloxía Funcional e Ciencias da Saúde, Fac.

Bioloxía, Universidade de Vigo; and ECIMAT. 2Dept. Fish Physiology and

Biotechnology, Inst. Acuicultura de Torre de la Sal (IATS-CSIC). $: Current

address: Natl. Institute of Aquatic Resources, Technical University of Denmark.

Stress is negatively affecting animal welfare in such a way that most behavioral

and physiological functions, such as food intake, are jeopardized. In fish, the

stress response initiates with the activation of the hypothalamus-sympathetic-

cromaffin (HPC), and the hypothalamus-pituitary-interrenal cells (HPI) axes

leading to increased plasma catecholamines and cortisol levels. In addition, our

previous results point to the important role played by both dopaminergic and

serotonergic systems in initiating the response to acute stress. However, little is

known regarding the involvement of both systems under chronic stress, but also

their influence on food intake. To address that question an experiment was

performed consisting on stressing rainbow trout (Oncorhynchus mykiss) by high

stocking density for 3 and 10 days. Food intake was evaluated all over the

experiment in non-stressed and stressed fish. Plasma cortisol, glucose and

lactate levels, the content of DA and 5-HT, and their main metabolites (DOPAC

and 5-HIAA) in different brain regions, and hypothalamic TPH1 mRNA

abundance were assessed. Our results reveal that stressed animals showed

reduced food intake, increased plasma cortisol levels, and enhanced

dopaminergic and serotonergic activities in telencephalon, hypothalamus, optic

tectum and hindbrain, independently of the stress duration. TPH1 mRNA

abundance also increased in stressed trout at both 3 and 10 days. Our present

results support the hypothesis of a key role played by both dopaminergic and

serotonergic systems in initiating and maintaining the neuroendocrine response

to stress. In addition their involvement in mediating the stress-related food

intake inhibition might be taken in consideration.

Acknowledgements: Funded by Ministerio de Economía y Competitividad and

European Fund for Regional Development (AGL2013-46448-3-1-R and

FEDER) and AEO-ECIMAT.

34

O19

PLASTIC PHYSIOLOGY, SOCIAL BEHAVIOUR AND ENDOCRINE

PROFILES OF AN INVASIVE CICHLID IN SOUTHERN PORTUGAL

Flávia B. Silva, J.L. Saraiva, M. Vargas, M. Silva, J. Soares, A.V.M. Canário

and Pedro M. Guerreiro. Email:fbsilva@ualg.pt

CEIB - Centro de Ciências do Mar, Universidade do Algarve, Portugal.

Invasive species are a growing concern for habitats worldwide. The chanchito,

Australoheros facetus, is a neotropical cichlid which displays high tolerance to

abiotic factors and marked social behaviours. The species is currently found in

the upper regions of the Guadiana, Sado and Odelouca basins in habitats with

striking seasonal variations in hydrological regimes. We studied its endocrine

physiology in the context of environmental acclimation, hierarchy formation

and reproductive behavior.

Fish were exposed to a range of salinities of upper estuaries during short and

long-term trials. A significant reduction in growth and in social behavior was

noticed at higher salinities (18 ppt), at which plasma osmolality, cortisol and

enzymatic activity were significantly elevated. Whether the rise in cortisol is

related to salinity acclimation mechanisms or a result of stress is still to be

determined.

Monogamous pairs establish and defend breeding territories. Social groups were

followed at different periods of the year. Dominant status was attributed to fish

that formed a territorial pair and a subsequent linear hierarchy ensued.

Dominance indexes and fish size are highly correlated (?: R=0.55; ?: R=0.84;

p<0.05) and even very small size differences account for social ranking. No

correlation to sex, GSI or HIS was found. Significant differences in initial and

final 11K-testosterone(11KT) levels occurred among dominant and subordinates

males but not in females of different status. Estradiol(E2) in non-territorial

males was slightly reduced after hierarchy formation. Females showed high but

variable E2 levels not related to status.

Size is key in hierarchy formation but 11KT may influence male status, while

the variance found in females E2 may reflect different maturation stages.

Members of the pair perform parental duties during offspring development but

males spend more time patrolling while females care for the young. Whether

such behaviors are under hormonal control is currently under investigation.

FBS has a Science Without Borders doctoral fellowship from the Brazilian

CNPq. Funded partially by FCT - Foundation for Science and Technology -

UID/Multi/04326/2013.

35

O20

TEMPERATURE IMPRINTING DURING EARLY DEVELOPMENT

AND THE ADULT STRESS AND IMMUNE RESPONSE

Ana Patrícia Mateus1,2

, R. Costa1, J.R. Cardoso

1, E. Gisbert

3, D.M. Power

1.

Email: apmateus@ualg.pt

1Comparative Endocrinology and Integrative Biology, Centre of Marine

Sciences, University of Algarve, Gambelas, 8005-139 Faro, Portugal, 2Health

Superior School, University of Algarve, Av. Dr. Adelino da Palma Carlos, 8000-

510 Faro, Portugal, 3IRTA, 43540 Sant Carles de la Ràpita, Spain

Despite the variations in the HPA axis organization in mammals and fish, the

regulatory loop is conserved. CRH is released in response to stress, stimulating

the production of POMC/ACTH in the pituitary and the subsequent release of

cortisol by modulation of StAR in the head kidney in fish. Since fish do not

thermoregulate, changes in early life temperatures are sub-lethal stressors which

have a strong impact on the development plasticity of fish.

The impact of thermal imprinting in early life on the plasticity of the HPI axis

and stress response in juvenile seabream (Sparus aurata) was assessed by

rearing fish under 4 thermal regimes: 2 constant (18-18ºC; 22-22ºC) and 2

variable temperatures between egg incubation and larval rearing (22-18ºC; 18-

22ºC); and exposing the resulting juvenile to acute confinement stress to

monitor plasma cortisol levels and genes of the HPI axis by qPCR. Changes in

the immune response, a common result of stress, was also evaluated using

histomorphometric measurements of MMCs in the head kidney and by

monitoring transcripts related with their immunological role.

Thermal imprinting caused a change in whole animal physiology and growth,

reflected by a significant increase in pituitary GH. Early thermal history

significantly modified HPI responsiveness as assessed by the amplitude of the

cortisol response, reflected by a significant shift in the relative abundance of

pituitary POMC and head kidney StAR. Furthermore, a modification in immune

responsiveness after an acute stress challenge was evident and there was a

decrease in the number of MMCs and a significant down-regulation of DCT

after acute stress. The results demonstrate the impact of early life history on

juveniles of the teleost sea bream.

Acknowledgements: This study was financed by the European Regional

Development Fund (ERDF) COMPETE and Portuguese funds through

Foundation for Science and Technology (FCT) under the project

UID/Multi/04326/2013 and the EC FP7 Program (2007-2013) grant agreement

nº 222719 – Lifecycle.

36

O21

ACTIVATION OF SOMATOSTATIN RECEPTOR 3 REDUCES CELL

VIABILITY AND HORMONAL SECRETION IN NON-FUNCTIONING

PITUITARY ADENOMAS THROUGH MAPK SIGNALING

Mª Carmen Vázquez-Borrego1, A. Ibáñez-Costa

1, E. Venegas-Moreno

2, A.

Toledano-Delgado3, A. Soto-Moreno

2, MA. Gálvez

4, MD. Gahete

1, JP. Castaño

1,

RM. Luque1. E-mail: z32vabom@uco.es

1Department of Cell Biology, Physiology and Immunology, University of

Córdoba (UCO); Hospital Universitario Reina Sofía (HURS); Instituto

Maimónides de Investigación Biomédica de Córdoba; CIBERobn and ceiA3,

Córdoba, Spain; 2Metabolism and Nutrition Unit, Hospital Virgen del Rocío,

Sevilla; 3

Service of Neurosurgery, HURS; 4Service of Endocrinology and

Nutrition, HURS/IMIBIC.

Non-functioning pituitary adenomas (NFPA) are characterized by the lack of

hormone hypersecretion and are associated to severe comorbidities related to

mass effects. Surgical resection of NFPAs is generally needed because

pharmacological treatments using currently available somatostatin analogs (SA)

are ineffective and therefore, identification of novel therapies deem necessary to

manage this pathology. One of the reasons of this lack of response to SA might

be related to the fact that NFPAs express high levels of sst3 and low sst2 and

sst5 levels. Thus, the aim of this study was to determine the effect of an sst3-

specific non-peptidic analogue (L-796,778; Merck) on key functional

parameters (cell viability, apoptosis/necrosis, hormone secretion and

intracellular signaling) in primary human NFPA cell cultures. Firstly, we

analyzed the expression profile of a series of 39 NFPAs and 10 normal

pituitaries (NP) by quantitative real-time PCR. We found a clear alteration in the

expression of key hormones (increased FSH levels) and receptors (increased

sst3 and truncated sst5TMD4 levels) in NFPAs compared to NP. Remarkably, in

vitro treatment of NFPA cultures with L-796,778 significantly reduced cell

viability and chromogranin-A secretion and increased necrosis by a Ca2+

-

independent mechanism. These direct effects of L-796,778 might be mediated

by the inhibition of MAPK signalling (ERK1/2-phosphorylation). In

conclusion, our study provides new evidence regarding the role and potential

clinical implications of sst3 in the pathophysiology of NFPAs, suggesting that

pharmacological treatment specifically targeting sst3 could be a promising

therapeutic alternative for this pathology.

Funding: CTS-1406, PI-0639-2012, BIO-0139, PI13-00651, BFU2013-43282-

R, CIBERobn and Merck Serono.

37

O22

LACK OF CORTISTATIN OR SOMATOSTATIN PLAYS

DIFFERENTIAL ROLES IN THE CONTROL OF MAMMARY GLAND

TUMORIGENESIS IN LEAN AND OBESE MICE

Alicia Villa-Osaba1,2,3

, MD Gahete1,2,3

, F López-López1,2,3

, AI Pozo-Salas1,2

, R

Sánchez-Sánchez4, R Ortega-Salas

4, M Álvarez-Benito

2,5, J López-Miranda

2,3,6,

RM Luque1,2,3

, JP Castaño1,2,3

. Email: b42viosa@uco.es

1Department of Cell Biology, Physiology, and Immunology, University of

Córdoba. 2Instituto Maimónides de Investigación Biomédica de Córdoba

(IMIBIC), Hospital Universitario Reina Sofía (HURS). 3CIBERobn.

4Anatomical Pathology Service, HURS.

5Mammary Gland Unit, HURS.

6Lipids

and Atherosclerosis Unit, HURS; Córdoba, Spain.

Somatostatin (SST) and cortistatin (CORT) are two peptides that exert common

biological actions, including the suppression of tumor cell proliferation,

offering a potential therapeutic target. However, attempts to apply SST analogs

in the treatment of breast cancer have been controversial and the specific role of

SST and/or CORT in mammary gland tumorigenesis remains uncertain,

especially in obesity. We studied the role of endogenous SST and CORT on

carcinogen-induced mammary gland tumorigenesis under normal-weight and

obesity conditions, by treating SST- and CORT-knockout (KO) and littermate-

controls mice, fed a low fat (LF) or a high fat (HF) diet, with 7, 12-

dimethylbenzaanthracene. Lack of SST did not impact DMBA-induced tumor

incidence under lean conditions, while lack of CORT aggravated DMBA-

induced tumorigenesis in LF-fed mice. These differences were not attributable

to altered mammary gland development or changes in relevant circulating

hormones. Interestingly, HF-diet increased the sensitivity to DBMA-induced

carcinogenesis in control and SST-KO mice while, tumor incidence was not

statistically altered in HF-diet compared with LF-diet CORT-KO; however,

tumor incidence was already significantly elevated in LF-fed CORT-KO, which

might preclude a further elevation in obesity. Therefore, our data indicate that

endogenous SST and CORT distinctly contribute to the control of DMBA-

induced tumorigenesis, which is strongly influenced by the metabolic/endocrine

milieu, suggesting that CORT, rather than SST, might represent a key inhibitor

factor of mammary tumorigenesis. Thus, these data invite to suggest that CORT-

like rather than SST-like molecules could be more promising tools for the

treatment of breast cancer tumors.

Funding: PI13-00651, BFU2013-43282-R, BIO-0139, CTS-1406, PI-0639-

2012 and CIBERobn.

38

O23

THE TRUNCATED SOMATOSTATIN RECEPTOR SST5TMD4 IS

OVEREXPRESSED IN PROSTATE CANCER AND INCREASES

AGGRESSIVENESS FEATURES THROUGH REGULATION OF

ANGIOGENIC FACTORS AND WNT/B-CATENIN PATHWAY.

Daniel Hormaechea-Agulla1, MD Gahete

1, A Ibáñez-Costa

1, E Gómez-Gómez

2,

JA Ramos-Fernández1, J Valero-Rosa

2, J Carrasco-Valiente

2, MM Moreno

3, MD

Culler4, MJ Requena

2, JP Castaño

1, RM Luque

1. Email:

hormaechea85@gmail.com

1Department of Cell Biology, Physiology and Immunology, University of

Córdoba, Hospital Universitario Reina Sofía (HURS), Instituto Maimónides de

Investigación Biomédica de Córdoba (IMIBIC), CIBERobn and, ceiA3;

Córdoba, Spain. 2Urology Service, HURS.

3Anatomical Pathology Service,

HURS; Cordoba, Spain. 4IPSEN Bioscience, Cambridge, MA, USA.

Somatostatin is a highly conserved, pleiotropic neuropeptide that binds to a

family of G protein-coupled receptors with seven transmembrane domains

(TMD), called somatostatin receptors (sst1-5), to exert its biological function.

Our group has identified and characterized in several species additional sst5

variants originated from alternative splicing that lack several TMDs.

Particularly, the human sst5TMD4 variant, a receptor with four TMDs, seems to

have pathological consequences since it is abundantly expressed in a subset of

pituitary tumors and breast cancer samples, wherein it could exacerbate the

malignant phenotype. Here, we analyzed the presence and role of sst5TMD4 in

human prostate cancer (PC). We found a clear overexpression of sst5TMD4 in

PC samples compared to control tissues and, its expression was higher in

tumors from patients with metastasis compared to those without metastasis.

Thus, the in vitro functional role of sst5TMD4 was explored by using androgen

dependent (VCaP) and independent (PC-3) PC cell lines. Specifically,

sst5TMD4 overexpression increased cell proliferation and migration in both cell

lines while, its silencing by siRNA decreased basal cell proliferation. Moreover,

several important pathways were deregulated after overexpression of

sst5TMD4, including a dramatic downregulation of two tumor suppressor

proteins from the Wnt/B-catenin pathway (APC and SFRP1) and an increase of

angiogenic factors such as VEGF and ANG1. Altogether, our results suggest a

relevant role of sst5TMD4 in PC where is associated to enhanced features of

aggressiveness through regulation of angiogenic factors and Wnt/B-catenin

pathway.

Funding: PI13-00651, BFU2013-43282-R, BIO-0139, CTS-1406, PI-0639-

2012 and CIBERobn.

39

O24

IN1-GHRELIN INCREASES MALIGNANCY FEATURES OF BREAST

CANCER CELL LINES

David Rincón-Fernández, MD Gahete, V Ruiz-Murillo, R Santamaría, RM

Luque, JP Castaño. E-mail: davidrfp@gmail.com

Department of Cell Biology, Physiology and Immunology, University of

Córdoba, Maimonides Institute for Research in Biomedicine of Cordoba

(IMIBIC), Reina Sofia University Hospital and CIBER Fisiopatología de la

Obesidad y Nutrición (CIBERobn). 14014-Córdoba, Spain

Aberrant alternative splicing is a hallmark of tumoral process that can worsen

cancer outcome by inducing the appearance of alternative proteins, which can

increase malignancy features of the tumoral cells. Our group has recently

discovered a splicing variant of the ghrelin gene, named In1-ghrelin, which

results from the retention of intron 1. In1-ghrelin variant is overexpressed in

breast cancer, and its expression correlates with proliferation markers,

suggesting the involvement of In1-ghrelin in the development and/or

progression of this pathology. The aim of this study was to determine the

functional role of In1-ghrelin (and native-ghrelin) in two breast cancer derived

cell lines: MDA-MB-231 and MCF7. Particularly, treatment with In1-ghrelin

peptides or native-ghrelin stimulated proliferation and migration in both cell

lines. Similarly, overexpression of native-ghrelin and In1-ghrelin increased the

proliferation rate, while only In1-ghrelin overexpression was able to increase

migration ability in MDA-MB-231 and MCF7 cells. These results were further

corroborated by silencing endogenous In1-ghrelin in MDA-MB-231 cells. In

addition, In1-ghrelin, but not native-ghrelin, overexpression induced an increase

in pERK1/2 and a decreased in pAKT. We then analyzed dedifferentiation

processes in response to native-ghrelin and In1-ghrelin by studying the

percentage of cells with mesenchymal-like phenotype and found that In1-

ghrelin, but not ghrelin, increased epithelial to mesenchymal transition in

MDA-MB-231 cells. In addition, we studied the capacity to form

mammospheres and found that only In1-ghrelin overexpression and treatment

led to the formation of more and bigger mammospheres. In summary, our data

demonstrate that In1-ghrelin splicing variant increases malignancy features of

breast cancer cells from two different cell models, suggesting a relevant role of

In1-ghrelin in breast cancer development and/or progression.

Funding: BFU2013-43282-R, PI13-00651, BIO-0139, CTS-1406, PI-0639-

2012 and CIBERobn.

40

025

EXPLORING ALLATOSTATIN-A, KISS AND GALANIN GPCRS IN

LOPHOTROCOZOANS

João C.R. Cardoso, N. Zaghdoudi-Allan, R.C. Félix, D.M. Power. E-mail:

jccardo@ualg.pt.

Comparative Endocrinology and Integrative Biology, Centre of Marine

Sciences, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro,

Portugal

The Allatostatin type-A (AST-A), kisspeptin (KISS) and galanin (GAL) G-

protein coupled receptor (GPCRs) and their activating peptides play a major

role in the regulation of feeding, energy metabolism and reproduction.

Receptors and peptides of this family emerged early in the bilaterians however

relatively few studies of their evolution and function in invertebrates exist. In

nematodes and arthropods, only the AST-A system exists, suggesting that KISS

and GAL receptors and peptides were lost during the protostome radiation. The

occurrence of the AST-A, KISS and GAL system in Lophotrocozoans, a major

group of the animal kingdom remains largely unstudied. The aim of the present

study was to exploit available molecular data for the Lophotrocozoans to study

the evolution of the AST-A/KISS/GAL system in protostomes and to compare it

with the deuterostomes. The existence of the peptides and receptors in the

genomes and transcriptomes of representatives of several Lophotrocozoan

clades (annelids, gastropods, bivalves, bryozoans, rotifers and

plathyhelminthes) was established. The Lophotrocozoan AST-A and KISS

receptors were compared with those in vertebrates and arthropods and revealed

that orthologues existed in the genomes of all the species analysed but that GAL

receptors were only present in annelid genomes. AST-A and KISS receptor gene

expansion was observed in some species suggesting that receptor gene

evolution was affected by lineage-specific events. The results from

Lophotrocozoans supported the recent hypothesis that AST-A/KISS/GAL

GPCRs were under distinct evolutionary pressures in the protostomes and that

the AST-A and the KISS receptors are more closely related than the GAL

receptors (Felix et al., 2015).

Acknowledgements: Funded through Foundation for Science and Technology

(FCT) through project UID/Multi/04326/2013 and European Union Seventh

Framework Programme (grant agreement n° 605051, CACHE-ITN). JCRC is

funded by an auxiliary research contract (UID/Multi/04326/2013), NA by

CACHE-ITN and RCF by FCT SFRH/BPD/89811/2012 grant.

41

O26

RESPONSIVENESS OF SEA BASS SCALES TO ESTRADIOL AND

GENISTEIN

M. Dulce Estêvão 1,2

, P. I. S. Pinto 1, A. Andrade

1, S. Santos

1 and D. M. Power

1. E-mail: mestevao@ualg.pt

1 CCMAR - Centre of Marine Sciences, Universidade do Algarve, Faro,

Portugal. 2Escola Superior de Saúde, Universidade do Algarve, Faro, Portugal

Phytoestrogens are plant-derived compounds with estrogen-like activity, to

which fish may be exposed in the wild due to aquatic contamination or in

aquaculture through ingestion of fish feeds enriched in plant-based ingredients.

While several endocrine-disrupting effects have been described for

phytoestrogens in fish reproductive functions, little is known about their

impacts on mineralized tissues. The present study focused on a fish scale model

because of their well recognized responsiveness to estrogen (E2). European sea

bass were exposed to estradiol and the phytoestrogen genistein (Gen) and their

effects on calcium and phosphorus plasma levels and the activities of alkaline

phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) markers of

osteoblast (OSB) and osteoclast (OSC) activity respectively, were evaluated.

The repertoire of nuclear and membrane ERs was characterized, plasma

vitellogenin (Vtg) was quantified and candidate E2 responsive genes were

analyzed in scales and liver. Results show that Ca and P plasma content

significantly increased 5 days after E2 injection although Gen had no effect. In

contrast, TRAP and ALP activities significantly increased 1 d after Gen

injection although 5 d after injection no significant effect was detected and E2

failed to stimulate these enzymes. Both Gen and E2 induced an increase in

plasma Vtg levels 1 d after the injection but after 5 d this effect was only

induced by E2. Both E2 and Gen induced significant changes in gene expression

in scales and an overlapping but not identical response was found in the liver,

suggesting different mechanisms of action and responsiveness to estrogens in

these tissues. In line with this observation different patterns of ERs expression

occurred in scales and liver. Overall the results suggest that exposure to

estrogens and phytoestrogens impacts on both liver (a classical E2 responsive

tissue) and mineralized tissues modifying their homeostasis and ultimately their

function.

Acknowledgements: Funded by FCT – Portuguese Science and Technology

Foundation through projects UID/Multi/04326/2013 and PTDC/AAG-

GLO/4003/2012 and grant FCT SFRH/BPD/25247/2005.

42

027

AN APPROACH TO STANNIOCALCIN SECRETION IN GILTHEAD

SEABREAM (Sparus aurata)

Ignacio Ruiz-Jarabo, S.F. Gregório, J. Fuentes. E-mail: irrocha@ualg.pt

Centre of Marine Sciences (CCMar), Universidade do Algarve, Campus de

Gambelas, 8005-139 Faro, Portugal

Calcium balance is of vital importance for vertebrates. In teleost fish, the most

important endocrine modulators of calcium homeostasis are stanniocalcin

(STC) and the parathyroid hormone-related protein (PTHrP), which act as

antagonists. In turn, both hormones are apparently regulated by a calcium-

sensing receptor (CaSR), and no other regulators are yet described for either

hormone. In this sense, teleost fish provide an unique model to study STC

regulation, as they have a specialized STC-producing gland called the

corpuscles of Stannius (CS). Here, using the seabream as a model, we have

developed an ex vivo assay to culture CS of fish and a competitive ELISA

method to measure STC concentrations. STC release from the CS is mediated

by a CaSR, as indicated by the effects of incubation with different calcium

levels, and the use of calcimimetic and calcilytic compounds. Moreover, we

describe for the first time the presence of two PTHrP receptors in the seabream

CS i.e. PTHR1 and PTHR3. Thus, ex vivo incubations revealed a dose-response

inhibitory effect of PTHrP on STC secretion under basal Ca2+

concentrations.

This inhibition is carried out through specific and reversible second messenger

pathways i.e. transmembrane adenylyl cyclases and phospholipase C. Together,

these results revealed a fascinating novel process of endocrine modulation

between two antagonist hormones, involved in calcium regulation, that

resembles the terrestrial regulation of calcium balance in the parathyroid gland.

Supported by PTDC/MAR/104008/2008, FCT, Portugal

43

O28

TARGETED PTH4 CELL ABLATION LEADS TO AN UNCOUPLING

OF BONE FORMATION AND MINERALIZATION

Paula Suarez-Bregua1,2

, A. Saxena3, M.E. Bronner

3, P. Moran

2 and J. Rotllant .

E-mail: paulasuarez@iim.csic.es

1Aquatic Molecular Pathobiology Lab, IIM- CSIC, Vigo, Pontevedra, Spain

.

2Dept. of Biochemistry, Genetics and Inmunology, University of Vigo.

Pontevedra, Spain. 3Division of Biology and Biological Engineering, California

Institute of Technology, Pasadena, United States.

Bone formation and mineralization are dynamic processes that occur during the

embryonic development but also along adult life through remodeling.

Mineralization is the most distinctive feature of the bone as it makes the

skeleton a great store of phosphate and calcium in order to contribute to the

mineral balance in vertebrates. Recent studies have provided evidence that the

central and peripheral nervous systems directly control bone homeostasis.

Therefore, the hypothalamus has been documented to be a potent regulator of

the skeletal mass through efferent neural signals and/or neuroendocrine signals.

Given its “sensing” properties, the hypothalamus is able to integrate

physiological information from the whole body and sends inputs to bone cells

that regulate skeletal mass accordingly. We have recently identified a new PTH

family member in zebrafish, Parathyroid hormone 4 (Pth4), produced by two

clusters of cells in the lateral hypothalamus. We found that Pth4 is involved in

bone mass accrual through phosphate homeostasis regulation in adult fish,

which indicates a new functional link between brain and bone. To better

understand Pth4 and its role during skeletal development, we have characterized

Pth4-expressing cells and performed single-cell resolution two-photon laser

ablation and confocal imaging in vivo. We found that hypothalamic cells

expressing pth4 are post-mitotic neurons. Targeting these neurons via two-

photon laser ablation resulted in impaired bone mineralization and altered

expression of mineralization and phosphate-regulating genes as well as

osteoblast differentiation markers. Collectively, these results suggest that Pth4-

expressing hypothalamic neurons may play a key in the regulation of bone

metabolism, possibly through regulating phosphate balance.

This work was funded by the Spanish Science and Innovation Ministry project

AGL2014-52473R to JR. Paula Suarez Bregua was supported by Campus Do

Mar-Xunta de Galicia PhD fellowship.

44

O29

THE GENE REGULATORY NETWORK UNDERPINNING DORSAL–

VENTRAL PIGMENTATION PATTERNING IN FISH. ANALYSIS OF

ASIP1 CIS-REGULATORY LANDSCAPE

Laura Cal1, I. Braasch

2, J.L.Gómez-Skarmeta

3, R. Kelsh

4, J.M. Cerdá-Reverter

5, J. Rotllant

1. E-mail: lauracal@iim.csic.es

1Institute of Marine Research, CSIC. Vigo, Spain.;

2Institute of Neuroscience,

University of Oregon, EE.UU.; 3CABR-CSIC;

4University Of Bath, UK.,

5Instituto de Acuicultura de Torre de la Sal (IATS-CSIC). Castellón, Spain.

Dorso-ventral pigment pattern differences are the most widespread pigmentary

adaptations in vertebrates. In mammals, ASIP have been reported as the key

factor in this process by regulating melanin types and levels in melanocytes. In

fish, studies of pigment patterning have focused on stripe formation, identifying

a core striping mechanism dependent upon interactions between different

pigment cell types. We have recently demonstrated that, in fact, zebrafish utilize

two distinct adult pigment patterning mechanisms -an ancient dorso-ventral

patterning mechanism, and a more recent striping mechanism based on cell-cell

interactions; remarkably, the dorso-ventral patterning mechanism also utilizes

Asip. We hypothesize that agouti have a conserved role in the countershading

pigment pattern formation in vertebrates. Yet the genetic mechanisms

underlying this dorso-ventral asip1 expression gradient remain largely

unexplored. Thus, we report here the asip1 expression in the holostei spotted

gar (Lepisosteus oculatus), one of Darwin's defining examples of 'living fossils'

member of a lineage diverged from teleosts before to Teleost Genome

Duplication (TGD), and through phylogenetic footprinting and transgenesis in

zebrafish, we examined the cis-regulatory landscape of Asip1.

Funded by MINECO (ALG2011-23581) and Xunta de Galicia (INCITE-09 402

193)

45

Posters

46

P1

INFLUENCE OF AVT AND CORTISOL TREATMENT ON STRESS AND

THYROID PATHWAYS IN THE GILTHEAD SEA BREAM (Sparus

aurata)

Juan Antonio Martos-Sitcha1,2

, I. Jerez2, G. Martínez-Rodríguez

1, J.M.

Mancera2. E-mail: juanantonio.sitcha@icman.csic.es

1Instituto de Ciencias Marinas de Andalucía, Consejo Superior de

Investigaciones Científicas (ICMAN-CSIC), 11510 Puerto Real, Cádiz, Spain. 2Department of Biology, Faculty of Marine and Environmental Sciences,

University of Cádiz, Campus de Excelencia Internacional del Mar (CEI-MAR),

11510 Puerto Real, Cádiz, Spain.

The endocrine system could be considered as a complex net of pathways for

which an interconnection between some of them cannot be ruled out. Cortisol,

the main corticosteroid in teleosts, is the final step on the hypothalamic-

pituitary-interrenal axis, and its functions are related to intermediary

metabolism, osmotic and ionic regulation, growth, stress and immunity. In turn,

the vasotocinergic, homologous to the mammalian vasopressinergic pathways,

and thyroid systems have also an important role in several physiological

processes in which cortisol plays a role (e.g. osmoregulation, metabolism or

stress). The aim of this study was to evaluate the possible interaction between

different endocrine axes (vasotocinergic, stress and thyroid pathways) in the

gilthead sea bream (Sparus aurata), through assessing the effects of exogenous

arginine vasotocin (AVT) and cortisol administration. Specimens acclimated to

seawater (38 ‰ salinity) were intraperitoneally implanted with vegetable oil

(mixture 5:1 coconut and seeds oils) alone (control) or containing different

doses of AVT (0.5 or 1.0 ?µg/g body weight) or cortisol (50?µg/g body weight)

and sampled at 12 hours, and 1 and 3 days after injection. Thus, both hormones

produced a significant decrease of hypothalamic corticotrophin releasing

hormone (CRH) mRNA expression from 12/24 hours onwards, while CRH-

binding protein (CRH-BP) mRNA levels did not altered its values in any of the

intraperitoneally injected specimens during the time that the experiment lasted.

Moreover, AVT and cortisol administration independently produced a

significant decrease in thyrotropin-releasing hormone (TRH) gene expression

from 12 h post-injection, whereas an up-regulation in thyroid receptor β (TRβ)

mRNA expression was observed just 12 h after hormonal administration. Our

results clearly suggest that both AVT and cortisol hormones independently co-

regulate the functions of these endocrine systems.

47

P2

STUDY OF THE SOMATOTROPIC HORMONES EXPRESSION

DURING THE EARLY DEVELOPMENT OF THICK LIPPED GREY

MULLET (Chelon labrosus)

Neda Gilannejad, J.A. Martos-Sitcha, V. de las Heras, M. Yúfera, G. Martínez-

Rodríguez. E-mail: neda.gilannejad@alum.uca.es

Instituto de Ciencias Marinas de Andalucía (ICMAN), CSIC, 11510 Puerto

Real, Cádiz.

Mullets are considered as potential species for sustainable aquaculture due to

their feeding characteristics. Among all the mullets found in the Spanish

territory, Chelon labrosus is having higher commercial interest because, in

comparison to the rest, it reaches larger sizes in a shorter period of time.

Recently hatched larvae go through a series of molecular, biochemical and

morphological changes during growth and development to juveniles. Some of

the processes involved in larval development, including the growth hormone

(GH) and insulin-like growth factor I (IGF-I), are subjected to endocrine

control. With the purpose of deepening the knowledge of the possible factors

involved in the early development and differentiation in this species, expression

of the two somatotropic genes, gh and igf1, was studied during the first 92 days

of life using real time PCR. These genes showed different expression patterns.

The expression of gh increased significantly, reaching to its maximum at 43

days post hatch (dph), and decreased drastically afterwards, maintaining

relatively low levels during the rest of the studied period. However, igf1 showed

a gradual raise, peaking at 50 dph, and maintaining thereafter relatively constant

levels. Moreover, the maximum growth rate, between 22 and 54 dph, coincides,

to a large extent, with elevated expression levels of gh and the beginning in the

increase of igf1 observed in this work. This can suggest that gh and igf1 are

expressed in an independent manner during the first 50 days of life, while the

GH/IGF-I axis starts to be functional afterwards.

Acknowledgments: This study has been partially funded by a grant for applied

innovation and transfer of knowledge in the professional formation of the

educative system from the Ministry of Education in Spain, entitled

“Optimización del Proceso de maduración y desove del mugílido Chelon

labrosus”, and co-financed by ESF. The authors acknowledge the IES Els

Alfacs (Sant Carles de la Rápita, Tarragona, Spain) for providing the eggs.

48

P3

LONG-TERM EFFECTS OF rBGH TREATMENT ON GH/IGFs AXIS IN

GILTHEAD SEA BREAM (Sparus aurata)

Emilio J. Vélez, M. Perelló, E. Lutfi, A. Moya, J. Fernández-Borràs, J. Blasco,

I. Navarro, E. Capilla, J. Gutiérrez. E-mail: evelezve@ub.edu

Department of Physiology and Immunology, Faculty of Biology, University of

Barcelona, Barcelona 08028, Spain.

Fish growth is mainly regulated by the growth hormone (GH)/insulin-like

growth factors (IGFs) axis. GH is secreted by the pituitary and stimulates the

synthesis of IGF-I, which is the largest growth inducer in muscle and bone

tissues. IGF-I binds to membrane receptors activating different signalling

pathways to regulate metabolism and to promote growth. The aim of this work

was to study the long-term effects of sustained-release of recombinant bovine

GH (rBGH) in fingerlings of gilthead sea bream, in order to improve knowledge

on the role of GH/IGFs axis in this species. Six weeks post-injection, liver, bone

and muscle tissue samples were collected. mRNA levels of IGFs (IGF-I and II),

growth hormone receptors (GHR-I and II), IGF-I receptors (IGF-IRa and Rb), as

well as some IGF binding-proteins (IGFBP-2, 4 and 5) important for controlling

IGFs bioavailability, were measured by quantitative PCR. Body-weight results

showed that rBGH-injected fish grew significantly more than control fish. Gene

expression analyses indicated increased expression of several genes of the

GH/IGFs system in liver, whereas only minor differences were observed in bone

and muscle. Liver expression of GHR-II and both peptides, IGF-I and IGF-II,

were increased in rBGH-treated fish, whereas no differences were observed in

these molecules in the other tissues studied. Regarding IGF-IRs, in rBGH-

treated fish an increased expression of IGF-IRb was found in liver, whereas in

bone and muscle this receptor was down-regulated, as also IGF-IRa in bone.

With respect to IGFBPs, only IGFBP-2 showed an increase in the liver of

rBGH-treated fish, while IGFBPs 4 and 5 remained stable in muscle and bone

tissues. These findings reveal the main effects of long-term GH treatment in this

species through hepatic IGFs, and provide new insights to better understand the

endocrine regulation of fish somatic growth, which may contribute to improve

aquaculture production.

Thanks to Elanco Animal Health for kindly providing the rBGH. Supported by

MINECO (AGL2012-39768) and Catalonian Government (2014SGR-01371).

49

P4

HYPER- AND HYPO-OSMOTIC CHALLENGES MODIFY

HORMONAL PATHWAYS IN THE GILTHEAD SEA BREAM (Sparus

aurata): A MICROARRAY APPROACH

Juan Antonio Martos-Sitcha1,2

, J.M. Mancera2, J.A. Calduch-Giner

3, J. Pérez-

Sánchez3, M. Yúfera

1, G. Martínez-Rodríguez

1. E-mail:

juanantonio.sitcha@icman.csic.es

1Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC), E-11519 Puerto

Real, Cádiz, Spain. 2Department of Biology, Faculty of Marine and

Environmental Sciences, University of Cádiz, Campus de Excelencia

Internacional del Mar (CEI-MAR), E-11519 Puerto Real, Cádiz, Spain. 3Nutrigenomics and Fish Growth Endocrinology Group, Institute of

Aquaculture Torre de la Sal (IATS-CSIC), Ribera de Cabanes, E-12595

Castellón, Spain.

Euryhaline teleost can live in a wide range of environmental salinities. This

ability is carried out by different osmoregulatory and non-osmoregulatory

tissues, being gills, liver and hypothalamus very important due to their

implications in ion and water exchange, metabolism and endocrine regulation,

respectively. Transcriptomic analysis in these tissues by means of a specific

oligo-microarray revealed new aspects related to the osmoregulatory processes

mediated by different endocrine pathways after hypo- (38‰ ? 5‰) or hyper-

(38‰ ? 55‰) osmotic challenges to gilthead sea bream juveniles. In liver,

aldosterone-related signalling pathways, represented by 8 heat shock proteins

(DNAJC8, DNAJC11, DNAJC17, DNAJC30, HSP90AB1, HSPA14, HSPD1 and

HSPE1), showed an up-regulation in both salinity challenges, while other

enzymes related to cell growth (PRKCA), division (KRAS) and maturation

(PIK3C3) were down-regulated. In gills, serotonin, melatonin and thyroid

hormone pathways were affected by the differential up-regulation of genes

involved in xenobiotic and protective metabolism facing transfer to 5‰

(CYP1A1, SULT1A1 and UGT1A1) or 55‰ (CYP1B1, UGT2A3). A similar

pattern was also observed for several genes involved in the melatonin

metabolism pathway (CYP2J2, CYP1B1 and SULT1A1) in the hypothalamus of

specimens transferred to 5‰, whereas hyperosmotic challenge induced a clear

down-regulation in another gene (UGT1A9) involved in xenobiotic metabolism.

Taking together, these results point out the importance of liver, gills and

hypothalamus in the response to osmolality change, showing the differentially

regulated endocrine components and pathways that process the harmful

metabolites related to osmotic stress.

Acknowledgments: project Aquagenomics (MINECO; CSD2007-00002).

50

P5

CLONING, SEQUENCING AND EXPRESSION OF GENES BRDT AND

TERT IN SEABREAM (Sparus aurata): POTENTIAL NEW SEX

MARKERS IN FISH.

M. Úbeda-Manzanaro, J.B. Ortiz-Delgado, C. Sarasquete. E-mail:

carmen.sarasquete@icman.csic.es

Instituto de Ciencias Marinas de Andalucía ICMAN-CSIC

Campus Universitario Río San Pedro. CEIMAR. Apdo oficial, 11510, Puerto

Real, Cádiz

Applying molecular amplification and cloning techniques obtained cDNA

sequences of Brdt and Tert genes, which has allowed to analyze levels of

differential expression in gonads, brain and somatic tissues, by q-PCR

quantitative, comparatively, using two reference genes (β-actin and 18S rRNA)

At the same time, its cellular-tissue distribution will be analyzed, and

phylogenetic analyses are performed.

Tert gene encodes a catalytic subunit of the enzyme telomerase reverse

transcriptase, which is which keeps the telomeric ends of chromosomes,

protecting them from abnormal folds and their degradation. In the majority of

cells, telomeres will shorten progressively as this divides, and after a certain

number of successive divisions, the telomeres are so short that stops cell

division or cell apoptosis is induced. In mammals, the activity of telomerase in

tissues of adults is limited to stem cells with high potential for proliferation,

finding expression in the cells of the germline, tumors and neoplastic cells.

However, in fish the Tert gene seems to express themselves, so ubiquitous, in

different gonad, brain and somatic tissues.

On the other hand, the Brdt gene encodes a protein regulating the transcription,

which in mammals is expressed almost exclusively in the testis, and is essential

for the proper sexual function of males. BRDT protein control reorganization

post-meiotic of the genome that occurs after the hiperacetilation of histones in

the phase of elongation of the spermatids, to be crucial in the development of

spermatogenesis and therefore, determinants of male fertility.

The results for both genes showed relative expression levels significantly higher

in testis, in relation to ovarian, and other tissues, corroborating their great

importance during the reproductive process in general, and in the

spermatogenesis in particular. It was shown expression in postmeiotic stages, as

in spermatocytes and spermatids, and negative expression in gametes.

51

P6

AVT AND IT SYSTEMS MEDIATED METABOLIC EFFECTS

INDUCED BY AIR EXPOSICION IN THE GILTHEAD SEA BREAM

(Sparus aurata)

Arleta Krystyna Skrzyńska1, M. Bastaroli

1, E. Maiorano

1, G. Martínez-

Rodríguez2, J.M. Mancera

1, J.A. Martos-Sitcha

2. E-mail:

arleta.skrzynska@uca.es

1Department of Biology, Faculty of Marine and Environmental Sciences,

Campus de Excelencia Internacional del Mar (CEI-MAR), University of Cádiz,

11510 Puerto Real, Cádiz, Spain; 2Instituto de Ciencias Marinas de Andalucía,

Consejo Superior de Investigaciones Científicas (ICMAN-CSIC), 11510 Puerto

Real, Cádiz.

Arginine vasotocin (AVT) and isotocin (IT) are considered as important players

in stress regulation. The present study assesses the response of both

vasotocinergic and isotocinergic systems in the gilthead sea bream (Sparus

aurata), through their specific receptors (AVTR V1a2-type, AVTR V2-type and

ITR), after an acute stress situation. Specimens were exposed to air for 3 min by

lifting the wire-net cage out of the tank and put back in their respective tanks

after that, being sampled in a time course response (15 min, 30 min, 1, 2, 4 and

8 hours post-stress). Plasma cortisol values increased after few minutes post-

exposure, decreasing during the experimental time while a metabolic

reorganization occurred in both plasmatic and hepatic levels. Thus, an

enhancement in hepatic glycogenolysis and lactate production, together with

higher plasma glucose and lactate levels, as well as a decrease in free plasma

proteins was observed. Acute stress response involves changes in gene

expression of both hypothalamic AVT and IT precursor levels as well as in their

specific hepatic receptors, which are differentially expressed depending on the

receptor type (AVTR V1-type, AVTR V2-type or ITR). This response suggests

that activation of both vasotocinergic and isotocinergic systems is important

during metabolic mobilization induced by an acute stress situation, which is

stated, at least, through changes in mRNA expression levels of these genes

analyzed.

52

P7

INFLUENCE OF GLUCOSE LEVELS ON LACTATE METABOLISM IN

BRAIN GLUCOSENSING AREAS OF RAINBOW TROUT

(Oncorhynchus mykiss)

Cristina Otero-Rodiño, M. Librán-Pérez, C. Velasco, J. Hernández-Pérez, R.

Álvarez-Otero, M. Conde-Sieira, J.L. Soengas. E-mail: cris.otero@uvigo.es

Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e

Ciencias da Saúde, Facultade de Bioloxía y ECIMAT, Universidade de Vigo

There is no direct evidence in fish brain demonstrating the existence of changes

in lactate metabolism in response to alterations in glucose levels to support the

existence of an astrocyte-neuron lactate shuttle (ANLS) in these areas.

Therefore, we induced in rainbow trout, through IP treatments, hypo-glycaemic

or hyper-glycaemic changes to assess the response of parameters involved in

putative ANLS in glucosensing areas like hypothalamus and hindbrain. To

distinguish those effects from those induced by peripheral changes in the levels

of metabolites or hormones, we also carried out ICV treatments with 2-deoxy-

D-glucose (2-DG, a non-metabolizable glucose analogue thus inducing local

glucopenia) or glucose. Finally, we also incubated hypothalamus and hindbrain

in vitro in the presence of increased glucose concentrations. As a whole, the

induction of changes in glucose levels in hypothalamus and hindbrain of

rainbow trout resulted in the activation of the glucosensing mechanism

dependent on glucokinase (GK) in these areas in agreement with similar

previous studies. However, when we assessed in these areas the response of

parameters related to lactate metabolism, the results obtained were

contradictory. The increase in glucose levels did not produce in general the

expected changes in those pathways with only a minor increase in their capacity

of lactate production. The decrease in glucose levels was however more clearly

related to a decreased capacity of the pathways involved in the production and

use of lactate, and this was especially evident after ICV treatment with 2-DG in

both areas. In conclusion, the present results only partially support the existence

of an ANLS in hypothalamus and hindbrain of rainbow trout relating glucose

availability to lactate production and use.

Acknowledgements. This study was supported by Ministerio de Economía y

Competitividad and European Fund for Regional Development (AGL2013-

46448-3-1-R and FEDER).

53

P8

ORGANOTIN ENDOCRINE DISRUPTORS ACT AS OBESOGENS IN

RAINBOW TROUT CULTURED ADIPOCYTES

Lutfi, E.1 Córdoba, M.

1 Nerín, G.

1 Porte, C.

2 Gutiérrez, J.

1 Capilla, E.

1

Navarro, I.1. E-mail: mnavarro@ub.edu

1Department of Physiology and Immunology, Faculty of Biology, University of

Barcelona, Barcelona 08028, Spain. 2Environmental Chemistry Department,

IDAEA-CSIC, 08034 Barcelona, Spain

In recent years, several environmental pollutants have been identified to act as

obesogenic compounds affecting endocrine signalling and lipid homeostasis.

Among them, organotins such as tributyltin (TBT) and triphenyltin (TPT) have

been the most widely studied in aquatic environments. In vitro and in vivo

studies have demonstrated that TBT and TPT promote adipogenesis trough

peroxisome proliferator-activated receptor gamma (PPARγ) and retinoid X

receptor (RXR) activation. The aim of the present study was to investigate in

vitro the effects of TBT and TPT on the development and lipid metabolism of

rainbow trout (Onchorynchus mykiss) primary cultured adipocytes. Results

obtained by oil red O staining indicated that TBT and TPT induce only slightly

lipid accumulation compared with the differentiation medium in the absence of

lipid mixture; but when present, lipid accumulation was significantly higher

with and without organotins. Moreover, triglyceride content measurement

revealed that TPT produces stronger lipid accumulation than TBT, although the

highest accumulation of triglycerides was observed when adding lipid mixture,

also in those cells exposed to TBT or TPT. Furthermore, protein expression of

two key regulators of adipogenesis was evaluated by immunofluorescence.

Results showed that TBT and TPT activate PPARγ and CEBPα protein

expression; however, the stronger effect was observed again when lipid mixture

was added to the differentiation medium. Overall, the activation of PPARγ and

CEBPα, together with the increase of triglyceride levels inside the cells

confirmed that TBT and TPT enhance adipocyte differentiation and act as

potential obesogens in rainbow trout. Moreover, our results highlighted that

lipid mixture is essential to achieve complete cell differentiation in cultured

adipocytes from this species. Additionally, quantitative real-time PCR analyses

are being performed to further clarify the effects of TBT and TPT on the gene

expression of lipid metabolism and cell proliferation/differentiation markers.

Supported by MINECO (AGL2011-24961) and DECO (2014SGR-01371).

54

P9

EFFECTS ON FEEDING RAINBOW TROUT WITH A LIPID-

ENRICHED DIET ON FATTY ACID SENSING, FOOD INTAKE

REGULATION AND CELLULAR SIGNALING PATHWAYS IN

HYPOTHALAMUS AND LIVER

Marta Librán-Pérez, C. Otero-Rodiño, C. Velasco, J. Hernández-Pérez, F.

Naderi, J.M. Míguez, J.L. Soengas. E-mail: marta.libran@uvigo.es

Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e

Ciencias da Saúde, Facultade de Bioloxía, Universidade de Vigo, E-36310

Vigo, Spain.

In previous studies carried out in rainbow trout, we have characterized in

hypothalamus and liver the presence and functioning of fatty acid-sensing

systems whose activation ultimately lead to decreased food intake. Since a

reduced food intake has been observed after feeding fish with lipid-enriched

diets, changes in fatty acid-sensing systems are expected in fish fed with diets

containing different lipid levels that have not been assessed yet. There is

evidence in rainbow trout for the presence and functioning of AMPK and

proteins involved in cellular signaling like mTOR and Akt but to date there is

no information in any fish tissue regarding the response of these sensors and

proteins to changes in the levels of nutrients like fatty acids. The aim of this

study in rainbow trout fed with low fat (LF) or high-fat (HF) diets was to

determine if the response of food intake, mRNA abundance of hypothalamic

neuropeptides, and fatty acid sensing systems in hypothalamus and liver is

similar to that previously observed when levels of specific fatty acid were raised

by injection and to determine if the phosphorylation state of AMPK, Akt and

mTOR display changes in hypothalamus and liver in response to changes in

dietary lipid levels. The increased levels of fatty acids in hypothalamus and liver

of rainbow trout fed the HF diet only partially activated fatty acid-sensing

systems with no changes in food intake allowing us to suggest that fatty acid-

sensing response in fish to increased levels of fatty acids is more dependent on

the presence of specific fatty acids rather than to the global increase in fatty

acids. In hypothalamus and liver, AMPK, Akt and mTOR are generally activated

in fish fed the HF diet, suggesting an enhanced response of the cellular

signaling pathways to the increased availability of fatty acids.

Funded by Ministerio de Economía y Competitividad and European Fund for

Regional Development (AGL2013-46448-3-1-R and FEDER).

55

P10

REGULATION OF ZEBRAFISH GAMETOGENESIS: A TRANSGENIC

APPROACH

Sandra Navarro1, R. Guillot

1, M. Mischitelli

2, E. Sánchez

1, R. Cortés

1, L.

Soletto1 & J.M. Cerdá-Reverter

1. E-mail: sandranavarro@iats.csic.es

1Department of Fish Physiology and Biotechnology, Institute of Aquaculture of

Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC),

Castellón, Spain. 2

Department of Biological and Environmental Sciences,

University of Messina, S. Agata (ME), Italy.

Early puberty adversely affects growth, food efficiency conversion and market

values. Conversely, the delayed puberty can also be a problem for the

aquaculture which involves an increase in costs and health risk to maintain

stocks of fish.

In Poecilid fish, the first sexual maturation is regulated by a unique sex-linked

Mendilian locus named locus P. Recent experiments have demonstrated that

locus P is occupied by multiple copies of the melanocortin receptor 4 (MC4R).

Melanocortins are small peptides encoded by a complex precursor called

proopiomelanocortin. These peptides include the melanocyte-stimulating

hormones and the adrenocorticotropic hormone. Five different receptors

(MC1R-MC5R) mediate their physiological actions. Melanocortin signaling is

regulated also by endogenous antagonists, agouti-signaling protein (ASIP) and

agouti-related protein (AGRP) that compete with the melanocortin peptides by

binding to MCRs. AGRP is mainly expressed in the hypothalamus and works as

an inverse agonist and/or competitive antagonist at MC4R. ASIP is mainly

expressed in the ventral skin and binds MC1R to inhibit ventral melanogenesis.

Very recently, we have developed a new transgenic zebrafish line ovexpressing

ASIP. This fish line exhibits profuse alteration of the dorso-ventral pigment

pattern but also exhibits faster and bigger growth than wildtype fish both in

length and weight. We have carried out experiments to evaluate the puberty

onset in zebrafish using same-aged embryos from different ASIP and wildtype

breeding mates. Our results demonstrated that ASIP males reach puberty earlier

than wildtype fish (unpublished data) however studies on female transgenic fish

remains to be done. In addition, we demonstrate the presence of MC4R in ovary

and testis by ISH. The involvement of melanocortin system in the puberty onset

will be discussed.

Funded by Ministerio de Economía y Competitividad and European Fund for

Regional Development (AGL2013-46448-3-3-R and FEDER).

56

P11

MRAPS PARTICIPATION IN THE FISH MELANOCORTIN SYSTEM

Raúl Cortés1, M.J. Agulleiro

1, M. Michel

2, S. Navarro

1, E. Sánchez

1, L. Soletto

1,

R.D. Cone2, J.M. Cerdá-Reverter

1. E-mail: raulcortesbarrera@gmail.com

1Department of Fish Physiology and Biotechnology, Institute of Aquaculture of

Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC),

Castellón, Spain.

. 2Department of Molecular Physiology & Biophysics,

Vanderbilt University. Nashville, Tennessee (USA).

Melanocortin system is composed of hormones derived from

proopiomelanocortin precursor (POMC), including adrenocorticotropic

hormone (ACTH) and melanocyte-stimulating hormones (MSHs), melanocortin

receptors (MC1R-MC5R), and endogenous antagonists, i.e. agouti signaling

protein (ASIP) and agouti-related protein (AgRP). Recently, a new member of

melanocortin system has be added, the melanocortin receptor accessory protein

(MRAP). Our studies have demonstrated the presence of three MRAPs in the

zebrafish genome named as MRAP1, MRPA2a and MRAP2b. Similar to

mammalian system, MRAP1 is necessary for the ACTH signaling via MC2R

but MRAP2s were unknown.

In order to explore possible interactions with MCRs we studied the expression

profiles of all zebrafish MRAPs and MCRs by qPCR. Both MRAP2s were

highly expressed in the brain together with MC4R. Immunoprecipitation studies

showed physical interaction MRAP2s/MC4R. Double in situ hybridization

showed MRAP2 and MC4R co-localization in the neurons of the tuberal

hypothalamus meanwhile MRA2b/MC4R are co-localized within the preoptic

area. Pharmacological experiments demonstrated that co-transfection of

MRAP2a/MC4R, transform zebrafish MC4R into an ACTH receptor whereas

MRAP2b/MC4R results in a reduction of the constitutive activity of the

receptor. We have also demonstrated that MRAP2s interact with MC1R, but no

effects at signaling level were observed. We have further demonstrated that

ACTH-induced inhibition of food intake requires a functional MC4R, thus

providing a plausible physiological role to the MRAP2/MC4R interaction. We

are now using genome editing techniques like CRISPR/Cas9 system to

corroborate this result. We have successfully deleted MRAP2a from zebrafish

genome and we have discarded the involvement of MRAP2a in the larval

growth as previously suggested.

Funded by Ministerio de Economía y Competitividad and European Fund for

Regional Development (AGL2013-46448-3-3-R and FEDER).

57

P12

POSSIBLE ROLE OF MELANOCORTIN IN THE CONTROL OF

CIRCADIAN ACTIVITY RHYTMS

Lucia Soletto1, S. Puchol

1, S. Navarro

1, R.. Cortés

1, E. Sánchez

1, J.M. Míguez

JM2, J.F. Rosel

3, J. Rotllant

4 , J.M. Cerdá-Reverter

1 E-mail:

lucia.soletto@csic.es 1Department of Fish Physiology and Biotechnology, Institute of Aquaculture of

Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC),

Castellón, Spain. 2Lab. Fisiología animal, Depto. Biol. Funcional y CC Salud,

Facultad de Biología, Universidad de Vigo, Spain. 3Departamento de

Psicología Evolutiva, Educativa, Social y Metodología, Universidad Jaume I,

Castellon, Spain. 4 Aquatic Molecular Pathobiology Lab, IIM- CSIC, Vigo,

Pontevedra, Spain.

Melanocortin system is formed by peptides derived from proopiomelanocortin

(POMC). Signaling is mediated by five different melanocortin receptors

(MC1R-MC5R). In addition, two different endogenous antagonists of MCRs

have been characterized: the agouti-signaling protein (ASIP) and agouti-related

protein (AGRP). ASIP is expressed in the ventral skin where it antagonizes the

effects of MSH on MC1R and is responsible for the dorso-ventral pigment

pattern in fish. AGRP is made in the hypothalamus and is involved in the

control of food intake by antagonizing central MC4R function. Zebrafish

genome exhibits an extra copy of AGRP named AGRP2. AGRP1 is expressed in

the hypothalamus whereas AGRP2 is mainly made in the pineal complex.

Previous studies have shown that ASIP functions as an antagonist for zebrafish

MC4R and ASIP over-expression in transgenic systems results in increased

growth and food intake. Our brain-pituitary transcriptomic analysis have

revealed two important sets of evidence concerning the involvement of the

circadian timing system in melanocortin function. A comparison of

differentially expressed genes (DEG) in the brain between wild type (WT)

males and female fish shows an enrichment of GO terms related with circadian

rhythms (p<5.2x10-9

) suggesting that males and females may show fundamental

differences in circadian timing system function. However, our comparison of

the brain-pituitary transcriptome of males and females over-expressing ASIP

(ASIP zebrafish) revealed that these differences were mitigated. A comparison

between ASIP vs WT males or females, revealed GO enrichment only in males

(p<1.7x10-3

) but not when comparing females of different genotypes. Results

suggest that WT males and females seem to exhibit differences in their

circadian timing system but that ASIP is able to mitigate such differences.

Funded by Ministerio de Economía y Competitividad and European Fund for

Regional Development (AGL2013-46448-3-3-R and FEDER).

58

P13

THE INVOLVEMENT OF GALANIN IN THE REPRODUTIVE CYCLE

OF SEA BASS.

Zélia Velez1, P. Pinto

1, R.S. Martins

1, S. Santos

1, A. Andrade

1, A. Gómez

2, S.

Zanuy2, A. Canário

1 . E-mail: ppinto@ualg.pt

1Laboratory of Comparative Endocrinology and Integrative Biology, CCMAR –

Centre of Marine Sciences, University of Algarve; 2 Instituto de Acuicultura de

Torre de la Sal, Castellon, Espana.

In commercial aquaculture systems the sea bass (Dicentrarchus labrax)

consistently presents high rates of precocious puberty males, which is correlated

with negative aspects to aquaculture production. Preliminary data suggest that

galanin (GAL), a neuropeptide associated with increased appetite, is potentially

involved in the control of the reproductive cycle. Taking in account that the

identification and characterization of regulators of the reproductive cycle in sea

bass would be extremely beneficial to commercial aquaculture systems, the aim

of the present work was the evaluation of the putative involvement of GAL in

the reproductive cycle of sea bass. Dispersed pituitary cells collected from fish

in different stages of the reproductive cycle were incubated with GAL and the

amount of released gonadotropins luteinizing hormone (LH) and follicle

stimulating hormone (FSH) was quantified and compared with that of control

cells. In addition, the effects of GAL in cAMP production was also accessed.

Our results showed that there was a peak of FSH release in September in two

year-old males (pre-pubertal) in response to GAL, while there were no

differences relative to control cells in November nor in January (spermiating

males). No effects of GAL were induced on pituitary LH release in September

and November, while in January there was a significant increase of LH release

in all the concentrations tested. Galanin alone had no effect on pituitary cells

cAMP production, however in cells previously stimulated with forskolin (an

activator of adenylate cyclase) there was an inhibition of cAMP production by

GAL, in September and October but not in January. RT-PCR allowed to detect

the expression of several subtypes of galanin receptors in pituitaries collected at

the analysed time points of the year. These results suggest the involvement of

galanin in the reproductive cycle of sea bass.

Acknowledgements: Funded by FCT – Portuguese Science and Technology

Foundation through projects UID/Multi/04326/2013 and PTDC/MAR-

BIO/3890/2012 and grant FCT SFRH/BPD/25247/2005 to PISP.

59

P14

SEASONAL SYNCHRONIZATION TO ENVIRONMENTAL CUES IN

SALMONIDS: ARE PHOTOPERIOD AND TEMPERATURE ACTING

THROUGH A COMMON PATHWAY?

Laura Gabriela Nisembaum1, A. Bantz

1, E. Magnanou

1, M. Fuentes

1, P.

Martin2, L. Besseau

1, J. Falcón

1. E-mail: nisembaum@obs-banyuls.fr

1Sorbonne Universités, UPMC Univ Paris 06, CNRS, Biologie Intégrative des

Organismes Marins (BIOM), Observatoire Océanologique, Banyuls/Mer,

France; 2Conservatoire National du Saumon Sauvage, Chanteuges, France.

The complex life cycle of the Atlantic salmon (Salmo salar) involves a

downstream migration from fresh to sea water. The timing of this physiological

and behavioral transformation (smoltification) is triggered by external cues such

as photoperiod and temperature. Their combination is currently changing with

global warming: ambient temperatures are rising while daily and seasonal

photoperiod variations remain the same. The secretion of the time-keeping

hormone, melatonin (MEL), is a nocturnal and Ca2+

dependent process that

takes place in the pineal and retinal photoreceptor cells. The duration of MEL

secretion reflects night length, while the amplitude of its surge is influenced by

ambient temperature. While the mechanisms of the light/dark regulation of

melatonin secretion are quite well understood, those of temperature remain

unelucidated. We hypothesize that thermoreception in salmonids might be

mediated by transient receptor potential channels (TRPs), that mediate

temperature sensing in mammals. TRPs are activated by different ranges of

temperature and are permeable to calcium. Therefore, light and temperature are

likely to act through the same intracellular messenger, Ca2+

in the

photoreceptors. In the present study, photoperiod and/or temperature were

manipulated during smoltification process in order to assess the effect of each

signal and/or mimic the predicted effect of global warming. Salmons were

sampled every two months from December to June of the following year, and

the gene expression of two receptors from the vanilloid subfamily (TRPV1 and

TRPV4) was quantified in the retina, pituitary gland and gills. A combined or

separated impact of temperature and photoperiod was observed on the

expression levels of TRPVs, in a tissue specific-way. These results support TRP

as good candidates for thermoreception in fish and provide clues to understand

temperature modulation of melatonin secretion and synchronization to seasonal

events.

60

P15

QUANTIFICATION OF PLASMA STEROIDS IN SOLE (Solea

senegalensis) BY ULTRA HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY COUPLED TO TANDEM MASS

SPECTROMETRY

Eduardo Beltrán1, V. Piquer

2, J.M. Guzmán

3, P. Swanson

3, E. Mañanós

2, R.

Serrano1. E-mail: evaristo@iats.csic.es

1Instituto Universitario de Plaguicidas y Aguas (IUPA), Universidad Jaume I,

12071-Castellón, España. 2Instituto de Acuicultura de Torre la Sal (IATS),

CSIC, 12595-Castellón, España. 3Northwest Fisheries Science Center, NOAA,

Seattle, Washington 98112, USA

Steroidal hormones control the reproductive functions of vertebrates. Their

study is crucial to understand the different stages of fish development

(ontogeny, gonad differentiation and reproductive periods). Steroids are present

in biological matrices at low levels (in plasma at ng ml-1

to pg ml-1

). Therefore,

a robust and sensitive analytical method for multiple detection is required.

Immunoassays are the preferred technique for steroid determination due to its

high sensitivity; however, they require processing one sample for each analyte.

Liquid chromatography coupled to tandem mass spectrometry is the alternative

technique which allows a sensitive and reliable simultaneous determination of

several hormones. In this work, an ultra high performance liquid

chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS)

method was developed for the analysis of 11 steroids (17β-estradiol, estrone,

androstenedione, testosterone, progesterone, pregnenolone, 11ketotestosterone,

cortisol, 11-deoxycortisol, 17α,20β-dihydroxy-4-pregnen-3-one and

17α,20β,21,trihydroxy-4-pregnen-3-one) in plasma samples of sole. Sample

treatment was based on protein precipitation with acetonitrile and subsequent

clean-up with OASIS HLB SPE cartridge. Target compounds were determined

in selected reaction monitoring mode, using a triple quadrupole analyser (TQS,

Waters) with a rapid chromatographic separation (6 min), using a reversed

phase column (Acquity BEH 1.7µm, 50 x 2.1 mm). The method was validated

by means of recovery experiments, obtaining satisfactory results of accuracy

and precision. Lowest level validated was 0.1 ng ml-1

. Limits of detection

(LODs) were in the range of pg ml-1

. Finally, the method was applied to the

analysis of plasma samples. The acquisition of two MS/MS transitions for each

compound allowed the unequivocal confirmation of positive samples.

Acknowledgements: Funded by Ministerio de Economía y Competitividad

(“Solembryo” AGL2013-49027-C3-3-R).

61

P16

THE ROLE OF LIPIDS AND FATTY ACIDS THROUGHOUT

SPERMATOGENESIS OF EUROPEAN EEL (Anguilla anguilla) AND

EFFECT OF DIETS ON REPRODUCTIVE PERFORMANCE

Rosa Baeza1, I.A.E. Butts

2, M.C. Vílchez

1, V. Gallego

1, D.S. Peñaranda

1, H.

Tveiten3, L. Pérez

1, J.F. Asturiano

1 . E-mail: jfastu@dca.upv.es

1Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología

Animal. Universitat Politècnica de València, 46022 Valencia, Spain. 2Technical

University of Denmark, National Institute of Aquatic Resources. Kavalergården

6, 2920 Charlottenlund, Denmark. 3Norwegian Institute of Fisheries and

Aquaculture, Muninbakken 9-13 Breivika, 6122, Tromsø, Norway

During the last decades, European eel populations have declined considerably

and this species is considered outside its safe biological limits. The life cycle of

the eel is quite complex including transoceanic migration where native

conditions are still unknown and the natural spawning process has never been

observed. Eels cease feeding during migration and that is why their energy

reserves, such as lipids, play a crucial role in obtaining good quality gametes

(eggs and sperm). In the last years, efforts have been directed at studying the

influence of fatty acids on the reproductive performance of females, while less

effort has been devoted to males.

The present study describes the results obtained in two experiments on

European eel males. The first experiment focused on the effect of fatty acids

throughout sexual maturation, their relationship with steroid hormones, and

their effect on sperm quality parameters. The second experiment involved the

comparison of different dietary regimes on sperm quality traits.

Results of the first experiment showed ARA, EPA and DHA maintenance in the

testis during eel spermatogenesis, while all other fatty acids decreased. ARA

and EPA maintenance may have a physiological function, i.e. as prostaglandins

precursors, while the maintenance of DHA levels may have a structural role for

spermatozoa membrane formation. On the other hand, EPA and DHA acted as

modulators of androgens synthesis particularly during the final phase of the

sperm maturation, and ARA had an effect on sperm velocity. With the acquired

knowledge, a second experiment was carried out in which we designed feeds

with different fatty acid percentages in order to evaluate the influence of these

diets on sperm quality. Together, this work demonstrated higher sperm motility

and increased milt volume from eels fed balanced diets.

62

P17

CHARACTERIZATION OF ALDEHYDE DEHYDROGENASES aldh1a2

AND aldh1a3 IN THE EUROPEAN SEA BASS

Paula Medinaac

, A. Gómezb, S. Zanuy

b, M. Blázquez

a. E-mail:

blazquez@icm.csic.es

aInstitut de Ciències del Mar (ICM-CSIC), Barcelona, Spain.

bInstituto de

Acuicultura de Torre la Sal (IATS-CSIC), Castellón, Spain. c

Universidad de

Antofagasta, Antofagasta, Chile

The aldh1a family is involved in the oxidation of aldehydes in different tissues.

Several members of this family are responsible for the synthesis of retinoic acid

(RA), known to act as a meiotic stimulating factor in higher vertebrates, but in

fish this is yet to be confirmed. The main goal of the study was the molecular

characterization of the aldh1a family in the European sea bass, Dicentrarchus

labrax. The study reports the isolation and characterization of aldh1a2 and

aldh1a3, as well as the identification of both genes in other teleost and non-

teleost genomes. Their tissue distribution in males and in females was also

assessed. In addition, testicular cultures were used to study the effect of

exogenous RA and FSH, an endocrine key regulator of germ cell development,

on the expression of aldh1a2 and aldh1a3. The study shows that both proteins

exhibit the main structural amino acid motifs that define them as active

NAD(P)-dependent catalytic enzymes. The phylogenetic and synteny analyses

of the proteins confirm that they are different orthologs further supporting that

the aldh1 family appeared before the divergence of teleosts from the

evolutionary lineage. Tissue-specific mRNA expression in juvenile sea bass

showed that aldh1a2 levels were higher than those of aldh1a3. Both mRNAs

were ubiquitously expressed in several tissues including testis and ovaries, with

a clear preference and female-specific expression for aldh1a3 in gonads and

brain. This sex-differential expression in gonads suggests a tight regulation on

the oxidation of aldehydes and the subsequent synthesis of RA, pointing at their

role in cell proliferation and differentiation during gonadal development.

Moreover, the expression in testicular cultures of both aldhs during early stages

of maturation was sensitive to exogenous RA and FSH, further suggesting an

important role of these enzymes in gonadal maturation and reproduction.

Funded by projects Reprosex (AGL2011-28890) and Reprobass

(PROMETEOII/2014/051). PM was sponsored by a grant MECE2 (ANT0806).

63

P18

EXPOSURE OF SOLE (Solea senegalensis) LARVAE TO DIFFERENT

ENVIRONMENTAL CONDITIONS AFFECT THE SYNTHESIS OF

REPRODUCTIVE HORMONES

Vanesa Piquer1, M. Aliaga

2, J.A. Paullada

2, J. Ramos

1, J.A. Muñoz-Cueto

2, E.

Mañanós1. E-mail: vanesa@iats.csic.es

1Instituto de Acuicultura de Torre la Sal (IATS-CSIC), Castellón, España.

2Departamento de Biología, Facultad de Ciencias del Mar y Ambientales,

Universidad de Cádiz, Puerto Real, España

In fish, environmental factors have profound effects on larval development. In

sole, we have recently shown that daily thermocycles, compared to constant

temperatures, affect gonad differentiation (GD) and change sex ratios. Also,

blue light affects positively several metabolic and reproductive parameters,

compared to other light spectra. The mechanisms involved in the integration of

environmental signals in fish larvae and the early establishment of the

reproductive axis are largely unknown. This study investigated the combined

effects of light and temperature during early development. Sole larvae were

exposed to, 1) daily thermocycle and blue light (18-21ºC; TCblue) or, 2)

constant temperature and white light (19.5ºC; cteW; routine aquaculture

conditions). In addition, and to determine a potential sensitive period, the

TCblue conditions were applied in different time frames, 1) until

metamorphosis (0-12 days post hatching (dph)), 2) post-metamorphosis (0-30

dph) or, 3) gonad differentiation (0-100 dph). Effects were studied by analyzing

steroid levels (ELISA) and gonadotropin gene expression (qPCR). Body weight

was similar in all TCblue groups and, from 30 dph onwards, higher than in

cteW larva. Metamorphosis happened at 13-20 dph in all groups. Expression of

follicle-stimulating hormone beta (fshb) and luteinizing hormone beta (lhb))

subunits increased at metamorphosis, but only under TCblue; they peaked again

at GD and, in most sampling points, expression was higher in TCblue groups

than cteW. Levels of estradiol, testosterone and 11-ketotestosterone increased at

metamorphosis and GD, whereas the progestrogen 17,20β-P increased after

metamorphosis and was maintained high until 138 dph; no clear effect of

treatments was evident. The results showed a significant synthesis of

reproductive hormones during larval ontogeny and GD which was influenced by

light and temperature regimes.

Work funded by the Spanish Ministry of Economy and Competitiveness

(AGL2010-22139-C03-02 and AGL2013-49027-C3-3-R).

64

P19

IN VIVO EXPRESSION OF NUCLEAR AND MEMBRANE ESTROGEN

RECEPTORS IN EUROPEAN EEL THROUGH SPERMATOGENESIS

Marina Morini1, D.S. Peñaranda

1, M.C. Vílchez

1, A.G. Lafont

2, L. Pérez

1, S.

Dufour2, J.F. Asturiano*

1. E-mail: jfastu@dca.upv.es

1Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología

Animal. Universitat Politècnica de València, Valencia, Spain. 2UMR BOREA

"Biology of Aquatic Organisms and Ecosystems", MNHN, CNRS, IRD, UPMC,

Paris, France

The sex steroid estrogens can bind nuclear receptors (ER) or membrane

receptors (GPR30 or GPER) to elicit a slow genomic action by modulating the

gene transcription and translation activity, or a rapid, non-genomic activation of

intracellular signal transduction pathways. The nuclear ER include ERα and

ERβ, with at least three distinct subtypes, including ERα, ERβ1 and ERβ2 in the

Actinopterigii species. The GPER (G-protein coupled ER) is recognized as a

membrane-bound ER binding estradiol that initiate subsequent signalling

cascade and present at least one copy in all the vertebrates.

In this study, we measured for the first time the expression in vivo of three

nuclear (ERα, ERβ1 and ERβ2) and two membrane (GPER1 and GPER2)

estrogen receptors simultaneously in the tissues of the brain-pituitary-gonad

axis, through the spermatogenesis, in an early teleost, the European eel

(Anguilla anguilla).

Male eel ER mRNA expression in the brain, pituitary and gonads, receptors

increased with the change from freshwater to sea water, suggesting that nuclear

ERs in the tissues of the gonadotropic axis play a critical part in osmoregulatory

adaptations. Gonad nuclear ERs, which were more expressed at spermatogonia

stage, are likely to play a role in the spermatogonia renewal; whereas both

membrane receptors (GPERs), which are more expressed at spermatozoa stage,

seems to be involved in the final spermatogenesis. Furthermore, brain and

pituitary nuclear ERs and pituitary GPERs seems to mediate autocrine or

paracrine communication of local estrogen production, through the

spermatogenesis. Finally, the expression of both nuclear ERs and GPERs in the

brain-pituitary-gonad axis indicates possible cooperation between genomic and

non-genomic estrogen actions in controlling reproduction.

Funded by IMPRESS (Marie Sklodowska-Curie Actions; Grant agreement nº:

642893) and MINECO (REPRO-TEMP; AGL2013-41646-R).

65

P20

INFLUENCE OF EXOGENOUS MELATONIN ON GROWTH,

GONADAL MATURITY AND kiss/gnrh GENE EXPRESSION

PATTERNS IN THE BRAIN OF MALE SEA BASS (Dicentrarchus labrax)

M. Victoria Alvarado, M. Carrillo, A. Felip. E-mail: afelip@iats.csic.es

1Instituto de Acuicultura Torre la Sal, Consejo Superior de Investigaciones

Científicas (CSIC), Castellón, Spain.

The influence of long-term melatonin administration, as an implant, on growth

performance and reproduction of adult male sea bass was studied in vivo. Two

years old male sea bass were organized into two groups: in the first, which acted

as a control group, the animals were administered empty implants, whereas in

the second, the animals were administered melatonin implants. Our results

provided that this hormone had an anorexic action, thus affecting the

performance of fish. Melatonin reduced the fish weight and condition factor,

with the mean weight of melatonin-treated fish amounting to only 77.7% as

compared to controls. Melatonin also affected gonadogenesis following

testicular maturation in adult sea bass when administered over an extended

period of time throughout their entire reproductive cycle. A decrease in the

gonadosomatic index after 150 days of treatment (29% lower than in controls)

and a lower percentage of running males during the spermatogenesis and full

spermiation stages (January-March) was observed in this species. Exogenous

melatonin also resulted in lower plasma levels of testosterone and 11-

ketotestosterone during the reproductive period, and showed a significant

decrease of circulating Lh and Fsh levels after 30 and 60 days of treatment,

respectively. Furthermore, the hypothalamic expression of kiss1 was

significantly higher in melatonin-treated fish than in controls after 30 days of

treatment, while a significant increase in kiss2 expression was detected on day

90 of treatment. By contrast, melatonin showed a significant decrease in

kisspeptin expression in the dorsal brain on day 150 of treatment and also

affected the expression of gnrh-1 and gnrh-3 as well as gnrhr-II-1a and 2b and

the fshβ gene in the pituitary. Taken together, these findings reveal that

melatonin evokes seasonal changes in key reproductive hormones that affect

testicular maturity. In addition, melatonin might presumably induce the

downregulation of kisspeptin-gnrh members on the dorsal brain thus affecting

fsh-beta ?transcription during early gametogenesis that appear to mirror

disturbances in spermatogenesis.

66

P21

REGULATORY RESPONSES TO SALINITY IN SEA BASS

Pedro S. Palma1, P. M. Guerreiro

1, D. M. Power

1, A. V. M. Canário

1. E-mail:

pfpalma@ualg.pt

1CCMAR - Centre of Marine Sciences, University of Algarve, Portugal.

Changes in salinity modifies the extracellular ionic environment which needs to

be regulated by multiple hormones. Calcium and sodium are key ions that need

to be regulated to maintain homeostasis as they are toxic and the two main

glands known to influence their extracellular levels are the pituitary and the

corpuscles of Stannius (CS). Calcium homeostasis is maintained by the

antagonistic actions of the parathyroid hormone-related protein (PTHrP) and

Stanniocalcin (STC). STC is a hypocalcemic hormone and is secreted by the

Corpuscles of Stannius (CS) which undergo changes in size/activity as water

calcium levels increase, and by the other hand PTHrP has hypercalcemic actions

promoting calcium uptake via gills and intestine. Prolactin (PRL) produced in

the pituitary is known to regulate sodium. Here we have analysed changes in

expression of genes that encode for these hormones as well as other genes

known or suspected to be involved in ion homeostasis during exposure of sea

bass, Dicentrarchus labrax, to salinity changes (transfer from 36 ppt to 3.6 ppt

starting at 2 hours after transfer and up to 30 days). Significant changes in gene

expression compared to control maintained at 36 ppt were found for PTHrPA

and STC1 in pituitary and CS, for PRL in the pituitary and for fibroblast growth

factor 23 (FGF23) in CS. There were no significant changes in calcium sensing

receptor and PTH receptors, with the exception of PTH3R in the pituitary.

Overall our results reflect coordinated and tightly controlled mechanisms of

gene expression underlying ion regulation during brackish water adaption at the

level of the pituitary and CS.

Acknowledgements: Funded by Portuguese Foundation for Science and

Technology projects PTDC/MAR/121279/2010 to AVMC and PDCT/BIA-

ANM/4225/2012 to PMG.

67

P22

VASOTOCINERGIC AND ISOTOCINERGIC CO-REGULATION IN

STRESS RESPONSE OF COMMON CARP (Cyprinus carpio L.)

Ismael Jerez1, J. M. Mancera

1, G. Flik

2 & M. Gorissen

2. E-mail:

ismael.jerez@uca.es

1Department of Biology, Faculty of Marine and Environmental Sciences,

University of Cadiz, Spain. 2Department of Animal Physiology, Institute for

Water and Wetland Research, Radboud University, Nijmegen, The Netherlands.

Under stress conditions, the teleostean hypothalamic-pituitary-interrenal (HPI)

axis is stimulated in order to cope with a disturbance of the homeostasis.

Endocrine regulation of this axis is controlled at different levels, involving

several pathways that activate the synthesis and release of cortisol. This axis

starts with the integration of the perception and appraisal of the stressor(s) by

the brain and the release of the corticotropin-releasing factor (CRF) from the

hypothalamic nucleus preopticus (NPO). CRF stimulates mainly the synthesis

of adrenocorticotropic hormone (ACTH) from the pituitary pars distalis, which

in turn stimulates the production and release of cortisol by the interrenal cells in

the head kidney. Furthermore, the NPO innervates pars intermedia cells from

the pituitary gland to produce acetylated endorphins and α-MSH (α-melanocyte-

stimulating hormone). The role of α-MSH in the stress response is not yet clear,

being either associated with pigmentation and food intake, and depending on

the species in the regulation of cortisol levels under chronic stress situations.

The picture becomes even more complex as arginine vasotocin (AVT) and

isotocin (IT), homologous to mammalian vasopressin and oxytocin, have been

demonstrated to be associated with both the central and peripheral regulation of

the stress response. In this study, we aim to assess the effect of AVT and IT on

the release of α-MSH and cortisol in common carp (Cyprinus carpio L.). We

performed an in vitro superfusion experiment using pituitary glands and head

kidneys, and incubated them with different combinations of either CRF or

ACTH, with / without AVT and / or IT. Our results suggest a potentiation of the

stress response by AVT and IT, by stimulation of α-MSH and cortisol release in

co-operation with CRF and ACTH, respectively.

68

P23

CHANGES IN PLASMA AND BRAIN STRESS-RELATED

PARAMETERS IN RAINBOW TROUT EXPOSED TO CONSPECIFIC

CHEMICAL SIGNALS

Jésus M. Míguez, F. Naderi, M.A. López-Patiño, J.L. Muñoz*, J. Hernández-

Pérez, R. Álvarez-Otero, M. Gesto¥. Email: jmmiguez@uvigo.es

Lab. Fisiología animal, Depto. Biol. Funcional y CC Salud, Facultad de

Biología, Universidad de Vigo, Spain. *,¥Current address: *Centro i?mar, Univ.

Los Lagos, Puerto Montt, Chile; ¥Section for Aquaculture, DTU Aqua, Tech.

University of Denmark, Hirtshals, Denmark.

Many fish species use chemical cues for predator recognition and defensive

behavior. These chemical signals are generally produced, or contained, within

the skin or in body fluids, and are only released through mechanical damage or

injuries, eliciting alarm reactions on conspecifics that comprise behavioral and

physiological changes. It has been demonstrated that olfaction is mainly

involved on the alarm response but it is still unclear whether an activation of the

neuroendocrine stress pathways participate in this reaction in fish. In the present

study we evaluated the physiological response of rainbow trout (Oncorhynchus

mykiss) to the presence in water of blood and skin extracts coming from

conspecifics. We exposed trout for 3 min and 15 min to 4 different scents:

“stressed blood”, “unstressed blood” (obtained from stressed and unstressed

trout, respectively), skin extract or distilled water (control). Trout from all

groups were sacrificed avoiding handling stress, and blood and brain samples

were obtained and assayed for plasma levels of stress-related hormones

(catecholamines, cortisol) and metabolites (glucose, lactate), as well as for brain

monoaminergic neurotransmitters (serotonin, dopamine). Trout exposed to

blood from stressed fish or to skin extract showed increased plasma

catecholamines and cortisol levels. At the brain level, we found decreased

dopaminergic and serotoninergic activity in telencephalon and hypothalamus of

trout exposed to unstressed blood, whereas increases were noted after exposure

to blood of previously stressed trout or to skin extract. Interestingly, the levels

of dopaminergic activity in the olfactory bulb were increased in all exposed

groups. Altogether, these data indicate that different chemical cues from

conspecifics can induce a mild but detectable stress response in trout, thus

demonstrating that these cues, which were already known to induce behavioral

alterations in fish, could also have a metabolic cost. The relevance this kind of

stressor may have in fish farms, where fish often suffer skin lesions, is yet to be

determined.

Funded by MEC AGL2013-46448-3-1-R and FEDER

69

P24

GHRELIN MODIFIES CLOCK GENES EXPRESSION IN THE LIVER

OF GOLDFISH VIA PLC-PKC PATHWAY.

Aida Sánchez-Bretaño, A.M. Blanco, A.L. Alonso-Gómez, M.J. Delgado, E.

Isorna. E-mail: eisornaa@bio.ucm.es.

Department of Animal Physiology, Faculty of Biology, University Complutense

of Madrid.

The liver has been proposed in mammals as a main oscillator which links the

circadian system with metabolism. Ghrelin plays a key role in energy balance

regulation, promoting food intake, carbohydrate utilization and adiposity, and

other physiological processes. In goldfish, an intraperitoneal injection of ghrelin

modifies clock genes expression in several structures including the liver,

suggesting that it could act as a metabolic/food intake signal. The objective of

this work was to investigate if ghrelin acts directly on the liver to modify clock

genes expression and which is the intracellular pathway employed. To this aim,

we studied ghrelin effects on clock gene expression using in vitro static cultures

of goldfish liver. Livers (n=6) were sampled and preincubated in 1 ml DMEM

(15 mg liver/well) during 2 h. Then, the samples were divided into 10

experimental groups: 1 control, 3 ghrelin-treated groups (0.1, 1, 10 nM), 3

groups with a ghrelin-antagonist ([D-Lys-3]-GPRH; 1, 10, 100 nM), and 3

groups with a ghrelin antagonist (1, 10, 100 nM) plus ghrelin (10 nM). Cultures

were maintained for 1 or 5 h. In a second experiment to study if PLC-PKC

pathway is the intracellular pathway used by ghrelin, cultured liver were

preincubated with a PLC inhibitor (U73122; 1, 10 µM) and incubated with or

without ghrelin (10 nM) for 1 and 5 h. Ghrelin induced (2-6 fold) per1a, per1b,

per2a, per3, bmal1a, clock1a and rev-erbα expression after 1 h of treatment,

and this effect was decreased or completely abolished after 5 h. In all the cases,

ghrelin effects at 1 h were partially or completely eliminated by the antagonist

of the ghrelin receptors and with U73122. Present results show for the first time

in teleosts the direct action of ghrelin on the hepatic oscillator by modulating

clock genes expression. Moreover, our data indicate that ghrelin acts via PLC-

PKC pathway. Taken together, these results evidence that ghrelin is one of the

direct links between the food intake and energy balance with the circadian

system in teleosts.

Acknowledgements: Supported by Spanish MINECO (AGL 2013-46448-C3-2-

R). A. Sánchez-Bretaño and A.M. Blanco are predoctoral fellowship from

Spanish MINECO (FPI) and MEC (FPU) respectively.

70

P25

PROLACTIN IN THE MEAGRE (Argyrosomus regius): MOLECULAR

CLONING AND EXPRESSION ANALYSIS UNDER DIFFERENT

ENVIRONMENTAL SALINITIES

Rubén Ayala Suárez1, A. Astola

1, J.A. Martos-Sitcha

2, J. M. Mancera

3. E-mail:

juanmiguel.mancera@uca.es

1Dpto. de Biomedicina, Biotecnología y Salud Pública. Facultad de Ciencias.

Universidad de Cádiz.2Instituto de Ciencias Marinas de Andalucía (ICMAN-

CSIC). 11510 Puerto Real, Cádiz.3

Dpto. de Biología. Facultad de Ciencias del

Mar y Ambientales. Campus de Excelencia Internacional del Mar (CEI-MAR),

11510 Puerto Real, Cádiz. Universidad de Cádiz.

The meagre Argyrosomus regius has gained good popularity for both

aquaculture producers and consumers due to its rapid growth rates, ease in

processing, low fat content and somewhat rigid texture. Recent studies have

shown the good euryhalinity capacity of this species. The role of prolactin (prl)

is well understood in many perciformes species as a fresh-water adapting

hormone, preventing ion loss and promoting water removal, but its role in A.

regius has not been inquired yet. Thus, understanding of this role will provide

knowledge assisting in the development of molecular strategies to identify

saline stress and optimal environmental salinity to improve the culture of this

species. Degenerated primers were designed from published cDNA prl

sequences in perciformes. cDNA synthesis was carried out using total RNA

extracted from pituitary gland, and a prl cDNA was amplified, cloned and

sequenced. prl sequence belongs to partial CDS (GenBank acc. no.:

KP984534), and it shared high sequence identity with its counterparts in other

teleosts. Finally, prl expression level was tested in juveniles specimens

acclimated for 21 days to 4 different environmental salinities: 5 ppt

(hyposmotic, 140 mOsm kg?1

H2O), 12 ppt (isosmotic, 364 mOsm kg?1

H2O), 38

ppt (hyperosmotic; normal seawater, 1090 mOsm kg?1

H2O) and 55 ppt

(extremely hyperosmotic, 1546 mOsm kg?1

H2O). This analysis was

accomplished by quantitative polymerase chain reaction. Generated data was

statistically analysed using one-way analysis of variance (ANOVA) and Tukey-

HSD Post-Hoc test. Significant values were considered when P < 0.01. Results

showed that prl mRNA levels decreased as salinity increased, showing a three-

fold gain at 5 ‰ related to seawater group. According to the hyperosmotic role

of PRL, our results indicated that prl seem to be important under hyposaline

environments, supporting the high euryhaline capacity of A. regius.

71

P26

CLONING, SEQUENCING, AND mRNA EXPRESSION PATTERNS OF

GH AND IGF-I GENES IN MEAGRE (Argyrosomus regius) JUVENILES

ACCLIMATED TO DIFFERENT ENVIRONMENTAL SALINITIES

Khaled Mohammed-Geba1,2,3

, J.A. Martos-Sitcha2, G. Martínez-Rodríguez

2,

J.M. Mancera1. E-mail: juanmiguel.mancera@uca.es

1Genetic Engineering and Molecular Biology Division, Department of Zoology,

Faculty of Science, Menoufia University, Shebin El- Kom, Menoufia,

Egypt.2Instituto de Ciencias Marinas de Andalucía, Consejo Superior de

Investigaciones Científicas (ICMAN-CSIC), 11510 Puerto Real (Cádiz),

Spain.3Department of Biology, Faculty of Marine and Environmental Sciences,

Campus de Excelencia Internacional del Mar (CEI-MAR), University of Cadiz,

11510 Puerto Real, Cadiz, Spain.

The Mediterranean meagre Argyrosomus regius exhibits a growing interest for

aquaculture, presenting a good euryhaline capacity as well as enhanced growth

under isosmotic environments. However, the role of growth hormone

(GH)/insulin-like growth factor-I (IGF-I) axis mediating this euryhalinity is still

completely unveiled. Therefore, and for the first time in this species, cDNAs of

meagre’s pituitary growth hormone (gh), hepatic insulin-like growth factor (igf-

1), and β-actin (actb) were cloned. Their mRNA expression patterns were tested

in juveniles acclimated to 4 different environmental salinities: i) 5 ppt

(hyposmotic, 140 mOsm kg?1

H2O), ii) 12 ppt (isosmotic, 364 mOsm kg?1

H2O),

iii) 38 ppt (hyperosmotic; normal seawater, 1090 mOsm kg?1

H2O), and iv) 55

ppt (extremely hyperosmotic, 1546 mOsm kg?1

H2O). All investigated

transcripts shared high sequence identities with their counterparts in other

perciformes. gh mRNA enhanced significantly in both 12 and 55 ppt salinity

groups in comparison to control group. However, igf-1 showed its maximum

expression levels under isosmotic salinity. The results indicated that growth

enhancement observed in A. regius specimens under isosmotic environment is

supported by the stimulation of GH/IGF-I axis.

Acknowledgments: This work was carried out as a part of the Spanish-Egyptian

joint project AP/039755/11 (Agencía Española de Cooperación Internacional

para el Desarrollo, AECID)

72

P27

PHYSIOLOGICAL CHANGES IN SILVER CATFISH (Rhamdia quelen)

TRANSPORTED WITH ESSENTIAL OIL OF Myrcia sylvatica

E.M.H. Saccol1, J.A. Martos-Sitcha

2, I. Jerez

3, T.S. Pês

1, R.H.V. Mourão

4, B.

Baldisserotto1, M.A. Pavanato

1, G. Martínez-Rodríguez

2, J.M. Mancera

3. E-

mail: etianehsaccol@gmail.com

1Departmento de Fisiologia e Farmacologia, Universidade Federal de Santa

Maria (UFSM), Santa Maria, Rio Grande do Sul, Brazil. 2Instituto de Ciencias

Marinas de Andalucía, Consejo Superior de Investigaciones Científicas

(ICMAN-CSIC), Puerto Real, Cádiz. 3

Departamento de Biología, Facultad de

Ciencias del Mar y Ambientales, Campus de Excelencia Internacional del Mar

(CEI-MAR), Universidad de Cádiz, Puerto Real, Cádiz, Spain. 4Instituto de

Biodiversidade e Florestas, Universidade Federal do Oeste do Pará (UFOPA),

Santarém, Pará, Brazil.

Aquaculture practices include several procedures as capture, handling and

transport, which cause stress in fish. Anesthetics or sedative substances have

been used to reduce this stress. The aim of this study was to evaluate the effects

of essential oil of Myrcia sylvatica (EOMS) in the water on stress system

activation in the silver catfish Rhamdia quelen summited to transport. Fish were

captured in the production ponds and transferred to a 250-L tanks (density of 54

kg/m3). After 24 hours, 10 fish were caught, euthanized by section of the spinal

cord and sampled (basal group). The remaining fish were placed in plastic bags

containing 5 L of water (density of 150 kg/m3) with different doses of EOMS

(0, 25 or 35 μL/L diluted in 315 μL/L ethanol), in triplicate, and transported for

6 h. After transportation, 10 animals of each group were captured, euthanized

and sampled. Cortisol and glucose levels in plasma, as well as hypothalamic

corticotrophin-releasing hormone (CRH) and pituitary proopiomelanocortin

(POMC) “a” and “b” mRNA expressions were determined. Cortisol levels and

CRH expression enhanced after 24h of handling, decreasing during the

transport with addition of EOMS. Expression of POMCa was higher in fish

transported with 25 μL/L respect to the rest of groups. Therefore, it is suggested

the use of EOMS for transporting fish in order to avoid the stress associated

with this procedure.

73

P28

CLOCK GENES AS TARGET FOR FEEDING IN THE FOOD

ENTRAINABLE OSCILLATOR OF LIVER IN GOLDFISH

Miguel Gómez-Boronat, A. Sánchez-Bretaño, A.M. Blanco, I. Redondo, M.J.

Delgado, N. De Pedro, E. Isorna. E-mail: miguelgomezboronat@ucm.es

Department of Animal Physiology, Faculty of Biology, University Complutense

of Madrid.

The circadian system consists of a net of oscillators whose molecular function is

based on circadian regulatory genes named clock genes. Although daily

photoperiod is the most well-known synchronizer of circadian oscillators,

scheduled feeding can also drive circadian rhythms, especially in peripheral

tissues, as the liver. However, very little is known about how food intake is

detected by the hepatic clock in mammals, and nothing in fish. The objective of

this study was to identify the clock genes that could be acting as possible targets

for feeding signalling in the liver clock of goldfish (Carassius auratus). To this

aim, the 24-h expression pattern of several clock genes was analyzed in the liver

of goldfish maintained under a 12L:12D photoperiod and scheduled feeding at

ZT 2. Secondly, we studied the periprandial fluctuations of hepatic clock genes

in fed and unfed fish. Finally, clock genes expression in liver was analyzed in

three fish groups: daily fed (1% bw), fasted (48 h), and fasted plus refed. In all

the experiments, liver samples were collected and clock genes (per1a, per1b,

per3, bmal1a, rev-erbα and pparα) expression was determined using real-time

RT-PCR. Significant daily rhythms were found for all the studied clock genes in

the liver. Results of the periprandial experiment showed that expression of

per1a, per1b and rev-erbα decreases 3 h after food intake in both experimental

groups (fed and unfed fish). Thus, these changes would be a consequence of

their daily rhythmic profiles, regardless of feeding. However, expression of

bmal1a increased only in fed fish, suggesting that the daily profile observed in

this gene is mainly due to food intake. In the last experiment, 48-h fasting

decreased per1a and bmal1a expression, and this effect was reverted by

refeeding, in agreement with the feeding bmal1a induction observed in the

periprandial experiment. Taken all together, present results suggest that bmal1a

could be a target in the food entrainment of hepatic oscillator in fish.

Acknowledgements. Supported by the Spanish MINECO (AGL 2013-46448-C3-

2-R). A.M. Blanco and M. Gómez-Boronat are predoctoral fellows from the

Spanish MEC and MINECO, respectively.

74

P29

INSULIN AND IGF-I PLAY A RELEVANT ROLE IN THE

REGULATION OF NORMAL AND TUMORAL PROSTATE CELL

FUNCTION

F. López-López, A. Sarmento-Cabral, M.D. Gahete, J.P. Castaño, R.M. Luque.

E-mail: ferll_@hotmail.com

Department of CellBiology, Physiology and Immunology, University of

Córdoba; Hospital Universitario Reina Sofía, Instituto Maimónides de

Investigación Biomédica de Córdoba; CIBER Fisiopatología de la Obesidad y

Nutrición;and, Campus de Excelencia Internacional Agroalimentario,

Córdoba, Spain.

Obesity (OB), a multifactorial chronic endocrine-metabolic disease, represents

one of the most serious globalhealth threats. OB is associated with an increase

in the incidence of certain cancertypes, such as prostate cancer (PC), a

hormone-related, heterogeneous, and complex cancer with a high incidence.

Unfortunately, the precise molecular/cellular and endocrine-metabolic

mechanisms underlyingthepathophysiological association between OB and the

higher incidence/aggressiveness of PC are stillnot well understood.Indeed,

although androgens play a primary, crucial role in PC development/progression,

there is growing evidence that other hormones and growth factors that are

significantly altered in OB, such asinsulin (INS) and IGFI, might also play a key

role in the development and progression of PC. However, the direct and relative

effectsof INS and IGFI or the combination of both (I+I) in controlling prostate

cell function has not been completely elucidated. Here, we used normal prostate

(NP) tissues and primary NP cell cultures from mice,as well as two human PC

cell lines (PC3 and LNCaP),as models to evaluate, in parallel, the presence of

INS/IGFI-axes and the direct role of INS and IGF-I alone or in combination. We

found that IGFIR was consistently more expressed than Ins-R in all

experimental models analyzed. Remarkably, both INS and IGFI treatment

increased IGFI/IGFI-R, but not Ins-Rexpression in prostate cells, whichcould be

considered as an indication of PC progression. In fact, IGFI, but not INS or I+I,

treatmentwas able to increase cell proliferation in PC3 and LNCap; whereas,

INS, IGFI and I+I treatments increased migration capacity in PC3. Interestingly,

INS and IGFI alonedecreased PSA secretion in LNCaP cells.Altogether, our

data indicate that INS and IGFI could contribute to the regulation of malignancy

featuresin normal and tumoral prostate cells.

Funding: PI13-00651, BFU2013-43282-R, BIO-0139, CTS-1406, PI-0639-

2012, and CIBERobn

75

P30

LONG-TERM CROWDING STRESS INCREASED CORTISOL ON

SERUM AND SKIN MUCUS OF THE GILTHEAD SEABREAM (Sparus

aurata L.)

Francisco A. Guardiola1, A. Cuesta

1, J.Meseguer

1, M.A. Esteban

1. E-mail:

faga1@um.es

1Fish Innate Immune System Group, Department of Cell Biology and Histology,

Faculty of Biology, Campus Regional de Excelencia Internacional “Campus

Mare Nostrum”, University of Murcia, 30100 Murcia, Spain.

One of the primary responses to stress is mediated by the hypothalamus-

pituitary-interrenal (HPI) axis, which releases cortisol, the dominant steroid in

stress physiology, into the circulatory system. Plasma cortisol levels have been

used to evaluate the stress response in a variety of cultured fish species.

Nonetheless, little is known about the cortisol skin mucus levels as stress

response of fish. To date, this is the first report about the cortisol level in fish

skin mucus. Thus, the aim of this study was to determine the gilthead seabream

(Sparus aurata) cortisol response in serum and skin mucus after long-term

crowding stress. Fish were subjected to 10 kg m-3

(low density, control group)

and 50 kg m-3

(high density as crowding group) during 2, 24 and 48 h. Then, the

cortisol level was evaluated in serum and skin mucus samples using commercial

kits (Calbiotech, USA) following the manufacturer's procedures. Ours results

demonstrated that the cortisol level in serum and skin mucus of gilthead

seabream showed a statistically significant increase in fish that were exposed to

crowding stress after all experimental times. In addition, the cortisol levels in

both samples (serum and skin mucus) followed the same pattern to each point

of sampling, being observed a higher increase in fish exposed to high density

for 24 hours. This study represents an advance on our knowledge about the

interaction between stress and cortisol response in fish skin mucus. Further

investigations are needed in the detection of this stress hormone in skin mucus

of fish subject to other stressor situations and their possible use as stress

bioindicator in intensive aquaculture systems.

Financial support by grants AGL2011-30381-C03-01 (Ministerio de Economía

y Competitividad) and 04538/GERM/06 (Fundación Séneca de la Región de

Murcia, Grupo de Excelencia) are gratefully acknowledged.

76

P31

17β-ESTRADIOL ALTERS THE SUSCEPTIBILITY TO NODAVIRUS

AND THE VIRAL IMMUNE RESPONSE OF EUROPEAN SEA BASS

Yulema Valero1, M. Arizcun

1, FA Guardiola

2, M.A. Esteban

2, A. Cuesta

2, E.

Chaves-Pozo1. E-mail: faga1@um.es

1Centro Oceanográfico de Murcia, Instituto Español de Oceanografía, 30860

Puerto de Mazarrón, Spain. 2Fish Innate Immune System Group, Department of

Cell Biology and Histology, Faculty of Biology, Campus Regional de

Excelencia Internacional “Campus Mare Nostrum”, University of Murcia,

30100 Murcia, Spain.

In addition to their role in fish reproduction, sex steroid hormones are known to

modulate the immune response in vertebrates, including fish. In teleost fish, the

increase on the serum level of 17β-estradiol (E2) has been correlated with

immunosuppression and increased disease susceptibility whilst some studies

have also demonstrated stimulation of some humoral activities. This fact

highlights the complexity of the role of E2 in the regulation of the immune

response and point to the necessity to make further studies to understand how

pathogens modulate E2 serum levels in order to modulate the immune system

and evade the immune response. Nodavirus (NNV) is a vertical transmitted

pathogen that is able to decrease or increase the E2 serum levels of infected

European sea bass (Dicentrarchus labrax) or gilthead seabream (Sparus

aurata), respectively, as revealed by some data obtained in our laboratory.

Taking into account that the European sea bass is very susceptible to NNV we

have experimentally altered the sex steroid serum levels of European sea bass

juveniles specimens by an intraperitoneal injection of E2 concomitantly with an

in vivo infection with in order to determine whether high levels of E2 in serum

might modify the kinetic of the infection by means of studying some humoral

activities and gene expressions relevant to the innate immune response.

Financial support by grants AGL2013-43588-P (MINECO and FEDER), RYC-

2009-05441 (MINECO), 04538/GERM/06 (Fundación Séneca, Región de

Murcia) and fellowship to Y. Valero (Instituto Español de Oceanografía) are

gratefully acknowledged.

77

P32

KISSPEPTINS PATHWAY IS ALTERED IN THE EUROPEAN SEA BASS

UPON NODAVIRUS INFECTION

Yulema Valero1, A. Cuesta

2, M. Arizcun

1, FA Guardiola

2, M.A. Esteban

2, J.

Meseguer2, E. Chaves-Pozo

1. E-mail: faga1@um.es

1Centro Oceanográfico de Murcia, Instituto Español de Oceanografía, 30860

Puerto de Mazarrón, Spain. 2Fish Innate Immune System Group, Department of

Cell Biology and Histology, Faculty of Biology, Campus Regional de

Excelencia Internacional “Campus Mare Nostrum”, University of Murcia,

30100 Murcia, Spain.

In vertebrates, the kisspeptins pathway in the brain is an essential upstream

regulatory element of the hypothalamus - pituitary - gonad (HPG) axis which

regulates reproduction. In European sea bass it has been described two forms of

kisspeptin genes, kisspeptin 1 (kiss1) and kisspeptin 2 (kiss2), and two forms of

G-coupled protein receptor for kisspeptins, gpr54-1b and gpr54-2b. The

kisspeptins pathway activation is involved in the release of GnRH in the brain,

which subsequently, is involved in the release of the gonadotropin hormones

(LH and FSH) in the pituitary, and in turn acts in the regulation of the

steroidogenesis and gametogenesis in the gonad. One of the pathogens that

greatly affects the brain is nodavirus (NNV), a known vertically transmitted

virus, which causes the viral encephalopathy and retinopathy (VER) disease.

Previous studies showed that, in addition to infect the brain and retina and

cause a considerable fish death in European sea bass specimens, NNV is able to

colonize and replicate into the gonad, altering the sex steroid hormone levels

and triggering a high immune response into the tissue. In this study, we have

determined the alteration on the expression levels of kiss1 and 2, gpr54-1b and -

2b, gnrh1, 2 and 3 genes and also the GnRH receptor (gnrhr2a) gene in the

brain and retina of European seabass males upon NNV intramuscular or

intravitreal infection. This study represents an advance on our knowledge about

the interaction between host and NNV, needed to understand how this virus is

able to avoid the immune response of adult fish to spread to the progeny.

Financial support by grants AGL2013-43588-P (MINECO and FEDER), RYC-

2009-05441 (MINECO), 04538/GERM/06 (Fundación Séneca, Región de

Murcia) and fellowship to Y. Valero (Instituto Español de Oceanografía) are

gratefully acknowledged.

78

P33

DIETARY Pi RESTRICTION IN SEA BASS AND HORMONAL

MODULATION OF PHOSPHORUS AND CALCIUM INTESTINAL

ABSORPTION

Alexandra Alves, S.C. Silva, A.V.M. Canario, D.M. Power, P.M. Guerreiro.

Email:alcalves@ualg.pt

CEIB - Centro de Ciências do Mar, Universidade do Algarve, Portugal.

The endocrine factors regulating inorganic phosphorus (Pi) homeostasis in

vertebrates are not completely understood and our knowledge comes from its

association with calcium (Ca) where parathyroid hormone (PTH) or 1,25-

dihydroxyvitamin D3 (VitD) play relevant roles. A novel factor, fibroblast

growth factor-23 (FGF23) was described as a key Pi regulator, with actions on

urinary Pi excretion but also on the expression of PTH and VitD. There is no

information on how these factors may interplay in fish but our studies show that

the Corpuscles of Stannius, the source of the anti-hypercalcemic stanniocalcin

also express PTHrP and FGF23. Does Pi availability determine hormonal

profiles and the response of absorption/excretion mechanisms?

We measured the rates of intestinal Pi and Ca absorption in sea bass,

Dicentrarchus labrax, fed with different Pi diets, and under several hormonal

treatments ex vivo. Fish (~70g) were allocated in 5 groups in tall 500L open

system tanks and fed a commercial control diet (1.1%P) for at least two weeks.

For the feeding trial four experimental diets containing 0.05, 0.3, 1.1 and 3.0

%P, plus the control diet, were used. Fish were fed (2-3% w/w) twice a day and

the remaining pellets collected 30 minutes after feeding to establish Pi

consumption and growth and feeding parameters. At 10 and 30 days into the

trial blood samples were collected and analyzed for electrolytes and energetic

substrates. Intestinal sections from fish in different conditions were dissected

and mounted in Ussing chambers and uni-direccional fluxes were measured in

symmetric and asymmetric conditions with or without hormonal exposure using

radioactive tracers.

Different dietary Pi had a marked effect (P<0.001) on circulating Pi but not on

Ca (P=0.176), and quantification of circulating hormone levels is underway.

Regional differentiation in intestinal absorption was observed for both Pi and

Ca, and this was further exacerbated by dietary conditions. Preliminary data

shows effects of VitD and PTHrP on absorption but it still uncertain whether

their actions or FGF23 were modulated by available Pi.

Funded by FCT - Foundation for Science and Technology through project

PTDC/BIA-ANM/4225/2012.

79

P34

DOES THE URINARY BLADDER PLAY A ROLE IN FISH RENAL

FUNCTION? SOME HINTS FROM PHOSPHATE AND CALCIUM

TRANSPORT STUDIES

Pedro M. Guerreiro, A. Alves, S.C. Silva, A.G. Becker, A.V.M. Canario,

Jonathan M. Wilson1 and J. Fuentes. Email:pmgg@ualg.pt

CEIB - Centro de Ciências do Mar, Universidade do Algarve, Portugal and 1CIIMAR, Portugal and Wilfrid Laurier University, Canada.

In contrast to the ectodermal amphibian bladder or the reptilian cloaca, and the

largely endodermal mammalian bladder, the teleost urinary bladder derives as

an expansion of mesonephric duct which gives origin to the kidney and it is

thus possible that it possesses cellular and functional properties of the distal

renal tubule. It has been shown that the bladder permeability to water can be

modulated in different environmental conditions by hormones such as prolactin

and cortisol. Additionally, teleosts show considerable diversity in the size, shape

and even localization of the bladder, which can correlate to important adaptive

functions. Here we aimed to evaluate a possible role in Ca/Pi balance during

urine formation.

We have used the Lusitanian toadfish Halobatrachus didactylus bi-lobulated

urinary bladder as an ex vivo model using several approaches. Histological

sections of the bladder show differential distribution of distinct cell types, some

consistent with high transepithelial transport activity. Gene expression studies

indicate that the receptors for relevant calciotropric or phosphatropic factors,

such as Parathyroid hormone related-protein (PTHrP) and fibroblast growth

factor 23 (FGF23) are present in the urinary bladder. Transepithelial transport

using unidirectional 33

P or 45

Ca fluxes in bladder sac preparations or in Ussing

chambers under symmetrical and asymmetrical clamped conditions indicate

relevant transport rates occur and are modified by the addition of intracellular or

membrane transport modulators such as Forskolin, Amiloride or Ouabain.

Although our current data is not sufficient to clearly establish a role in Ca/Pi

balance, the relatively high variability observed in transport amplitude among

individuals suggests this tissue may be regulated and such differences may be

related to nutritional status, as relevant differences were observed in plasma and

urine phosphate levels. Further experiments in different environmental

conditions are being carried out.

Funded by FCT - Foundation for Science and Technology through project

PTDC/BIA-ANM/4225/2012.

80

P35

EXPRESSION OF FGF23/KLOTHO SYSTEM IN THE SEA BASS:

TISSUE DISTRIBUTION AND REGULATION BY DIETARY Pi

Sandra C. Silva, A. Alves, A.V.M. Canario, D.M. Power, P.M. Guerreiro. E-

mail:scsilva@ualg.pt

CEIB - Centro de Ciências do Mar, Universidade do Algarve, Portugal.

Fibroblast growth factor-23 (FGF23) is a key regulator of phosphorus (Pi)

homeostasis, secreted in response to increased serum Pi. FGF23 binds to FGF

receptor 1 (FGFR1) when coupled to the transmembrane protein αKLOTHO,

decreasing PTH secretion and increasing urinary phosphorus excretion. Given

its crucial physiological role, abundant amounts of Pi are provided in fish feeds,

with deleterious effects to either fish or the environment. Here we aimed to

characterize the presence and regulation of the genes encoding the main players

in Pi uptake, regulation and excretion in Dicentrarchus labrax.

Tissue distribution was performed in 3 male and 3 female sea bass (~250g).

RNA extraction and cDNA synthesis were performed using the E.Z.N.A. Total

RNA kit from Omega Biotek and Ribolock/RevertAid from Life Technologies.

Fish were pooled by gender and expression evaluated by PCR using primers

designed upon the sea bass genome. For the diet experiment, fish (~70g)

allocated in 5 groups fed commercial control diet: 1.1%P, and four

experimental diets: 0.05, 0.3, 1.1 and 3.0 %P. At 10 and 30 days into the

feeding trial tissues were collected in RNA later and processed as above. Gene

expression was performed by QPCR using EvaGreen® Supermix and a iCycler

thermocycler (Bio-Rad).

FGF23 and FGFR1 are expressed in all the tissues analyzed but αKLOTHO

expression occurs only in liver, kidney, CS, pyloric ceca and pituitary, indicating

these may be targeted as a FGFR1/ αKLOTHO complex is required for

transactivation. The sodium-phosphate cotransporter NaPi-IIa was abundantly

expressed in kidney while intestinal expression of the different NaPi forms is

inconclusive at this point. The evaluation of dietary effects on gene expression

is on-going. We expect high dietary P to reduce renal reabsorption while

increasing Pi excretion, probably by up-regulation of the FGF23 system, and

dietary P restriction to increase expression of NaPis both in intestine and kidney.

The elevated expression of FGF23 and its receptor complex in the CS, which

secretes Stanniocalcin and expresses PTHrP suggests it may be a central player

in Pi regulation.

Funded by FCT - Foundation for Science and Technology through project

PTDC/BIA-ANM/4225/2012.

81

P36

CAN SALINITY MODULATE AN INTERPLAY BETWEEN PTHRP AND

STANNIOCALCIN THROUGH CALCIUM AND THE CALCIUM

SENSING RECEPTOR IN THE SEA BASS (Dicentrarchus labrax)?

Alexssandro G. Becker, P.M. Guerreiro, P.F. Palma and A.V.M.Canário. E-

mail:agbecker@ualg.pt

CEIB – Center of Marine Sciences, University of Algarve, Portugal.

Stanniocalcin 1 (STC) and Parathyroid Hormone related Protein A (PTHrP) are

endocrine factors that regulate calcium in fish, the first being anti-

hypercalcemic and the latter hypercalcemic and their expression coincides in

pituitary (Pit) and Corpuscles of Stannius (CS). Previous studies indicated that

both STC and PTHrP secretion may be regulated by environmental salinity but

the onset mechanism is not yet elucidated. We aimed 1) to characterize the

effects of salinity in plasma calcium, STC secretion by CS and on the

expression of the STC, PTHrP and CaSR at different salinities (in vivo) and 2)

to determine STC and PTHrP secretion in CS and Pit incubated in vitro at

various Ca2+

or calcimimetics conditions.

For Task 1, fish (~200g) were acclimated to different water salinities (0.4 (FW),

12.8 (BW) and 35 (SW)) to determine physiological, biochemical and molecular

responses. Blood and tissues (CS, Pit, gill and kidney) were collected. Plasma

ion levels and osmolality were determined by colorimetric kits and a Vapour

pressure osmometer. Na+/K+-ATPase activity was evaluated on an enzymatic

assay and STC and PTHrP expression via QPCR (ongoing). Branchial activity

was higher in FW and SW conditions and reduced in BW. Plasma Na+ was

reduced in lower salinities but no evident changes occurred in Ca2+

indicating

this ion was tightly regulated.

In Task 2, fish (~700g) maintained in seawater conditions were sacrificed and

CS and Pit carefully removed to a perifusion setup. In dynamic conditions, CS

were perifused with 0.1, 0.25, 0.5, 1.25, 3.0, 5.0, 8.0 mM Ca2+

or 10, 50 and

500nM; NPS2143 in oxygenated Hank’s medium and 20 min perifusate

fractions collected for ~3 hours. In the static system, CS and Pit were incubated

as above in 48-well plates and the medium containing putative secreted proteins

was collected and substituted for fresh medium every 60 min also up to 3 hours.

Fractions and tissues were collected to determine hormone levels and gene

expression (ongoing analyses).

AGB has a postdoctoral grant from Portuguese Foundation for Science and

Technology SFRH/BPD/96187/2013. Funded by FCT projects

PTDC/MAR/121279/2010 and PTDC/BIA-ANM/4225/2012.

82

P37

GONADOTROPIN-INHIBITORY HORMONE IN THE FLATFISH Solea

senegalensis: MOLECULAR CLONING, BRAIN LOCALIZATION AND

PHYSIOLOGICAL EFFECTS.

María Aliaga-Guerrero1, P. De Terry-Castro

1, V. Gallego-Recio

1, J.A. Paullada-

Salmerón1, E. Mañanós

2, J.A. Muñoz-Cueto

1. E-mail: maria.aliaga@uca.es

1 Department of Biology. Faculty of Marine and Environmental Sciences.

University of Cádiz. 11510-Puerto Real, Spain

2 Institute of Aquaculture of Torre de la Sal. CSIC. 12595-Ribera de Cabanes,

Spain

Gonadotropin-inhibitory hormone (GnIH) is a recently discovered neuropeptide

that plays a very important role in the regulation of reproduction in birds and

mammals. This neuropeptide from the RFamide family has neuromodulatory

functions and controls the synthesis and/or release of brain gonadotropin-

releasing hormone (GnRH) and pituitary gonadotropins. Scientific and

technological advances on the GnIH system in fish are, up to date, scarce,

contradictory and inconclusive. For this reason, research on fish GnIH system

appears necessary to better clarify its role in the neuroendocrine and

environmental control of reproduction in this important group of vertebrates. In

this context, we have cloned a full-length sequence for the GnIH precursor of

Senegal sole (ssGnIH), which codifies for three putative GnIH peptides. We

have also generated specific antisera against ssGnIH peptides, and used them to

elucidate the localization of GnIH cells and their projections in the brain and

pituitary of sole. ssGnIH-immunoreactive cell bodies were present in the

olfactory bulbs, ventral telencephalon, caudal preoptic area, dorsal tegmentum

and rostral rhombencephalon. These GnIH cells innervated profusely the brain

and pituitary of sole. Intramuscular injection of ssGnIH-3 provokes a significant

reduction in GnRH-3 (dose of 1.0μg/g) and LH (doses of 0.1μg/g and 1.0μg/g)

expression at 4 hours post-injection. In contrast, no effect of ssGnIH-2 on the

transcript levels of the main reproductive genes was evident. Our

immunohistochemical and physiological results reveal that GnIH could

represent an important actor in the neuroendocrine control of reproduction of

sole. Further studies are being directed to elucidate if this inhibitory

neurohormone is involved in the reproductive dysfunctions that occur in the F1

generation of aquacultured sole.

Funded by MINECO (AGL2010-22139-C03-03 and AGL2013-49027-C3-2-R

grants to JAM-C and BES-2011-047276 Fellow to MA-G).

83

P38

THE ONTOGENY OF SOLE (Solea senegalensis) NEUROENDOCRINE

SYSTEMS IS AFFECTED BY ENVIRONMENTAL AND CULTURE

CONDITIONS.

María Aliaga-Guerrero1, V. Piquer

2, E. Mañanos

2, J.A. Muñoz-Cueto

1. E-mail:

maria.aliaga@uca.es

1 Department of Biology. Faculty of Marine and Environmental Sciences.

University of Cádiz. 11510-Puerto Real, Spain. 2 Institute of Aquaculture of

Torre de la Sal. CSIC. 12595-Ribera de Cabanes, Spain

Aquaculture of sole is seen as an excellent choice for diversification and

economic expansion of the European aquaculture. Reproduction of wild sole

breeders in captivity is successful whilst sole specimens from F1 generation

present reproductive dysfunctions. Fish farming in captivity introduces

variations in environmental conditions compared to natural ecosystems, which

could affect the ontogeny of the brain and neuroendocrine systems and,

therefore, the reproductive performance and behavior. However, information on

how environmental and culture conditions affect the establishment of fish

reproductive systems is rather scarce. For this purpose, sole larvae were exposed

to: 1) daily thermocycle and blue light (18-21ºC; TCblue) or, 2) constant

temperature and white light (19.5ºC; cteW; routine aquaculture conditions). In

addition, and to determine a potential sensitive period, the TCblue conditions

were applied in different time frames: 1) until metamorphosis (0-12 days post-

hatching or dph), 2) post-metamorphosis (0-30 dph) or, 3) gonad differentiation

(0-100 dph). In other experiment, development of sole embryo and larvae under

routine aquaculture conditions and mesocosm conditions was compared. Effects

were determined by analyzing gonadotropin-releasing hormone (GnRH),

kisspeptin and gonadotropin-inhibitory hormone (GnIH) gene expression by

qPCR. The results obtained demonstrated that under routine aquaculture

conditions, developing sole exhibited higher GnIH transcript levels in critical

ontogenetic periods (sex differentiation) compared to specimens reared under

natural thermocycles and blue lights. Mesocosm culture conditions also affected

GnIH, sGnRH and kisspeptin ontogenetic expression patterns. Our results

evidence that routine conditions used in aquaculture practice can disturb the

natural organization of sole neuroendocrine systems, reinforcing the importance

of mimicking natural environmental conditions during fish rearing.

Funded by MINECO (AGL2010-22139-C03-03 and AGL2013-49027-C3-2-R

grants to JAM-C and BES-2011-047276 Fellow to MA-G).

84

P39

ONTOGENY OF LIGHT-SENSOR SYSTEMS OF SENEGAL SOLE

EXPOSED TO DIFFERENT LIGHT PHOTOPERIOD AND SPECTRA

Isabella Paradiso, S. Frau, J.A. Muñoz-Cueto, Á.J. Martín Robles. E-mail:

sara.frau@uca.es

Department of Biology. Faculty of Marine and Environmental Sciences.

University of Cádiz. 11510-Puerto Real, Spain

Light represents the main environmental factor synchronizing biological

rhythms and entraining melatonin production, which is dependent on light

intensity, spectra (wavelength) and duration (photoperiod). The light that

penetrates into the seawater column changes in intensity and wavelength due to

selective absorption. As a result, most of the wavelengths are absorbed in the

upper waters, while the blue light reaches greater depths. The Senegal sole

undergoes a real metamorphosis between days 12 and 19 after hatching and

during this process it changes its life habits from pelagic to benthonic and from

diurnal to nocturnal. However, it is unknown if this metamorphic process is

accompanied by a differential expression pattern of opsins with different

photosensitivity. In the present study we investigated the expression of four

classes of opsins (UV opsin 5, green sensitive pigment, red opsin and teleost

multiple tissue opsin or TMT) during the development of this flatfish species by

using quantitative real time PCR. The eggs were collected immediately after

spawning during the night and exposed to light-dark (LD) 12L:12D cycles of

white (LDW), blue (LDB, λ peak = 463 nm), red (LDR, λ peak = 685 nm),

continuous white light (LL) and continuous darkness (DD). Our results show a

peak of expression of green sensitive pigment between day 6 and 12 in all the

conditions, decreasing thereafter. Red opsin expression shows a peak at D12 in

all the conditions and decreases during metamorphosis, but in LL, LDW and

LDB it increases again in post-metamorphosis (D25). UV opsin expression

presents a first peak at day 1 in all the conditions, increasing during

metamorphosis (D19) in LDB, LDW and LL and decreasing in post-

metamorphic animals (D25). Finally, TMT opsin transcript levels peaked at D12

in all conditions. Day-night difference in opsin expression was only evident

under blue lights. These results show a differential pattern of expression of

different opsins before, during and after the completion of metamorphosis in

Solea senegalensis and suggest that its bottom settlement is accompanied by

changes in photopigments.

Funded by a grant from MINECO (AGL2013-49027-C3-2-R) to JAM-C.

85

P40

PHOTOPERIODIC SIGNALLING MEDIATES THE CONTROL OF

KISS1 AND GNRH2 NEURONAL POPULATIONS ON THE

REPRODUCTIVE AXIS IN A TELEOST FISH, EUROPEAN SEA BASS

(Dicentrarchus labrax)

Felipe Espigares, A. Rocha, A. Gómez, M. Carrillo, S. Zanuy. E-mail:

s.zanuy@csic.es

Department of Fish Physiology and Biotechnology, Institute of Aquaculture of

Torre de la Sal (IATS), Spanish National Research Council (CSIC), Ribera de

Cabanes s/n, 12595, Castellón. Spain.

There are a wide variety of environmental factors that change seasonally and

that may modulate the activity of the reproductive axis. Of all, photoperiod is

the main environmental signal that most temperate fish species use to predict

the changing seasons and therefore anticipate spawning time through activation

of neuroendocrine pathways which, in turn, stimulate gonadotropic axis and

gonadal growth. However, many molecular and endocrine mechanism involved

in the onset of puberty and the influence of environmental conditions, such as

photoperiod signalling, are not well understood in fish. In this study, 1 year-old

male European sea bass (Dicentrarchus labrax) were exposed to three different

photoperiod conditions, natural photoperiod (NP), advanced photoperiod (AP)

and continuous light (LL) in combination with size-sorting and studied from

August to February. The obtained results clearly indicated that AP and LL were

able to train the rhythms of both kiss1 and gnrh2 mRNA levels in the forebrain-

midbrain (FB-MB), suggesting that the photoperiodic signalling may modulate

the activity of the habenular Kiss1 and the synencephalic Gnrh2 neurons to

drive reproductive axis. Also AP and LL regimes affected both plasma Fsh and

11-KT profiles, which in turn would explain the phase shift and reduction of

gonadal recrudescence under AP and LL, respectively. In conclusion, the

present study reveals a strong relation between environmental control of puberty

and the Kiss1 and Gnrh2 systems in agreement with previous ?ndings observed

in other teleost species, including European sea bass. We also hypothesize that

very likely, as a result of the modulatory role of the photoperiod on the Kiss1

and Gnrh2 activity, this is able to shift the hormone profiles and hence advance

the onset and progression of gametogenesis under AP, whereas under LL, the

unbalanced production of 11-KT, regulated by an altered Fsh profile, could be

limiting the stimulation of germ cell proliferation at the testicular level and thus

prevent the normal progression of the spermatogenesis.

Supported by GV (PROMETEO/2010/003 to S.Z.), EU (LIFECYCLE,

FP7222719 to S.Z.). F.E. was supported by a JAE Predoctoral fellowship

(CSIC).

86

P41

SPECIATION IN THE EVOLUTION OF ALLATOSTATIN-TYPE A

RECEPTORS (AST-AR) IN DIPTERA

Rute C Félix, Joao CR Cardoso and Deborah Power. Email. rcfelix@ualg.pt

Comparative Endocrinology and Integrative Biology, Centre of Marine

Sciences, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro,

Portugal

The Allatostatins type-A (AST-A) receptors are a group of invertebrate G-

protein coupled receptors (GPCRs) that share ancestral origin with the

vertebrate receptors for the peptides Kiss and Galanin. In arthropods, the

evolution of AST-AR was recently resolved and in insects, a single gene was

found but in dipterans two AST-ARs (AST-AR1 and AST-AR2) with distinct

functions co-exist as the result of gene duplication (Felix et al., 2015).

Anopheles mosquitoes have a rapid rate of speciation and a rapidly evolving

genome and in A. gambiae (PEST strain) both AST-ARs map to a highly

modified chromosome but the impact of this on receptor gene structure and

function has not been determined. The aim of this study was to characterize the

evolution of AST-A receptor genes in Anopheles mosquitos and to determine

how adaptive evolutionary pressure has contributed to receptor gene divergence

in diverse Anopheles species. AST-AR genes were retrieved from 17 Anopheles

species genomes and receptor gene structure and sequence was compared

between species and with the orthologues isolated from Drosophila genomes.

Analysis revealed that in Anopheles, orthologue receptor gene structure was

different and exon tandem duplication and exon inversions modified the AST-

AR2 gene in some species. The potential effect on AST-AR gene evolution of

geographic localization of Anopheles, its efficiency as a malaria vector, host

feeding preference and taxonomic characterization will be analysed and

discussed.

Acknowledgements: This study was financed by the European Regional

Development Fund (ERDF) COMPETE and Portuguese funds through the

Foundation for Science and Technology (FCT) under the project

UID/Multi/04326/2013. RCF is in receipt of FCT SFRH/BPD/89811/2012 grant

and JCRC by an auxiliary research contract under the project

UID/Multi/04326/2013.Felix et al, 2015, PLoS One. 10, e0130347.

87

P42

SOMATOLACTIN α AND β SPECIFIC REGULATORY ELEMENTS

AND DIFFERENTIAL SPACIAL EXPRESSION IN PITUITARY OF

Cyprinus carpio

Valenzuela-Nieto, G.E., Stolzenbach, M.F., Henriquez, N., Vega, M., Romero, A.,

Figueroa, J., Kausel, G. E-mail:gkausel@uach.cl

Instituto de Bioquímica y Microbiología, Universidad Austral de Chile,

Valdivia, Región de Los Ríos, Chile.

Environmental changes affect gene expression that we addressed in the

pituitary, a central regulatory organ at the interface between the central nervous

system and the endocrine system where endocrine disruptor effects are still

poorly understood.

Somatolactin (SL), a fish specific pituitary hormone belonging to growth

hormone (GH) and prolactin (PRL) superfamily, is involved in background

adaptation, osmoregulation, reproduction and fatty acid metabolism. In carp,

transcripts of two SL genes (α and β) were detected in pituitary gland and when

treated with potential endocrine disruptor compounds differential response was

detected by RT-qPCR. Adult male carp responded with decrease of expression

of both SL genes to ZnCl2 treatment. However, expression of SLα and SLβ was

modulated differentially in pituitary of male adult carp in response to estrogen,

which might imply differential binding of transcription factors to the promoters

of these duplicated genes. With the aim to characterize in detail the divergent

regulation of the two SL genes, promoters of both genes were analyzed.

Therefore, the proximal promoter region of SLα and β were cloned applying

inverse PCR, transcription start site was determined with 5’RACE for 5´-UTR

and sequences complemented by in silico cloning. In the proximal promoter of

both genes several putative binding sites for Pit-1 were identified and confirmed

in vitro by electro mobility shift assays suggesting regulation of SL by this

pituitary specific transcription factor. In addition, clearly, specific binding to an

estrogen response element that was identified only in SLβ promoter, might

explain differential regulation of both SL in response to estrogen. Comparison

of the complete coding sequences of slα and slβ revealed 61.6% identity at the

nucleotide level and 46.2% between the derived aminoacid sequences. We

prepared antibodies against a carp SLα- specific oligopeptide and double

immunostaining with an anti-chum salmon SL and the specific anti-carp SLα

antibody revealed spacially specific expression patterns in pars intermedia of

carp pituitary sagittal sections.

Taken together, here we show for the first time a cis-regulatory element related

to estrogen response in SL which is absent in SLα promoter and a clear

spacially different expression pattern in pituitary of male carp. These data

88

highlight the importance to study regulation of duplicated genes in carp for

evaluation of environmental changes including effects of endocrine disrupters

of estrogenic potential in the aquatic environment.

Supported by: DAAD 50750108, DAE-UACh, Mecesup AUS1203, UACh-DID-

SE2015-02 (GK), CONICYT 21999 (GV), Fermelo S.A

89

PRESENTING AUTHORS

Oral Communications

Alves Costa R O15

Blanco AM O8

Cal L O29

Cardoso JCR O25

Castaño JP OL

Dulce Estêvão MD O26

Felip A O4

Gil Solsona R O9

Guerreiro PM O19

Gutiérrez J JP'C

Hernández-Pérez J O6

Hormaechea-Agulla D O23

Kah O CL

Mateus AP O20

Medina P O3

Morini M O2

Nácher-Mestre J O16

Naderi F O18

Peñaranda DS O1

Pérez-Sánchez J O12

Pinto PIS O14

Rincón-Fernández D O24

Ruiz-Jarabo I O27

Sarmento-Cabral A O11

Simó P O10

Skrzyńska AK O17

Suarez-Bregua P O28

Valdivieso A O5

Vázquez-Borrego MC O21

Velasco C O7

Villa-Osaba A O22

Zaghdoudi-Allan N O13

90

Poster

Aliaga-Guerrero M P37, P38

Alves A P33

Asturiano JF P16

Becker AG P36

Cortés R P11

Felip A P20

Félix RC P41

Frau S P39

Gilannejad N P2

Gómez-Boronat M P28

Guardiola FA P30, P31, P32

Guerreiro PM P34

Isorna E P24

Jerez I P22

Librán-Pérez M P9

López-López F P29

Mancera JM P25, P26, P27

Mañanós E P15

Martos-Sitcha JA P1, P4

Medina P P17

Míguez JM P23

Morini M P19

Navarro I P8

Navarro S P10

Nisembaum LG P14

Otero-Rodiño C P7

Palma PS P21

Pinto P P13

Piquer V P18

Sarasquete C P5

Silva SC P35

Skrzyńska AK P6

Soletto L P12

Valenzuela-Nieto GE P42

Vélez EJ P3

Zanuy S P40

91

AUTHOR INDEX

Agulleiro MJ P11

Aliaga M P18

Aliaga-Guerrero M P37, P38

Alonso-Gómez AL P24

Alvarado MV P20

Alvarez-BenitoM O22

Álvarez-Otero R O7, P7, P23

Alves Costa RA O15, O20, P33, P34, P35

Andrade A O26, P13

Arizcun M P31, P32

Astola A P25

Asturiano JF O1, O2, P16, P19

Ayala Suárez R P25

Baeza R P16

Baldisserotto B P27

Bantz A P14

Bastaroli M O17, P6

Becker AG P34, P36

Beltrán E P15

Berntssen MHG O16

Besseau L P14

Blanco AM O8, P24, P28

Blasco J JP'C,P3

Blázquez M O3, P17

Braasch I O28

Bronner ME O29

Butts IAE P16

Cal L O29

Calduch-Giner JA O9, O10, O12, P4

Canário AVM O19, P13, P21, P33, P34, P35, P36,

Capilla E JP'C,P3, P8

Cardoso JCR O20, O25, P41

Carrasco-Valiente J O23

Carrillo M P20, P40

Castaño JP OL, O11, O21, O22, O23, O24, P29

Ceinos R O6

Cerdá-Reverter JM O6, O28, P10, P11, P12

Chaves-Pozo E P31, P32

Churcher A O13

Clark M O13

92

Conde-Sieira M P7

Cone RD P11

Córdoba M P8

Cortés R P10, P11, P12

Cuesta A P30, P31, P32

Culler MD O23

de las Heras V P2

De Pedro N P28

de Terry-Castro P P37

Delgado MJ O8, P24, P28

Dufour S P19

Espigares F O4, P40

Esteban MA P30, P31, P32

Estêvão MD O26

Falcón J P14

Felip A O4, P20

Félix RC O13, O25, P41

Fernández-Borràs J JP'C,P3

Figueroa J P42

Flik G P22

Frau S P39

Fuentes J O27, P34

Fuentes M P14

Gahete MD OL, O11, O21, O22, O23, O24, P29

Gallego V O1, P16

Gallego-Recio V P37

Gálvez MA O21

Gesto M O6, P23

Gil Solsona R O9

Gilannejad N P2

Giménez I O1

Gisbert E O20

Gómez A O4, O3, O4, P13, P17, P40

Gómez-Boronat M O8, P28

Gómez-Gómez E O23

Gómez-Skarmeta JL O29

Gorissen M P22

Gracia-Navarro S OL

Gregório, SF O17

Guardiola FA P30, P31, P32

Guerreiro PM O19, P21, P33, P34, P35, P36

Guillot R P10

Gutiérrez J JP'C, P3, P8

93

Guzmán JM P15

Henriquez N P42

Hernández F O9, O16

Hernández-Pérez J O6, P7, P9, P23

Hormaechea-Agulla D OL, O23

Ibáñez M O16

Ibáñez-Costa A OL, O21, O23

Isorna E P24, P28

Jerez I P1

Jerez I P27

Jerez I P22

Kah O CL

Kausel G P42

Kelsh R O28

Lacalle L O9

Lafont AG P19

Librán-Pérez M O6, O7, P7, P9

López-López F OL, O11, O22, P29

López-Miranda J O21

López-Patiño MA O6, O7, O18, P23

Luque RM OL, O11, O21, O22, O23, O24, P29

Lutfi E JP'C,P3, P8

Magnanou E P14

Maiorano E O17, P6

Mancera JM O17, P1, P4, P6, P22, P25, P26 P27

Mañanos E P15, P18, P37, P38

Martin P P14

Martín Robles AJ P39

Martínez-Fuentes, A OL

Martínez-Rodríguez G O17, P1, P2, P4, P6, P26, P27

Martins RS P13

Martos-Sitcha JA O17, P1, P2, P4, P6, P25, P26, P27

Mateus AP O20

Medina P O3, P17

Meseguer J P30, P32

Michel M P11

Míguez JM O6, O17, P9, P12, P23

Mischitelli M P10

Mohammed-Geba E P26

Moran P O28

Moreno MM O23

Morini M O2, P19

Mourão RHV P27

94

Moya A P3

Muñoz-Cueto JA P18, P37, P38, P39

Muñoz JL O6, P23

Nácher-Mestre J O9, O16

Naderi F O6, O7, O18, P9, P23

Navarro I P3, P8

Navarro S JP'C,P10, P11, P12

Nerín G P8

Nisembaum LG P14

Nourizadeh-Lillabadi R O2

Ortega-Salas R O22

Ortiz-Delgado JB P5

Otero-Rodiño C O7, O18, P7, P9

Palma PF P36

Palma PS P21

Paradiso I P39

Paullada-Salmerón JA P18, P37

Pavanato MA P27

Pedraza-Agüera, A OL

Peñaranda DS O1, O2, P16, P19

Perelló M P3

Pérez L O1, O2, P16, P19

Pérez-Sánchez J O9, O10, O12, O16, P4

Pês TS P27

Piferrer F O5

Pinto PIS O14, O26, P13

Piquer V P15, P18, P38

Planas JV JP'C

Porte C P8

Portolés T O16

Power DM O13, O14, O15, O11, O20, O25, O26,

P21, P33, P35, P41

Pozo-Salas AI O22

Puchol S P12

Ramos J P18

Ramos-Fernández JA O23

Redondo I O12, P28

Requena MJ O23

Ribas L O5

Rincón-Fernández D O24

Rivero-Cortés, A.J OL

Rocha A P40

Romero A P42

95

Rosel JF P12

Rotllant J O28, O29, P12

Ruiz-Jarabo I O27

Ruiz-Murillo V O24

Saccol EMH P27

Sánchez E P10, P11, P12

Sánchez-Bretaño A P24, P28

Sánchez-Sánchez R O22

Sancho JV O9

Santamaría R O24

Santos S O16, P13

Saraiva JL O19

Sarasquete C P5

Sarmento-Cabral A OL, O11, P29

Saxena A O28

Serrano R O16, P15

Silva FB O19

Silva M O19

Silva SC P33, P35, P35

Simó P O10

Sitjà-Bobadilla A. O12

Skrzyńska AK O17, P6

Soares J O19

Soengas JL O19, P7, P9

Soletto L P10, P11, P12

Soto-Moreno A O17

Stolzenbach MF P42

Suarez-Bregua P O28

Swanson P P15

Thorne MAS O14

Toledano-Delgado A O21

Tveiten H P16

Úbeda-Manzanaro M P5

Valdivieso A O5

Valenciano, AI O8

Valenzuela-Nieto GE P42

Valero Y P31, P32

Valero-Rosa J O23

Vargas M O19

Vázquez-Borrego MC OL, O21

Vega M P42

Velasco C O7, O18, P7, P9

Vélez EJ JP'C,P3

96

Velez Z P13

Venegas-Moreno E O21

Vieira F O15

Vílchez MC O1, O2, P16, P19

Villa-Osaba A OL, O22

Weltzien FA O2

Wilson JM P34

Yarra, T O13

Yúfera M P2, P4

Zaghdoudi-Allan N O13, O25

Zanuy S O3, O4, P13, P17, P40