www.elsevier.com/locate/cbpa

Abstracts / Comparative Biochemistry and P

Society for Experimental Biology Annual Main Meeting

11th–15th July 2005, Universitat Autonoma de Barcelona, Barcelona, Spain

A2–GENOMICS IN AQUACULTUREOrganised by Nic Bury (King’ College London), Patrick Prunet (INRA SCRIBE), Deborah Power an Juan Fuetes (Universidade do

Algarve)

A2.1Utilization of QTL mapped in livestock

Pascale Le Roy, (Institut National de la Recherche Agronomique,

UMR Genetique Animale, Agro Campus de Rennes, 65 rue de

Saint Brieuc, 35042 Rennes Cedex, France)

Genomics allows the tracing of the transmission of genome

fragments between generations, and location and identification of

genes the polymorphism of which explains a part of quantitative

trait variability (QTL). This information coming from QTL

mapping will allow an increase of the selection efficiency in

livestock. This approach is useful for a better evaluation of

reproducers genetic values, in particular when traits cannot be

measured at a large scale for technical and/or economical reasons. It

is also useful for reducing the generation interval, through an early

choice of reproducers and for increasing the selection intensity.

Examples of applications are described in pig and ruminant species.

Keywords: QTL; Livestock; Selection

A2.2Gene expression profiling of gilthead sea breamduring early development and detection ofstress-related genes by the application of cDNAmicroarray technology

E. Sarropouloua,c, G. Kotoulasa, D.M. Powerb and R. Geislerc,

(aHellenic Center for Marine Biology, Institute for Marine Biology

and Genetics, Crete, P.O. Box 2214, 710 03 Iraklio, Crete, Greece,bCenter for Marine Science (CCMar), Universidade do Algarve,

8000-117 Faro, Portugal, cAbt. III (Genetik), Max-Planck-Institut

fur Entwicklungsbiologie, Spemannstrasse 35, 72076 Tubingen,

Germany, sarris@her.hcmr.gr)

Large-scale gene expression studies were performed for the

gilthead sea bream Sparus aurata L, one of the main European

aquaculture species. This Perciformes species with its relatively

small genome size is a member of the Sparidae family, which

comprises many economically important species and is therefore of

fundamental importance for aquaculture. Due to several advantages

like its taxonomic position sea bream is attractive not only for

doi:10.1016/j.cbpb.2005.05.007

aquaculture but also for evolutionary biology and comparative

genomics. As for most commercial fish species there is a

substantial lack of information on gene sequences, function and

expression patterns. In this study we describe the application of a

high-throughput approach, the cDNA microarray technique, in

order to address the following two questions: the study of gene

expression changes during early development and the investigation

of alteration of gene expression after stress, stimulated by cortisol

injections. For these purposes a cDNA microarray containing

10,176 clones was constructed containing ¨7000 unique EST

sequences out of a cDNA library of mixed embryonic and larval

stages.

The creation of gene expression profiles of sea bream by

microarray hybridisation will contribute to the isolation of novel

genes involved in multifactorial traits and certain regulatory

pathways, and also to a better understanding of the genetic

background of fish physiology that will help to improve

aquaculture practices.

Keywords: Gene-expression; cDNA microarray; Development;

Stress response; Sparus aurata

A2.3A functional genomic approach towards analysisof confinement and salinity stress responses inrainbow trout

P. Prunet, B. Auperin, L. Dengreville, S. Mativet, G. Leborgne and

O. Lepage, (INRA-SCRIBE, Group on Physiology of Stress and

Adaptation, campus de Beaulieu, 35042 Rennes cedex, France)

Whether living in natural environment or reared under intensive

conditions, fish are episodically exposed to stressors for which they

develop biological stress responses. Many studies have been

devoted to analysis of these responses at the level of specific

mechanisms and revealed complexity of these responses. The

recent development of functional genomic tools allowed us to

provide an integrated overview of the global responses at the level

of gene expression. Within a European-funded research project

(STRESSGENES), we have been involved these last years in

studying two different stress situations related to Salmonid

aquaculture practises, confinement stress and salinity stress.

hysiology Part A 141 (2005) S85 – S93

Abstracts / Comparative Biochemistry and Physiology Part A 141 (2005) S85–S93S86

Whereas trout never acclimate to confinement stress, direct

transfer to seawater induces short-term stress responses followed

by acclimation to hyperosmotic environment which develop

within 3 weeks. Gene profile analysis were carried out using

9000 probes microarrays from clones isolated from 19 SSH

cDNA libraries constructed from different tissues collected on

fish exposed or non-exposed to various stressors. Thus, we have

been using microarrays enriched in genes potentially involved in

stress response in trout. This largely comprises genes involved in

metabolism, cellular physiological process, cell communication

and response to stimulus. These arrays have been used to carry

out short-term (2 h) and long-term (21 days) expression analysis

of gene regulated in the trout gill by salinity stress exposure.

Using data from 2 h salinity exposure, we have selected a list of

¨367 clones corresponding to 46 different contigs which include

6 mitochondrial genes whereas with data from 21 days exposure,

the 104 clones which have been selected correspond to 68

different contigs including 29 mitochondrial genes. A similar

study have been conducted for analysing gene expression in

interrenal glands collected between 4 h and 21 days in fish

exposed to confinement stress until 21 days and we have

compared gene expression profiles. This analysis allowed us to

define 2 major clusters of gene over-expressed after confinement

stress exposure. Gene ontology annotations of selected genes in

both experiments provided us a functional overview of common

or stress-specific responses and indicated their biological signifi-

cance. (This project was supported by the 5th Frame Work of the

European Commission.)

A2.4Stress-regulated expression of glucocorticoidreceptor (GR) and mineralocorticoid receptor(MR) in carp (Cyprinus carpio)

E.H. Stoltea,b, K.M. Leon-Kloosterziela, B.M.L. Verburg-Van

Kemenadea and G. Flikb, (aCell Biology and Immunology,

Wageningen University, Wageningen, The Netherlands, bAnimal

Physiology, Institute for Neuroscience, Radboud University,

Nijmegen, The Netherlands, Ellen.Stolte@wur.nl)

Homeostasis in vertebrates depends critically on bi-directional

communication of the neuro-endocrine and immune system. Both

systems monitor the environment to adequately adapt the organism

to stress. The teleostean Hypothalamus Pituitary Interrenal (HPI)

axis is analogous to the mammalian stress axis.

In fish, stress causes the head kidney to produce glucocorticoids

(cortisol) and prepares the organism to cope with the stressor by

increasing metabolic activity and glucose levels. Secondly,

glucocorticoids regulate ion balance through effects on chloride

cell proliferation and differentiation. Finally, glucocorticoids

modulate immune function. In carp, cortisol causes redistribution

of leukocytes, induces apoptosis of B-lymphocytes, and inhibits

apoptosis of neutrophils. Thus different cell types respond differ-

entially to cortisol.

Mammals have divided the three functions of glucocorticoids in

fish between glucocorticoids (metabolism and modulation of

immune function) and mineralocorticoids (osmoregulation) and

have separate receptors for these two classes of steroid hormones.

In fish, the mammalian ligand for the mineralocorticoid receptor

(MR), aldosterone, is not found. Cortisol, however, can bind both

the glucocorticoid receptor (GR) and the MR in fish. Moreover, the

MR has a higher affinity for cortisol than GR. The physiological

effect of cortisol after stress-induced release therefore remains to be

evaluated.

We cloned and sequenced both GR and MR in carp and determined

relative expression levels of both receptors in different tissues. Fish

were exposed to different stressors (restraint and acclimation

temperature) to determine importance of receptor expression in

stress regulation of metabolism and immunity.

Keywords: Glucocorticoid/mineralocorticoid receptor; Stress;

Immune system; Osmoregulation

A2.5Proteomics analysis of the differentialdevelopmental stages of fish oocytes

A. Admon, T. Gattengo, V. Chapovetsky and E. Lubzens,

(Department of Biology, Technion-Israel Institute of Technology

and Israel Oceanographic and Limnological Research, Haifa

32000, Israel. E-mail: admon@tx.techniopn.ac.il)

Proteomics is a new scientific field aimed at simultaneously

characterizing entire protein repertoires of biological systems.

Using proteomics, comprehensive lists of the protein constituents

of cells and organisms can be established. Even more importantly

for developmental biology, changes in the relative amounts of

proteins and in the patterns of their posttranslational modifica-

tions can be followed. The most significant proteomics technol-

ogies used for the study of oocytes are based on two-dimensional

polyacrylamide gel electrophoresis (2D-PAGE), multi-dimen-

sional high-performance liquid chromatography (HPLC), protein

arrays and mass spectrometry. Here we describe a study of

proteomes of the different maturation stages of fish oocytes. The

study reveals similarities and differences between oocytes of

different organisms in their protein content, variability in the

protein content between seemingly phonotypic identical oocytes

by proteomics analyses of single oocytes. The shift in the pattern

of modifications of the vitellogenin was studied in the different

staged oocyte, revealing a pattern of cleavages and processing

unique to each of the fish species. The effects of cryopreservation

were also studied by proteomics attempting to determine the

effect of the different chilling regimes and of the cryopreservants.

Protein catalogues have been created for oocytes of both gilthead

sea bream and zebra fish. This way the involvement of the

identified proteins in the maturation process can be followed and

molecular staging can be implemented based on correlation with

protein patterns.

Keywords: Fish oocyte; Proteomics; Vitellogenin

A2.6The omics era and the fight against disease inaquaculture

A. Figueras and B. Novoa, (Instituto Investigaciones Marinas,

CSIC, Eduardo Cabello 6, 36208 Vigo, Spain)

Diseases are a key factor in the profitability of any animal

production activity. Pathologists focused in aquaculture have

concentrated their efforts on diagnosis of ‘‘serious’’ pathogens

using state of the art techniques as they became available. Lately,

their efforts have switched towards functional immunology and

recently to understanding the molecular basis of the immune

Abstracts / Comparative Biochemistry and Physiology Part A 141 (2005) S85–S93 S87

response. However, almost no research has been done on disease

resistance selection programmes using modern molecular biology

tools and the few selection programmes are mainly based on

classic genetic approaches.

In the near future biotechnology could add value to aquaculture

production. Research on antimicrobial peptides or molecules with

biological activity obtained from these species may have a

potential beneficial impact on the production of these relatively

easy and low cost production species.

A series of developments both in bivalve and fish pathology are

vaccine development, genes related with immune response and

maybe resistance and immune response enhancement that may take

the advantage of the recent developments in the molecular biology

field.

A2.7PLEUROGENE: flatfish genomics and proteomicsfor aquaculture

J. Cerdaa, S.E. Douglasb, C. Buesac, P. Canavated, J. Lopez-Bareae,

M. Manchadod, G. Martınezf, J.I. Navasg, F. Piferrerh, J.V. Planasi,

F. Pratj, M. Reithb, M. Ruız-Rejonk and M. Yuferaf, (aLab IRTA-

CSIC, CMIMA, 08003-Barcelona, Spain, bInstitute for Marine

Biosciences, Halifax, Nova Scotia, Canada B3H 3Z1, cOryzon

Genomics, Parc Cientıfic de Barcelona, 08028-Barcelona, Spain,dCentro de Investigacion y Formacion Acuıcola y Pesquera ‘‘El

Toruno’’, IFAPA, Junta de Andalucıa, 11500-Puerto de Santa

Marıa, Cadiz, Spain, eDepartmento de Bioquımica y Biologıa

Molecular, Universidad de Cordoba, 14071-Cordoba, Spain,fInstituto de Ciencias Marinas de Andalucıa (CSIC), 11510-Puerto

Real, Cadiz, Spain, gCentro de Investigacion y Formacion

Acuıcola y Pesquera ‘‘Agua del Pino’’, IFAPA, Junta de Andalucıa,

21450-Cartaya, Huelva, Spain, hInstitut de Ciencies del Mar

(CSIC), 08003-Barcelona, Spain, iDepartament de Fisiologia,

Facultat de Biologia, Universitat de Barcelona, 08028-Barcelona,

Spain, jInstituto de Acuicultura de Torre de la Sal (CSIC), 12595-

Torre de la Sal, Castellon, Spain, kDepartamento de Genetica,

Facultad de Ciencias, Universidad de Granada, 18071-Granada,

Spain, jcerda@icm.csic.es)

Senegal sole (Solea senegalensis) and Atlantic halibut (Hippo-

glossus hippoglossus) are two flatfish yielding high value market

products with good potential for aquaculture in Mediterranean

Europe and eastern North America, respectively. Production-

related problems in these two phylogenetically related species

may be addressed with improved knowledge of important basic

biological processes such as reproduction, development, nutrition,

genetics and immunity. The use of genomic and proteomic

approaches to thoroughly characterize these processes will translate

into knowledge that can be used to overcome the production

obstacles and create (for Senegal sole) or expand (for Atlantic

halibut) solid aquaculture industries. PLEUROGENE is a new

research programme funded by Genome Spain and Genome

Canada with two main goals: the analysis of global gene

expression during sex differentiation, reproduction, larval develop-

ment, immunity and nutrition, and the construction of genetic

linkage maps for use in the selection of improved broodstock.

High-throughput genome- and proteome-based technologies will

be applied to establish gene expression profiling during these

processes and to discover novel genes. All genetic, molecular and

morphological information obtained in this project will be

integrated into an interactive bioinformatics platform specifically

developed, the Solea-mold. The knowledge generated by the

PLEUROGENE project will ultimately lead to the establishment of

new technologies for the control of reproduction and optimization

of larval health and nutrition in the Senegal sole, Atlantic halibut,

and other related flatfish species under intensive culture conditions.

Keywords: Flatfish, Reproduction; Larval development;

Genomics; Proteomics

A2.8Marine Genomics Europe, resources foraquaculture

Adelino V.M. Canarioa, Joao Cardosoa, Eleftherios Zourosb and

Filip A.M.J. Volckaertc, (aCentre of Marine Sciences, University of

Algarve, Campus de Gambelas, P-8005-139 Faro, Portugal,bUniversity of Crete, Iraklio, Crete, Greece, cKatholieke Universi-

teit Leuven, Laboratory of Aquatic Ecology, Ch. de Beriotstraat 32,

B-3000 Leuven, Belgium. E-mail: acanario@ualg.pt)

‘‘Marine Genomics Europe’’ (MGE) is devoted to the develop-

ment, utilization and spreading of high-throughput genomic

approaches for the study of the biology of marine organisms

and the functioning of marine ecosystems. The MGE joint

research programme is broken down into three sections: Com-

parative, Functional and Environmental Genomics. Traditional

research lines are nested within each of these sections, leading to

four nodes: Microbial, Algal, Evolution Development and

Diversity, and Fish and Shellfish nodes. In the ‘‘Fish and

Shellfish’’ node (F&S), aquaculture takes a special role as it

provides an ‘‘economical model’’ for development and application

of genomics resources. A major objective of the initial F&S plan

of activities is the production of an extensive ‘‘genomic tool

box’’, which will include cDNA, bacterial artificial chromosome

and subtractive libraries, expressed sequence tags, type I and type

II markers, microarrays, etc., all integrated in a bioinformatics

platform. These tools can be used to address societal issues such

as ecosystem biodiversity and health, fisheries and stock assess-

ment, aquaculture, human health, consumer safety and legislation.

Networking with established genomics centres and among

partners, through workshops and courses, are crucial to increase

the level of expertise. Participation in MGE initiatives, especially

in educational activities, is open to third parties.

Keywords: Gene mapping; Fish and shellfish; Selective breeding;

High throughput

Acknowledgements: MGE has the financial support of the

Commission of the European Union, Sixth Framework Pro-

gramme, under priority 1.1.6, ‘‘Sustainable Development, Global

Change and Ecosystems’’ (contract no. 505403).

A2.9The 10,000 gene canine RH map

Francis Galibert, (UMR 6061 CNRS-Universite de Rennes 1,

France)

With more than 300 registered breeds, considered as genetic

isolates, the canine species present unique characteristics for the

identification of susceptibility genes and their alleles in complex

traits. To this aim, genomic resources such as maps and sequence

are essential.

Abstracts / Comparative Biochemistry and Physiology Part A 141 (2005) S85–S93S88

We have recently developed a comprehensive canine radiation

hybrid (RH) map constructed on a new 9000 rad panel

(RHDF9000) which has a calculated resolution power of 200 kb

thus allowing the positioning of markers at 12,000 unique positions

(Hitte et al. submitted).

This RH map contains 10,348 markers, all corresponding to a dog

gene for which a human ortholog has been identified. In addition, it

contains 545 ordered anchor corresponding to sequence ends of

BAC clones, previously mapped by FISH and RH on the

RHDF5000 panel. Map construction was carried out with the

rh_tsp_map2 package of CONCORDE algorithm. Computation of

the RH vectors allowed the construction of 88 linkage groups

unambiguously assigned to the 38 plus X CFAs and the ordering of

the markers within each linkage group. Synteny comparison

between the canine map and the human sequence (NCBI Build

34) allowed to identify 264 human/canine conserved segments

comprising 2 to 332 markers, the size of which ranges from 500 kb

to 124 Mb.

With this work we demonstrated that associated with a light

genome shotgun sequence maps represent resources that offer all

the information needed for further genomic studies.

A2.10Molecular markers and phenotypic characteristicsfor selecting breeding strains of Marble trout inSouth Tyrol (northern Italy)

A. Meraner, S. Baric and J.G. Dalla Via, (Research Centre for

Agriculture and Forestry Laimburg, 39040 Auer/Ora, Italy,

josef.dallavia@provinz.bz.it)

Populations of the Marble trout (Salmo trutta marmoratus C.)

have been declining since the beginning of the last century. One

of the main reasons for this occurrence is the alteration of the

genetic composition of locally adapted populations due to

stocking with allochthonous hatchery-reared strains. While in

the Soca river basin (Slovenia) it was possible to identify pure

Marble trout populations and establish indigenous breeding

strains, this attempt has so far failed in other parts of the

species’ distribution range. In South Tyrol 20 trout populations

were investigated by means of molecular genetic methods and

no single population unaffected by stocking was found. More-

over, it was shown that stocked hatchery-reared fishes hybridised

with fishes from local populations. Thus, to be able to select

pure Marble trout strains for future repopulation programs it is

necessary to establish reliable methods for distinguishing Marble

trout individuals from hybrids. Our approach included pheno-

typic information, sequence analysis of the mitochondrial DNA

control region and microsatellite DNA loci. The comparison of

each of the methods showed that morphological characteristics

revealed the highest number of incongruent results, while

analyses of microsatellite data in combination with a Bayesian

individual assignment approach were most efficient. However, it

was not sufficient to rely exclusively on the latter technique

because of the variance of the assignment values and the

influence of the theoretical model on the Bayesian approach. We

conclude that the best tool for the identification of purebreds is a

combined use of both molecular methodologies and phenotypic

information.

Keywords: Salmo trutta marmoratus; Mitochondrial DNA;

Microsatellite DNA

A2.11Genomic tools in aquaculture: use and utility ofgenetic linkage maps

L. Bargelloni, (Department of Public Health, Comparative Pathol-

ogy, and Veterinary Hygiene, University of Padova, Italy,

luca.bargelloni@unipd.it)

Genetic linkage maps represent a valuable tool for genetic

management of aquacultural species. The construction of a linkage

map consists of several steps: choice and preparation of a mapping

panel, isolation and optimization of genetic markers, genotyping of

the mapping population, and computer-assisted reconstruction of

linkage groups and genetic distances between markers. To illustrate

the above experimental stages as well as the possible uses of genetic

maps in aquaculture, the construction of a linkage map for the

gilthead sea bream (Sparus aurata L.) will be presented. The F1

progeny (2000 fish) of a single cross between two outbred

individuals was produced and reared to a weight of 30–50 g.

Two hundred F1 individuals were then sampled and stored for DNA

extraction. Fifty individuals were then used as mapping population.

Hypervariable genetic markers (short tandem repeats (STRs) or

microsatellites) were isolated using various standard or STR-

enriched genomic libraries. Markers that were polymorphic for this

panel were then used to genotype all the mapping populations. In

total, 207 microsatellite markers were scored. Linkage groups and

genetic distances between loci were reconstructed with the

computer programs CRIMAP and MAPMAKER. Twenty-three

linkage groups were found, which included 199 STRmarkers. Eight

loci remained unlinked. The female map was 1870.3 centiMorgan

(cM) in length, while the male map was 1666.9 cM.

Present and future applications of the obtained sea bream map such

as parentage assessment, genetic traceability, and genome scanning

for QTL analysis will be discussed.

Keywords: Gilthead sea bream; Linkage map; Microsatellites;

Marker assisted selection

A2.12The ArkChip—a multi-species mitochondrialgenome DNA ‘‘re-sequencing’’ strategy forbiodiversity and conservation genomics

S.M. Carr and H.D. Marshall, (Department of Biology, Memorial

UniversityofNewfoundland,St. John’s,NL,Canada, scarr@mun.ca)

In the study of Species-at-Risk, the degree to which the loss or

decline of local populations affects others and threatens species

extinction depends critically on the spatial scale of population

differentiation and gene flow. Studies of intraspecific phylogeog-

raphy, the analysis of genetic relationships in geographic context,

based on complete mtDNA genome sequences from multiple

individual Atlantic cod, harp seals, and descendants of the founding

human population of Newfoundland, have provided extremely

detailed information on the fine-scale population structure of

species, including previously unsuspected historical phenomena.

Population genomics by conventional methods remains laborious

and time-consuming. A new biotechnology, DNA ‘‘re-sequencing’’,

uses a DNA microarray to generate 30–300 kb of DNA sequence

information in a single experiment. Experiments with a first-

generation human mtDNA ‘‘Gene Chip’’ show that the method is

extremely efficient, accurate, and cost-effective. We propose to

Abstracts / Comparative Biochemistry and Physiology Part A 141 (2005) S85–S93 S89

develop DNA re-sequencing technology as a practical method for

assessing the population genomic structure of species at risk of

extinction or extirpation. We will design a DNA microchip for the

Atlantic Cod mtDNA genome and associated nucDNA holoenzyme

markers, and use it to describe the species’ fine-scale stock

structure. A second-generation, multi-species ‘‘ArkChip’’ will

permit simultaneous analysis of up to 20 species’ mtDNA genomes;

costs of each component project are reduced proportionately. A

breakthrough into high-throughput genomics would enable cost-

effective, co-ordinated investigation of multiple species of interest

to Species-at-Risk agencies, managers, and recovery teams.

[http://www.mun,ca/biology/scarr/ArkChip_for_COSEWIC.html].

A2.13Application of gene polymorphism in fish toaquaculture

Bruria Funkenstein and Ricardo Almuly, (Israel Oceanographic

and Limnological Research, National Institute of Oceanography,

Tel-Shikmona, Haifa 31080, Israel, bruria@ocean.org.il)

Sparus aurata, the major cultured species in the Mediterranean

region and a member of the family Sparidae, has a slow growth rate

and growth selection poses difficulties due to its reproduction as a

group spawner. A selection program to increase growth rate in sea

bream using mass selection showed response of 5–10% per

generation. Mass selection for growth rate can be improved by

employing genome level markers. One approach to identifying

genome level markers associated with traits of interest is the

candidate gene approach in which polymorphic sites at genes related

to a trait are tested for association with trait value. Since growth

hormone (GH) is a major growth regulator, it is a natural candidate

gene for growth rate. S. aurata GH gene (saGH) contains VNTRs

(micro- and minisatellites) in its introns and proximal promoter,

resulting in high polymorphism. Segregation of three polymorphic

loci revealed Mendelian inheritance. The microsatellite in the

promoter region was significantly associated with growth rate and

hence may be used for growth selection. Minisatellite alleles in

saGH 1st and 3rd introns in S. aurata fish from a hatchery

population selected for growth were found to be shorter and more

homogenous relatively to a non-selected hatchery population.

Similar VNTRs to those found in the saGH gene were also identified

at the same location in GH genes from other Sparids and other fish

species (olive flounder, barramundi), showing polymorphism.

Finally, longer 1st intron minisatellites were found to inhibit reporter

gene expression in both fish and mammalian cell lines.

Keywords: Growth hormone; Minisatellites; Microsatellites;

Sparidae

A2.14Immune–endocrine interaction after an immunechallenge in gilthead sea bream (Sparus aurata)

L. Aceretea, S. MacKenziea and L. Torta, (aDpt. of Cellular

Biology, Physiology and Immunology, Universitat Autonoma de

Barcelona, Facultat de Ciencies, 08193 Bellaterra, Spain, laura.

acerete@uab.es)

Cortisol is the most important corticosteroid in teleost fish, playing

roles mainly as glucocorticoid. This interrenal glucocorticoid

regulates the inflammatory response inhibiting the production of

cytokines after an immune challenge. It is produced in response to

stress, thus has been used as the principal indicator of stress

response. It also participates in metabolic pathways to maintain

homeostasis. In the liver, specifically, glucocorticoids increase the

transcription of genes involved in the gluconeogenesi, in the

aminoacid catabolism and in the acute phase response.

The biological effects of corticosteroid hormones are mediated

through intracellular receptors that act as ligand-dependant tran-

scription factors. Hepatic gene expressions are also controlled

through the glucocorticoid receptor (GR).

In the present study, a glucocorticoid receptor was cloned from

head kidney and brain mRNA of gilthead sea bream (Sparus

aurata), by polymerase chain reaction (PCR) with different pair

of primers designed for the hormone binding domain (HBD) and

for the DNA binding domain (DBD) based on mRNAs of other

teleost species. This new receptor shows high homology with

other glucocorticoids receptors including mammalian species.

The role of cortisol in the regulation of different immune and

endocrine processes has also been studied in S. aurata. We have

used primary cultures of gilthead sea bream (S. aurata) hepato-

cytes that have been stimulated with LPS and cortisol. The results

show the response of the GR and its interaction with the expression

of inflammatory genes such as TNFa or IL-1.

Keywords: Sparus aurata; Glucocorticoid; Receptor; Stress; Liver

A2.15Genetic characterization of European eels usingAnguilla anguilla and A. japonica microsatellites

D.S. Penarandaa, J.S. Vicenteb, L. Pereza, M.P. Viudes de Castroc,

M. Jovera and J.F. Asturianoa, (aGrupo de Investigacion en

Recursos Acuıcolas, Departamento de Ciencia Animal, Universi-

dad Politecnica de Valencia, Camino de Vera, s/n 46022 Valencia,

Spain, bGrupo de Mejora Animal, Laboratorio de Biotecnologıa de

la Reproduccion, Departamento de Ciencia Animal, Universidad

Politecnica de Valencia, Camino de Vera, s/n 46022 Valencia,

Spain, cCentro de Investigacion y Tecnologıa Animal, Instituto

Valenciano de Investigaciones Agrarias, Ctra. Naquera-Moncada

Km 4,5, 46113 Moncada, Valencia, Spain, jfastu@dca.upv.es)

The European eel, Anguilla anguilla, is a panmictic species which

distribution is around Europe and North Africa, but during the last

years the natural population has showed a dramatic decrease and

intense research is being developed on the control of reproduction

of this species in captivity. The use of microsatellites can be useful

in order to develop paternity tests and to make able the genetic

characterization of cryopreserved sperm samples.

Forty European eels were genetically characterized assaying four A.

anguilla microsatellites previously described by Daemen et al.

(1997) and five Japanese eel microsatellites (Sathoshi et al., 2001).

Three of the European species markers amplified correctly and

were used for the study of the samples, while the fourth showed

abnormal results. One of the Japanese eel microsatellites was not

amplified, while a second one was impossible to read because of

the bands of different sizes with equal intensity. The use of the

three other Japanese eel microsatellites allows a larger genetic

study of the European eel, a species in which the availability of

molecular markers is limited.

The number of allele, the heterozygosity and expected hetero-

zygosity, and the range of product size will be described in every

microsatellite loci.

Abstracts / Comparative Biochemistry and Physiology Part A 141 (2005) S85–S93S90

Acknowledgements. Supported by the Generalitat Valenciana

(GV04A-508, and a grant to DSP), Universidad Politecnica de

Valencia (20030488) and Spanish Ministry of Science and

Technology (AGL2003-05362-C02-01, including FEDER funds).

JFA has a Ramon y Cajal research contract, co-financed by the two

last organizations.

Keywords: European eel; Anguilla; Microsatellite

A2.16Evidence for involvement of chemicalcommunication in reproductionof the eel Anguilla anguilla

M. Huertasa, P.C. Hubbardb, A.P. Scottc, A.V.M. Canariob and J.

Cerdaa,d,T, (aCenter of Aquaculture-IRTA, 43540-Sant Carles de laRapita, Tarragona, Spain, bThe Centre for Environment, Fisheries

and Aquaculture Science (CEFAS), Barrack Road, Weymouth,

Dorset, DT4 8UB, UK, cCentro de Ciencia do Mar, Universidade

do Algarve, 8000-810 Faro, Portugal, dLab IRTA-CSIC, CMIMA,

Passeig Marıtim 37-49, 08003-Barcelona, Spain. *Corresponding

author: jcerda@icm.csic.es)

The European eel (Anguilla anguilla) is characterised by long trans-

oceanic migration to spawning sites during which the fish become

sexually mature. A previous study demonstrated that mature eels

may induce physiological and behavioural changes in immature

conspecifics. This raises the possibility that chemical communica-

tion may be involved in this process. The aim of the current study

was, therefore, to assess the olfactory sensitivity of eels to

conspecific-derived odours and to establish whether these odours

differ according to sex and/or state of maturity.

Solid-phase water extracts (using C18 cartridges), and bile and

mucus samples, were collected from mature and immature fish of

both sexes. Olfactory sensitivity to these samples was assessed by

recording the electro-olfactogram (EOG) from immature males. Of

the water extracts, those from spermiating males and pre-ovulatory

females evoked the largest responses. Both bile and mucus proved

to be highly potent odorants, evoking EOG responses at dilutions as

low as 1:107 and 1:106 respectively. Cross-adaptation studies

suggest that bile from males and females is qualitatively different

and that mucus from mature and immature fish of both sexes is

qualitatively and quantitatively different.

Taken together, these results provide strong support for a role for

chemical communication in this species, suggesting that a number

of different odorants and routes of release may be involved.

However, the chemical identities of these compounds and their

exact biological roles remain to be elucidated.

Keywords: Electro-olfactogram (EOG); Anguilla; Chemical

communication

A2.17The effect of temperature on the biology, survivaland viability of the fish parasite, Argulus japonicusThiele

P.D. Walker, I.J. Russon, R. Duijf and S.E. Wendelaar Bonga,

(Radboud University, Nijmegen, The Netherlands, P.Walker@

science.ru.nl)

With the global increase and diversification of fish farming

enterprises pathogens of fish are becoming a major factor in

determining the success of individual operations. Despite their

importance, basic information regarding the pathogens themselves

is often lacking, particularly information derived from controlled

scientific investigations. In this study we aimed to investigate the

effect of temperature on a well-known crustacean ectoparasite,

Argulus japonicus.

This piscicolous crustacean attaches to the skin of its fish host

using hook-like appendages (larvae) and/or suckers (adults) and

feeds on the blood of its host. It is well documented that increases

in ambient temperature have an effect on the development time of

this parasite and infection intensities often show an increase closely

correlated with rising water temperatures. We aimed to demonstrate

that temperature can also affect other aspects of this parasites

biology such as survival and viability.

Results showed that temperature has a significant impact on the

off-host survival times of larval, juvenile and adult lice. In contrast,

for the temperatures investigated, there was no significant effect of

temperature on parasite attachment success (viability). We con-

clude that this animal has evolved to tolerate wide temperature

ranges typical of the temperate regions in which it is predominantly

found. However evidence from this study suggests that temperature

may have an impact on this species metabolism and this is reflected

by the differences in off-host survival time at different temper-

atures, i.e. the animals utilize food reserves quicker at certain

temperatures and hence starve quicker.

Keywords: Temperature; Parasite; Viability; Survival; Argulus

A2.18The laboratory culture of the prawn Palaemonelegans as a live food source for aquaculture:osmoregulation and determination of the optimumsalinity

S. Khodabandeh* and B. Abtahi, (Faculty of Marine Sciences,

University of Tarbiat Modarres, Tehran, Iran. E-mail: surp78@

yahoo.com)

Rockprawn, Palaemon elegans Rathke 1837, is a decapod

crustacean that widely spread throughout the world and it has

adapted to habitats of constant or variable salinity. The ultra-

structure of the cells, and the localization of Na+, K+-ATPase were

examined in the branchial chambers of P. elegans from the Caspian

Sea exposed to different salinities. Ultrastructurally, typical

ionocytes were observed in the epipodites and in the inner side

of the branchiostegites. They have a common number of

cytological features including apical microvilli and basal mem-

brane infoldings associated with mitochondria. The gill filaments

are formed by a single layer epithelium with a thin lateral

expansion and a basal lamina limiting hemolymph lacunae.

Na+,K+-ATPase was detected through immunofluorescence in the

basolateral sides of the epipodites and in the inner side of the

branchiostegites cells. No immunoreactivity was detected in the

gill filaments. When this prawn is transferred to low salinity (8–12

ppt), the cytological features of the epipodites and branchiostegites

undergo marked ultrastructural changes, such as an increase in

height of the apical microvilli and an increase in the development

of basolateral infoldings and the number of mitochondria. The

concentration of Na+,K+-ATPase reactivity also increased in this

condition. In conclusion, the acquisition of the ability to tolerate a

large salinity range originates from the appearance of ionocytes on

the epipodites and branchiostegites. They participate in osmor-

Abstracts / Comparative Biochemistry and Physiology Part A 141 (2005) S85–S93 S91

egulation through active ion exchanges as evidenced by the

immunolocalization of Na+,K+-ATPase.

Keywords: Palaemon elegans; Na+,K+-ATPase; Branchial cham-

ber; Osmoregulation

A2.19Anatomical and histological study of ovarydevelopment stages of big-eye kilka(Clupeonella grimmi)

B. Abtahia, M.A. Khorashadi Zadeha and R. Kazemib, (aTarbiat

Modarres University, Faculty of Marine Sciences, Iran, bInterna-

tional Research Institute of Sturgeon Fishes, Iran, abtahibm@

modares.ac.ir)

Certain biological characteristics of female big-eye kilka, Clupeo-

nella grimmi, including body weight, fork length, age, gonad

development stages, gonad weight, Gonado Somatic Ratio (GSR)

were studied by sampling of 808 samples from the Southern Caspian

Sea-Babolsar Area from December 2002 to May 2003. Gonad

development stages were assessed through tissue sectioning.

Results revealed a spawning peak in early January. Other biological

features found were: body weight, 8.88T0.08 g; fork length,

111.03T0.26 mm; age, 3.6T0.03 years; gonad development stage,

3.67T0.03; gonad weight, 0.5T0.01 g; and Gonado Somatic Ratio

(GSR), 5.39T0.1. A total of five age classes dominated by the 4+ years

were distinguished and 1+ years were not observed. The abundance of

stages 2–5 was 18.19%, 14.60%, 48.89% and 18.32%, respectively.

Macroscopic (visual) and microscopic (tissue section) observations of

oocyte revealed the same results for gonad development.

Keywords: Clupeonella grimmi; Gonad histology; Caspian Sea;

Iran

A2.20Population changes of sturgeon fishes in theCaspian Sea

B. Abtahi, M. Rahnema and A. Monadi, (Is. Azad University-

Zandjan, Iran, behroozabt@yahoo.com)

The sturgeons enjoy a special place among the available biological

resources of the Caspian Sea with almost 85% of these fishes

natural resources living in this sea. The Caspian Sea caviar,

particularly Iranian, is well known throughout the world.

The sturgeon species caught on Iranian coasts include: Huso huso

(Great sturgeon), Acipenser persicus (Iranian sturgeon), A.

guldenstadti (Russian sturgeon), A. stellatus (Stellate sturgeon)

and A. nudiventris.

The catch figures for the whole of the Caspian Sea have increased

from 420 MT in 1943 to maximally 3571 MT in 1969 and by 1992

catch had leveled out at 2000 MT. From 1993 to 2000 the catch

figures have been tumbling reaching 1000 MT, which has

culminated in a catch of 640 MT in 2002. The percentage of

sturgeon caught in Iranian shores was shifted from 6.8% in 1942

up to 92.5% in 1964.

Catch figure of three species, A. persicus, A. guldenstadti and A.

nudiventris, fluctuated from almost 280 MT as a minimum in 1942

up to maximum of 1799 MT in 1969 and then showed 1000 MT

diminution by the end of 1992 and reached to 600 MT between

1993 and 1997.

Keywords: Sturgeon fishes; South Caspian Sea; Iran.

A2.21The effects of elevated dietary copper on theAfrican walking catfish

I. Hoyle and R.D. Handy, (School of Biological Sciences,

University of Plymouth, Drake Circus, Plymouth, PL4 8AA,

United Kingdom. E-mail: rhandy@plymouth.ac.uk)

Dietary copper (Cu) uptake is relatively well known in

salmonids, but there are few studies on warm water fish. This

study aimed to determine the Cu accumulation pattern and sub-

lethal effects in growing African walking catfish fed excess

dietary Cu (1500 mg Cu/kg food) for 30 days, compared to a

control of normal diet (15 mg Cu/kg food). The experiment was

triplicated, and fish were fed twice a day to satiation. Catfish

were sampled every 10 days for metal analysis, haematology,

biochemistry, and histology. Data were analysed by ANOVA for

treatment/time effects (data are meansTS.E., n =7 fish). Cu

showed a statistically significant increase (ANOVA, P <0.05) in

the intestine (20.24T2.23 Amol g�1 dw) and liver (7.59T0.81Amol g�1 dw) of Cu-fed fish compared to controls (intestine,

0.99T0.18; liver, 1.60T0.27 Amol g�1 dw, respectively), while

contamination in the gills remained low throughout the experi-

ment (about 0.3 Amol g�1 dw or less, ANOVA, P >0.05). The

Cu diet caused only a minor reduction in specific growth rate

(SGR, controls, 1.38; exposed, 0.93) and no overt pathologies.

There were no treatment-dependant changes in red cell counts,

blood haemoglobin concentration, haematocrit, or tissue electro-

lytes after 30 days of dietary Cu exposure. Na+,K+-ATPase

activities were also unaffected at day 30 (t-tests, P >0.05) in

the gill (control, 2.5T0.5; exposed, 3.5T0.7 Amol Pi mg�1 h�1)

and intestine (control, 3.4T0.63; exposed, 4.5T0.44 Amol

Pi mg�1 h�1).

Keywords: Dietary copper; Sub-lethal effects; Intestine; Sodium

A2.24HSP mRNA expression in response to acute stressconditions in Dicentrarchus labrax

L. Maccatrozzo, C. Poltronieri, C. Simontacchi and G. Radaelli,

(Department of Experimental Veterinary Sciences, Faculty of

Veterinary Medicine, University of Padua, Italy, giuseppe.

radaelli@unipd.it)

Heat shock proteins (HSP) represent a family of highly

conserved cellular proteins expressed in all organisms, including

fish. They usually consist of members that are induced under

various stress conditions, including heat shock, UV irradiation

and treatment with heavy metals, whereas the constitutive

members play important roles in unstressed cells. In aquaculture,

fish are subjected to different stress conditions mainly due to

manipulation, transport, overcrowding. Here we report on the

expression of HSP70 in several tissues of Dicentrarchus labrax

in relation to transport stress determined by using an RT-PCR

approach. Our data demonstrate clearly that the levels of mRNA

for inducible HSP70 are much higher in the different tissues

(brain, liver, skeletal muscle, heart, gills, skin, gonads) of

stressed animals compared to those of control ones. This data

indicates that HSP70 represents a suitable and useful marker for

stress in fish.

Abstracts / Comparative Biochemistry and Physiology Part A 141 (2005) S85–S93S92

Keywords: HSP; Stress; Expression; mRNA; Fish

References:

Gornati et al., 2005, Gene 341, 111–118; Iwama et al., 1998,

Reviews in Fish Biology and Fisheries 8, 35–56; Yamashita et al.,

2004, Gene, 336, 207–218.

A2.30Protein synthesis in juvenile barramundi, Latescalcarifer, at different temperatures

R.S. Katersky and C.G. Carter, (University of Tasmania, Tasma-

nian Aquaculture and Fisheries Institute, School of Aquaculture,

Locked Bag 1-370, Launceston, TAS 7250 Australia, robink@utas.

edu.au)

Temperature is recognized to be the most important environ-

mental factor affecting growth and protein synthesis in fish. The

optimal temperature for growth of juvenile barramundi is 27 -C,although culture often occurs at temperatures which are above

and below this optimum. Juveniles (11.10T3.72 g) were held at

three different temperatures, 21, 27 and 33 -C, and fed once a

day (504.5 g kg�1 crude protein, 156 g kg�1 lipid, 128.5 g kg�1

ash, 20.1 GE MJ kg�1). Protein synthesis was measured at 0, 4,

8, 12 and 24 h after feeding to determine when the peak of

protein synthesis occurred in white muscle, liver and whole body.

At the optimal temperature white muscle protein synthesis rates

peaked between 4 and 8 h after feeding; however, protein

synthesis remained significantly elevated over the initial 12 h

after feeding when compared to rates at 24 h. Understanding how

different temperatures affect the protein synthesis rates of juvenile

fish will provide a better understanding of processes that optimize

growth efficiency.

Keywords: Temperature; Protein turnover; Growth efficiency

A2.33Does circadian changes influence physiologicalvariables in the sea bream Sparus aurata?

L. Ribas, B. Barton, S. MacKenzie and L. Tort, (Dep. Biologia

Cellular, Immunologia i Fisiologia, Universitat Autonoma de

Barcelona, Edicifi Cs. Campus Bellaterra, Bellaterra 08193,

Barcelona, Spain, Laia.Ribas@uab.es)

Stress situations may present a threat to the fish health and fish

may experience stress under mild stress stimulus. It is well

known that cortisol is the principal corticosteroid in teleost fish

and its plasma concentrations rise dramatically during stress.

There is evidence that cortisol directly and/or indirectly plays an

important role in intermediary metabolism, ionic and osmotic

regulation and immune function, all of which argues for an

adaptive role for cortisol during stress situations in fish. The aim

of this study was to determine the changes in selected

physiological responses such as plasma cortisol, glucose, lactate

and osmolality, taking place during a circadian cycle. For this

experiment 36 fish (mean weight 150 g) were held in six

rectangular tanks containing 175 l each at a density of 7.5 kg/m3.

Fish were subjected to normal conditions of laboratory stock

maintenance (temperature, salinity and water quality parameters)

and a light photoperiod of 7:00 am–7:00 pm. Fish were sampled

every 4 h (8 h, 12 h, 16 h, 20 h, 24 h and 4 h next day). Plasma

cortisol was measured by radioimmunoassay, glucose and lactate

using commercial colorimetric kits and osmolality in a freezing-

point osmometer. The results revealed that plasma cortisol levels

increased significantly just after 1 h of the onset of darkness

(8:00 pm) and recovered 1 h after lighting (8:00 am sampling).

Glucose, lactate and osmolality did not show any significant

change during the cycle. We can conclude that sea bream is

susceptible to circadian changes and that cortisol is released in

concordance with daily light/darkness changes in laboratory

conditions. These results should be considered during exper-

imental procedures, as circadian changes may interfere with the

variables measured at particular sampling times.

Keywords: Cortisol; Stress; Circadian changes

A2.34A EC research project: development of quantitativeand qualitative molecular biological methods toidentify fish species in foods

O. Degen, (Genescan Analytics GmbH, EUROFINS GENESCAN,

D-79108 Freiburg, Germany. E-mail: o.degen@genescan.com)

One of the main tasks of the European Union is to strengthen

consumer confidence in the safety and authenticity of food

products. In recent years many cases of food-related fraud were

reported. Thus there is clear demand for new molecular methods to

proof authenticity of food. DNA-based methods for detection of

plants and genetically modified organisms could serve as an

example of how to control animal species in food. Molecular

methods were applied and validated in a recent EU project

(acronym DNA-IQ) on fish and seafood coordinated by Genescan

Analytics GmbH. Six partners out of four European countries were

involved in the DNA-IQ project. The project included three

aspects: Sequencing of new target regions, development of

qualitative methods which are useful to identify a broad variety

of different species (including commercially important ones and

species of regional interest) and application of real-time PCR

methods. Quantitative methods have been developed with respect

to threshold values for supporting the surveillance of legal

requirements. Salmon and flatfish detection systems were reviewed

in international ring trail studies.

The project was co-financed by grants of the European Union

under FP5.

Keywords: Species identification; Molecular methods; PCR; EC

project

References:

[1] O. Degen (2004). Seafood and consumer protection. In:

European Commission (Ed.), Genomics research in livestock:

what does it offer?, EUR 21031, Luxembourg, p. 31.

[2] B. Horstkotte, H. Rehbein (2003). J Food Science 68(9),

2658–2666.

A2.35Gene expression analysis in activated sea bream(Sparus aurata) immune cells

B. Castellana1, N. Roher2, S. MacKenzie2 and J.V. Planas1,

(1Department de Fisiologia, Facultat de Biologia, Universitat de

Barcelona; 2Unitat de Fisiologia Animal, Facultat de Ciencies,

Universitat Autonoma de Barcelona, bcastellana77@ub.edu)

Abstracts / Comparative Biochemistry and Physiology Part A 141 (2005) S85–S93 S93

The kinetics of lipopolysaccharide (LPS)-induced expression of

different immune genes like interleukin-1h ( IL-1h), Mx protein,

cathepsin D, PPAR-g and TNF-a in sea bream (Sparus aurata)

immune cells has been studied. We have studied immune gene

expression in LPS (10 Ag/ml)-stimulated peripheral blood

leukocytes (PBLs) in vitro for 12 h as well as in tissues of

LPS (8 mg/kg)-injected fish such as head kidney, spleen,

intestine and gills. Semiquantitative RT-PCR analysis revealed

that the expression of cathepsin D was up-regulated by LPS in

both PBLs and head kidney and also showed two obvious

bands, suggesting the existence of an incompletely spliced

variant. In addition, Mx protein expression was induced by LPS

in most of the tissues examined, but it was down-regulated in

PBLs. Furthermore, PPAR-g was up-regulated by LPS stimula-

tion in head kidney and especially in intestine after 12 h;

however, no expression in PBLs was detected. IL-1h expression

quickly increased following stimulation with LPS in head

kidney, spleen, intestine and gills, with transcripts being

detectable at 6 h post-stimulation, increasing at 24 h post-

stimulation and declining by 72 h. Surprisingly, LPS did not

induce IL-1h expression in PBLs. A slight increase of TNF-a

was found in both head kidney and intestine from LPS-

stimulated fish, although LPS had no effect in PBLs. In

conclusion, our study indicates that typical inflammatory stimuli

such as LPS modulate the expression of immune-relevant genes

in sea bream immune cells and tissues.

Keywords: Lipopolysaccharide, Gene expression, Immune cells,

Sea bream