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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
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