Primordial Germ Cells (PGCs)
identification and manipulation
present considerable potential
for hatchery practice and
surrogate broodstocks.
To carry out the PGCs
characterization a specific
molecular marker is required.
The vasa gene is a good
candidate to identify PGCs and
others germinal cells (Nagasawa
et al., 2009).
The aim of this study was the
cloning of the Solea senegalensis
vasa cDNA and its expression
pattern during early development
and adulthood

Cabrita, Elsa

Cross, I.

Gavaia, Paulo J.

Herráez, M. P.

Leite, Ricardo

Ortiz-Delgado, J. B.

Pacchiarini, T.

Rebordinos, L.

Sarasquete, C.

Cabrita, E.

Universidade do Algarve

CCMAR, University of the Algarve
 Faro, Portugal
5-7 April 2011
Sponsored by:
     
(Reproduced with permission of Intervet Schering Plough)
ABSTRACTS
TWO ISOFORMS OF vasa MATERNAL FACTOR IN SENEGALESE SOLE: 
BIOTECHNOLOGICAL APLICATIONS.
T. Pacchiarini1, E. Cabrita1, I. Cross2, JB. Ortiz-Delgado1, R. Leite3, PJ. Gavaia3, MP. Herráez4, L. 
Rebordinos2, C. Sarasquete1*.
1ICMAN.CSIC, Pol. Rio San Pedro, 11510 PuertoReal, Cádiz, Spain.
2Lab. de Genética, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain.
3CCMAR, Universidade do Algarve, 8005-139 Faro, Portugal. 
4Dept. Molecular Biology, University of León, 24071 León, Spain.
*carmen.sarasquete@icman.csic.es
In the last decade vasa homologs have been cloned in several teleosts and their expression patterns 
during oogenesis, spermatogenesis and development have been analyzed. These previous studies 
show that the vasa gene is a germ cells molecular marker with a high biotechnological potential for 
identification and manipulation of germ cells in surrogate broodstocks. In teleosts different 
expression patterns were observed between males and females (Kobayashi et al., 2000) and during 
embryonic and larval development (Krøvel and Olsen, 2004) for vasa isoforms. In this study two 
isoforms of vasa gene in S. senegalensis were identified, characterized and the expression pattern 
was described. The Senegalese sole vasa cDNA 1 (Ssvasa1) was 2374bp while the Senegalese sole 
vasa cDNA 2 (Ssvasa2) was 1993bp. Ssvasa isoforms showed high homology with others vasa 
cDNAs when submitted at Blast analysis. Resulting amino acidic sequences of Ssvasa isoform were 
clustered in the VASA subfamily  when compared with others DEAD box protein by neighbour-
joining analysis. The RT-PCR and Q-PCR analysis performed in different tissues of adult  fishes 
showed that Ssvasa gene expression was restricted to ovary and testis. Our results showed that the 
Ssvasa1 RNA is maternally provided since expression was only detected until mid-blastula 
transition stage. Expression patterns of Ssvasa1 and Ssvasa2 were different during embryonic and 
larval development, similar with the observed in zebrafish (Krøvel and Olsen, 2004). Our results 
demonstrate that Ssvasa1 RNA could be used as a molecular marker for testicular germ cells while 
Ssvasa2 is more appropriate for the study of primordial germ cells during embryonic and larval 
development in Senegalese sole. 
Acknowledgements: M.T. Dinis (CCMAR), P. Pousão-Ferreira (IPIMAR) and M. Manchado 
(IFAPA) for their scientific assistance and M. Cunha (A. Coelho e Castro, Lda) for biological 
material supply. Supported by: LARVANET COST action FA0801, MICINN projects 
(AGL2008-02172, AGL2006-13777), T. Pacchiarini (JAE pre-doc fellowship), E. Cabrita 
(RYC-2007-01650).
References
Kobayashi T, Kajiura-Kobayashi H, Nagahama Y. 2000. Differential expression of vasa homologue 
gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis 
niloticus. Mech. Dev. 99, 139-142.
Krøvel A.V and Olsen LC., 2004. Sexual dimorphic expression pattern of a splice variant of 
zebrafish vasa during gonadal development. Dev. Biol. 271, 190-197.
7
Skeletal quality in Senegalese sole: characterization of fish resulting from environmental and 
nutritional trials  
P.J. Gavaia1, G. Dionísio1, J. Cardeira1, M.E. Cunha3, P. Pousão-Fereira3, LEC Conceição1, M. L. 
Cancela1,2
1 CCMAR - Centre for Marine Sciences, and 2Dept of Biomedical Sciences and Medicine,, University of 
Algarve, 8005 – 139 Faro, Portugal. 
3Estação Piloto Piscicultura de Olhão, IPIMAR/CRIP Sul, Av. 5 Outubro s/n 8700-302 Olhão, Portugal
The Senegalese sole (Solea senegalensis) is characteristic from southern Europe and 
Mediterranean. It has recently been adapted for aquaculture production since it is well accepted by 
consumers and reaches high commercial values. After the initial description of the ontogenic 
events of  skeletogenesis, systematic evaluation of the  incidence of skeletal malformations by 
several groups revealed high levels of  skeletal deformities reaching up to 90% of the population. 
These high levels may impair development, growth and acceptability by the consumers, thus 
representing a constraint for commercial production of this species. A comparison on the 
intensiveness on rearing methodologies revealed a low  incidence of skeletal deformities in early 
life stages captured in nature (0-23%) and a relatively higher number of deformed individuals when 
raised under extensive conditions in mesocosms (20-50%), as opposed to 60-80% of deformed 
fish obtained from intensive rearing. Data suggests that deformities occur naturally and have other 
causes than just those induced by rearing in captivity. Modulation of temperature during the 
autotrophic period caused both an increase of deformities and an increase in the anomaly charge 
paralleling the increase in incubation temperature.. Our results clearly demonstrate a significant 
effect of water temperature on skeletal development of Senegalese sole, indicating that 
temperature control is crucial for larval rearing. Nutritional imbalances during early larval stages 
have been shown to affect bone development and promote the occurrence of skeletal deformities. 
Vitamin K (VK) is a liposoluble vitamin classically involved in blood coagulation, and until recently 
largely disregarded in aquaculture nutrition. VK plays an important role in bone metabolism by 
acting over gamma carboxylation of matrix proteins. The effects of vitamin K in Solea senegalensis 
skeletogenesis were evaluated in larvae from three different dietary regimes supplemented with 
graded levels of phylloquinone (VK1) (0mg/kg, 50 mg/kg, 250 mg/kg) using enriched Artemia as 
carrier. A positive effect on bone quality was observed with VK1, with a reduction in both number of 
deformed fish and number of deformities per fish.
In order to characterize the alterations resulting from spine curvature vertebral deformities in 
Senegalese sole, we have analysed vertebral deformities in juvenile and adult fish using 
histological, histochemical and immunochemical approaches to verify the physiological response at 
tissue and cellular levels. We have observed an adaptive response to increased strain, with 
affected vertebrae presenting an increase in bone formation and in mineral as well as the 
development of chondroid at the articular surfaces and chondroid bone on the vertebral centra.
Even though relatively high numbers of deformities were observed under different environmental 
and nutritional conditions, the external anatomy was usually not severely affected, meaning that 
the aquaculture success of this species might not be constrained by skeletal deformities. Still, 
advances in nutritional and zootechnical conditions should be made to improve the overall skeletal 
quality of Solea senegalensis.
EFFECTS OF ENVIRONMENTAL CYCLES (LIGHT AND TEMPERATURE) ON SOLE LARVAE 
DEVELEOPMENT
Borja Blanco Vives
To be inserted
8


Two isoforms of vasa maternal factor in Senegalese sole: Biotechnological applications

Primordial Germ Cells (PGCs)
identification and manipulation
present considerable potential
for hatchery practice and
surrogate broodstocks.
To carry out the PGCs
characterization a specific
molecular marker is required.
The vasa gene is a good
candidate to identify PGCs and
others germinal cells (Nagasawa
et al., 2009).
The aim of this study was the
cloning of the Solea senegalensis
vasa cDNA and its expression
pattern during early development
and adulthood.LARVANET COST action FA0801;  MICINN projects (AGL2008-02172, AGL2006-13777);  (RYC-2007-01650)

Sapientia

CCMAR, University of the Algarve Faro, Portugal5-7 April 2011Sponsored by:     (Reproduced with permission of Intervet Schering Plough)ABSTRACTSTWO ISOFORMS OF vasa MATERNAL FACTOR IN SENEGALESE SOLE: BIOTECHNOLOGICAL APLICATIONS.T. Pacchiarini1, E. Cabrita1, I. Cross2, JB. Ortiz-Delgado1, R. Leite3, PJ. Gavaia3, MP. Herráez4, L. Rebordinos2, C. Sarasquete1*.1ICMAN.CSIC, Pol. Rio San Pedro, 11510 PuertoReal, Cádiz, Spain.2Lab. de Genética, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain.3CCMAR, Universidade do Algarve, 8005-139 Faro, Portugal. 4Dept. Molecular Biology, University of León, 24071 León, Spain.*carmen.sarasquete@icman.csic.esIn the last decade vasa homologs have been cloned in several teleosts and their expression patterns during oogenesis, spermatogenesis and development have been analyzed. These previous studies show that the vasa gene is a germ cells molecular marker with a high biotechnological potential for identification and manipulation of germ cells in surrogate broodstocks. In teleosts different expression patterns were observed between males and females (Kobayashi et al., 2000) and during embryonic and larval development (Krøvel and Olsen, 2004) for vasa isoforms. In this study two isoforms of vasa gene in S. senegalensis were identified, characterized and the expression pattern was described. The Senegalese sole vasa cDNA 1 (Ssvasa1) was 2374bp while the Senegalese sole vasa cDNA 2 (Ssvasa2) was 1993bp. Ssvasa isoforms showed high homology with others vasa cDNAs when submitted at Blast analysis. Resulting amino acidic sequences of Ssvasa isoform were clustered in the VASA subfamily  when compared with others DEAD box protein by neighbour-joining analysis. The RT-PCR and Q-PCR analysis performed in different tissues of adult  fishes showed that Ssvasa gene expression was restricted to ovary and testis. Our results showed that the Ssvasa1 RNA is maternally provided since expression was only detected until mid-blastula transition stage. Expression patterns of Ssvasa1 and Ssvasa2 were different during embryonic and larval development, similar with the observed in zebrafish (Krøvel and Olsen, 2004). Our results demonstrate that Ssvasa1 RNA could be used as a molecular marker for testicular germ cells while Ssvasa2 is more appropriate for the study of primordial germ cells during embryonic and larval development in Senegalese sole. Acknowledgements: M.T. Dinis (CCMAR), P. Pousão-Ferreira (IPIMAR) and M. Manchado (IFAPA) for their scientific assistance and M. Cunha (A. Coelho e Castro, Lda) for biological material supply. Supported by: LARVANET COST action FA0801, MICINN projects (AGL2008-02172, AGL2006-13777), T. Pacchiarini (JAE pre-doc fellowship), E. Cabrita (RYC-2007-01650).ReferencesKobayashi T, Kajiura-Kobayashi H, Nagahama Y. 2000. Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus. Mech. Dev. 99, 139-142.Krøvel A.V and Olsen LC., 2004. Sexual dimorphic expression pattern of a splice variant of zebrafish vasa during gonadal development. Dev. Biol. 271, 190-197.7Skeletal quality in Senegalese sole: characterization of fish resulting from environmental and nutritional trials  P.J. Gavaia1, G. Dionísio1, J. Cardeira1, M.E. Cunha3, P. Pousão-Fereira3, LEC Conceição1, M. L. Cancela1,21 CCMAR - Centre for Marine Sciences, and 2Dept of Biomedical Sciences and Medicine,, University of Algarve, 8005 – 139 Faro, Portugal. 3Estação Piloto Piscicultura de Olhão, IPIMAR/CRIP Sul, Av. 5 Outubro s/n 8700-302 Olhão, PortugalThe Senegalese sole (Solea senegalensis) is characteristic from southern Europe and Mediterranean. It has recently been adapted for aquaculture production since it is well accepted by consumers and reaches high commercial values. After the initial description of the ontogenic events of  skeletogenesis, systematic evaluation of the  incidence of skeletal malformations by several groups revealed high levels of  skeletal deformities reaching up to 90% of the population. These high levels may impair development, growth and acceptability by the consumers, thus representing a constraint for commercial production of this species. A comparison on the intensiveness on rearing methodologies revealed a low  incidence of skeletal deformities in early life stages captured in nature (0-23%) and a relatively higher number of deformed individuals when raised under extensive conditions in mesocosms (20-50%), as opposed to 60-80% of deformed fish obtained from intensive rearing. Data suggests that deformities occur naturally and have other causes than just those induced by rearing in captivity. Modulation of temperature during the autotrophic period caused both an increase of deformities and an increase in the anomaly charge paralleling the increase in incubation temperature.. Our results clearly demonstrate a significant effect of water temperature on skeletal development of Senegalese sole, indicating that temperature control is crucial for larval rearing. Nutritional imbalances during early larval stages have been shown to affect bone development and promote the occurrence of skeletal deformities. Vitamin K (VK) is a liposoluble vitamin classically involved in blood coagulation, and until recently largely disregarded in aquaculture nutrition. VK plays an important role in bone metabolism by acting over gamma carboxylation of matrix proteins. The effects of vitamin K in Solea senegalensis skeletogenesis were evaluated in larvae from three different dietary regimes supplemented with graded levels of phylloquinone (VK1) (0mg/kg, 50 mg/kg, 250 mg/kg) using enriched Artemia as carrier. A positive effect on bone quality was observed with VK1, with a reduction in both number of deformed fish and number of deformities per fish.In order to characterize the alterations resulting from spine curvature vertebral deformities in Senegalese sole, we have analysed vertebral deformities in juvenile and adult fish using histological, histochemical and immunochemical approaches to verify the physiological response at tissue and cellular levels. We have observed an adaptive response to increased strain, with affected vertebrae presenting an increase in bone formation and in mineral as well as the development of chondroid at the articular surfaces and chondroid bone on the vertebral centra.Even though relatively high numbers of deformities were observed under different environmental and nutritional conditions, the external anatomy was usually not severely affected, meaning that the aquaculture success of this species might not be constrained by skeletal deformities. Still, advances in nutritional and zootechnical conditions should be made to improve the overall skeletal quality of Solea senegalensis.EFFECTS OF ENVIRONMENTAL CYCLES (LIGHT AND TEMPERATURE) ON SOLE LARVAE DEVELEOPMENTBorja Blanco VivesTo be inserted8

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Two isoforms of vasa maternal factor in Senegalese sole: Biotechnological applications

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