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Additional file 2: GO terms associated to R. philippinarum transcripts represented in the microarray using "Generic GO slim" in Blast2GO software. Details about "Biological process", "Molecular function" and "Cellular component" GO terms. (DOC 158 KB)

Authors’ original file for figure 2

Additional file of Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring

Authors’ original file for figure 3

Background: MEF2C is a transcription factor with important roles in differentiation and developmental processes, in particular in endochondral bone development. An association between human MEF2C gene mutations and particular phenotypes affecting craniofacial formation has been shown. Recent studies have shown that the zebrafish palate is under a genetic control similar to that controlling amniotic palatal skeleton, demonstrating the utility of this organism to study craniofacial development. Zebrafish has two mef2c genes and different lines of mutants are available. Therefore we have used zebrafish in an attempt to correlate specific MEF2C mutations with the abnormal human craniofacial phenotypes.

Temperature is a factor known to have a marked influence on metabolism and consequently on development, inducing ontogenetic plasticity in fish larvae. In this study, three different temperatures (15, 18 and 21°C) were assayed during Solea senegalensis embryonic development prior to hatching. After hatching all larvae were reared at the same temperature (21.0 ± 0.1°C) until 30 days. We observed that lowering the incubation temperature from 21 to 15°C reduced the incidence of skeletal deformities from 80 to 60%. Intermediate incubation temperature (18°C), resulted in larvae that displayed a rate of abnormalities close to that observed at 15°C (64%). A similar typology of deformities was observed in fish from all rearing temperatures, with caudal vertebra being the most affected structures and with a low incidence of cranial and appendicular deformities. The effect of temperature in developmental plasticity will lead to osteological abnormalities mainly affecting the caudal vertebrae at 18°C (30%) and particularly the preural vertebra at 15°C (55%). Our results, clearly demonstrate a significant effect of water temperature during egg incubation on the skeletal development of Senegalese sole, indicating that 18°C is the most appropriated temperature. These findings highlight the importance of tightly controlling temperature regimes during the early development of S. senegalensis, and the need for optimizing rearing protocols in order to avoid skeletal disorders or impaired growth. published

Lordosis and kyphosis are curvatures of the vertebral column that commonly affect Senegalese sole (Solea senegalensis) under intensive rearing conditions, constituting a problem for the aquaculture industry by increasing labor and operation costs and consequently reducing profits. However, no studies have yet addressed microanatomical changes in vertebral column curvature in this species. We have conducted histological analyses in order to determine the alterations at tissue and cellular levels in these deformities. Development of an ectopic cartilage-like tissue within bone matrix at the growth regions (contact area between two vertebrae) of affected vertebrae was observed in lordotic-kyphotic fish. As previously suggested, the increase in strain supported by deformed vertebrae may trigger the onset of metaplastic tissue formation through an osteochondroprogenitor precursor from the condensation of mesenchymal cells or by trans-differentiation of (pre-)osteoblastic cells to a chondrocytic lineage. The possibility of a notochord contribution, as well as the molecular events related to bone stress and bone types in terms of cellularity, are also discussed.

Vitamin K (VK) is a liposoluble vitamin known to be essential for bone metabolism by two different pathways: (i) by its role as a coenzyme in the gamma-carboxylation of some skeletal proteins (e.g. osteocalcin (OC) and matrix Gla protein (MGP); Price et al., 1998); and (ii) through its role in skeletal gene transcription via binding to the pregnane X receptor (PXR; Azuma et al., 2010). PDTC-MAR-105152-2008

Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are defining components of farmed fish feeds. They are known effectors of bone metabolism and may reduce the occurrence of skeleton deformities in farmed fish, given that dietary levels are optimized. Underlying mechanisms of bone-related effects of PUFAs are however poorly understood. An in vitro cell system developed from gilthead seabream vertebra and capable of mineralization was used to assess the effect of AA, EPA and DHA on cell proliferation, extracellular matrix (ECM) mineralization and expression of a selection of marker genes. While all fatty acids promoted morphological changes and stimulated cell proliferation, AA and EPA inhibited ECM mineralization and DHA had the opposite effect. When fatty acids were combined, DHA apparently compensated for the inhibitory effect of AA and EPA. qPCR data indicated that, while expression of COL1A1, ON, MGP and RUNX2 genes remained unaltered, that of OP, BMP2, TNAP and COX2 genes was significantly regulated upon exposure to individual PUFAs. The concomitant increase in BMP2 and TNAP gene expression in DHA-treated cells and the strong decrease in OP transcript in AA- and EPA-treated cells may explain the pro- and anti-mineralogenic effects of these fatty acids, respectively. An increase in COX2 gene expression was also observed in EPA- and DHA-treated cells but the biological significance of this result remains ambiguous. This work also demonstrated the suitability of our in vitro cell systems to get insights into bone-related effects of PUFAs and to investigate the intracellular pathways involved in their proliferative and mineralogenic effects.

Matrix Gla protein (MGP) is a secreted vitamin K-dependent protein (VKD) located in the extracellular matrix and capable of binding calcium through its -carboxyglutamate residues. Although identified in 1983, transcriptional and post-transcriptional mechanisms regulating its expression remain unclear.

Cholesterol is found in all animal tissues and is an important component of biological cell membranes with functions such as precursor to bile acids, hormones and vitamins. Fish meal and fish oil are cholesterol-rich ingredients. Replacement of these marine-derived ingredients by plant proteins and vegetable oils tends to reduce dietary cholesterol levels.

The meagre (Argyrosomus regius) is a new species for aquaculture diversification, presenting a high economic value and a high growth rate, reaching 1 kg year)1. The occurrence of deformities, which generally appear in cultured larvae at early stages, is problematic for aquaculture since they reduce fish fitness and profitability. Knowledge about the stages of skeletal formation is essential to accurately assess deformities in hatchery produced fish. Here, we describe the osteology of the axial skeleton and unpaired fins of the meagre. Based on this data, the osteological development of meagre larvae reared at the IPIMAR and IRTA-SCR, under different rearing conditions, was compared. Although little literature is available on the osteology of teleosts from the same taxonomic group, the skeletal composition of the meagre is comparable to that of other Sciaenidae. All skeletal structures considered appeared at earlier stages in larvae reared at IPIMAR, which presented a more advanced skeletal development in comparison to larvae of comparable length reared at IRTA-SCR that were submitted to lower temperature and light intensity, and reduced photoperiod regimes.

Fish have recently been recognized as a suitable model and a promising alternative to mammalian systems to study skeletogenesis. In this regard, several fish bone-derived cell lines have been developed and are being used to investigate mechanisms associated with insulin-like action of vanadium on extracellular matrix (ECM) mineralization. Although proliferative and mineralogenic effects of vanadate, insulin-like growth factor 1 (IGF-1), and insulin have recently been evaluated in a fish prechondrocyte cell line, no data are available in fish bone-forming cells, the osteoblasts. Using fish preosteoblast cells, we showed that IGF-1, but not insulin or vanadate, stimulated cell proliferation through the mitogen-activated protein kinase (MAPK) pathway, while both IGF-1 and vanadate inhibited cell differentiation/ECM mineralization through the same mechanism. Our data also indicated that the phosphatidyl inositol-3 kinase (PI-3K) pathway stimulates differentiation/ECM mineralization in osteoblasts and could represent a way to balance MAPK pathway action. The comparison of these new data obtained in fish with those available in mammals clearly evidenced a conservation of regulatory mechanisms among vertebrate bone-derived systems, although different players are involved.

Despite the low incidence of morpho-anatomical abnormalities occurring in natural populations, this pose a serious problem for cultivated fish causing increased mortality, reduced growth and conversion rates, thus limiting production and profits. In this study we made an osteological evaluation in the white seabream (Diplodus sargus) and in the common two-banded seabream (Diplodus vulgaris), two sparid species currently under adaptation to intensive aquaculture. Commission of the European Communities - 2007-2013 Atlantic Area Transnational Programme; Project “SEAFARE - Sustainable and environmentally friendly aquaculture for the Atlantic region of Europe”

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.

Skeletogenesis is a continuous process occurring during fish larval development that is influenced by vitamin K VK, an essential factor for the correct formation of coagulation factors and skeletal matrix proteins. High levels of skeletal deformities are recorded in most commonly cultured species like the sparids, sea bass and flatfish like the Senegalese sole. The appearance of malformations takes place during the larval stages, when skeletal structures are forming. This work was funded by project PDTC-MAR-105152-2008 (SPECIAL K) from the Foundation of Science and Technology.

Vertebral deformities are frequent and constitute a major drawback for many aquaculture produced species. Lordosis and kyphosis, which are severe curvatures of the vertebral column, are particularly problematic, compromising fish marketing and profitability. Senegalese sole is a species with increasing interest in this industry and, like many others, presents high frequencies of vertebral deformities under rearing conditions. project SEACASE (Nº 044483; FP6-2005-SSP5A) and by project SPECIAL_K (PTDC-MAR-105152-2008)

Replacement of significant amounts of marine fish oils by vegetable oils is a major trend in the aquaculture feed industry. However, knowledge on the mechanisms underlying the nutritional regulation of bone metabolism is extremely scarce in fish. We speculate that changes in the dietary ratio of fatty acids may modulate tissue eicosanoids production and affect bone formation in fastgrowing gilthead seabream, an important fish species for aquaculture in the Mediterranean region.

In the last decade, fish has emerged as an important organism for studies on skeletal development in vertebrates, and evidence has been accumulated showing that zebrafish is a suitable system to perform phenotype-based drug screens. The ability to regenerate epidermal injuries is a general feature of most organisms yet only a few can fully regenerate severed appendages comprising several different tissues. Zebrafish is one of the most used models for regeneration studies, creating a powerful tool to study de novo bone formation without affecting vital development processes. Operational Program Science and Inovation 2010 (POCI 2010); European Community Fund FEDER

The meagre (Argyrosomus regius) is a new species for aquaculture with high economical value, presenting growth rates of 1 kg/year. Skeletal deformities generally appear in cultured larvae at early stages, reducing the value of the fish. The present study describes the osteological development of the vertebral column and fins in meagre larvae. Meagre larvae were obtained from wild broodstocks, kept at IRTA and IPIMAR, and induced to spawn using GnRHa injection. AQUACOR 31-03-05-FEP-0003; INIA predoctoral fellowship

The Senegalese sole (Solea senegalensis) is a highly valued species, with particular interest for the southern European aquaculture industry. For the last two decades this species has been transposed for industrial production, but it still presents some constraints as the high frequency of vertebral deformities observed under intensive rearing. The patterns of osteological deformities have previously been described and found to affect mostly the haemal and caudal vertebrae, but no studies have been conducted in order to identify causes for such deformities.

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Senegalese sole is distributed along the Mediterranean and southwestern Europe, reaching a market price of 20-25 €/kg - Promising species for aquaculture diversification - Control and intensification of culture is reasonably achieved - The high incidence of skeletal anomalies in intensively reared fish represents a constraint to commercial production - Samples from Portugal and Greece were analyzed towards identifying skeletal differences due to rearing conditions

The meagre (Argyrosomus regius) is a marine teleost with high growth rates and excellent biological characteristics for rearing, making it a proper candidate for diversification of aquaculture in Southern Europe. The description of skeletal development is essential to establish chronometric relationships between skeletal developments, types of ossification, processes and different development stages, contributing to the improvement of rearing conditions. POTC-MAR-10515 2-2008; PTDC/MARl0S313(2008)

Matrix γ-carboxyglutamic acid (Gla) protein (Mgp) is a vitamin K-dependent protein normally found associated with the organic matrix of cartilage and bone in vivo. After the discovery of Mgp in various soft tissues, this protein was proposed to act as a local inhibitor of mineralization although its molecular mechanisms of action remain incompletely understood.

Vitamin K-dependent gamma carboxylation (VKGC) is crucial for posttranslational modification of glutamate residues to form α-carboxy glutamic acid (Gla) in the presence of reduced vitamin K, molecular oxygen, and carbon dioxide (1). This modification has important implications, mainly physiological, like homeostasis, signal transduction and bone calcification. This mechanism ensures complete carboxylation of coagulation factors, and proteins like bone Gla protein (BGP) and matrix Gla protein (MGP), being essential for their biological activity. Warfarin is a known anticoagulant that inhibits the action of VKGC (2), inhibiting consequently the activity of the referred proteins.

The appearance of skeletal deformities is a major problem associated to intensive, large-scale culture of fish species, affecting growth, development and ultimately, survival of produced fish. Although the mechanisms working behind it are still unclear, it is known that environmental, genetic and nutritional factors play fundamental roles. Given its importance as a vertebrate model to study skeleton-related diseases, the selection of an appropriate dietary protocol is a critical step in order to diminish the nutritional effect on bone and cartilage development. To better understand the effect of diet on skeletal development and deformities, as well as on larvae and juvenile global performance, we have tested four distinct dietary regimes, composed of rotifers Brachionus sp., Artemia nauplii and dry, formulated food.

Vanadium is a transition metal that occurs naturally in a variety of minerals and exhibits an exceptional complex chemistry in solution, e.g., several oxidation states ranging from ?2 to ?5, and formation of vanadium oligomers such as decameric vanadate (?5) species [1–4]. Besides its metallurgical role in steel alloys, vanadium is also an ultra trace element known to participate in many biological processes and considered to be essential for living organisms [5, 6]. It accumulates in a variety of organisms ranging from microbes to vertebrates, where it modulates the activity of an array of key enzymes or participates as a cofactor in the active centre of others [1, 2, 5–9]. In mammals, vanadium compounds can mimic insulin action and may prevent chemical carcinogenesis, most probably through the inhibition of cellular tyrosine phosphatases and subsequent activation of signalling pathways, suggesting their use as pharmacological tools to treat human diabetes mellitus and cancer, respectively [10–14]. Anti-tumoral action of vanadium is, however, controversial as several studies have proposed that vanadium could act as a mitogen, tumor promoter and co-carcinogen (see [15] and references therein). Other studies have reported an osteogenic role for vanadium compounds and suggest that vanadium could also have a therapeutic application in bone-related diseases, such as osteoporosis [16–18]. Decades of research have thus provided evidence for vanadium’s physiological and pharmacological properties, supporting the claim that it may represent a promising therapeutic agent for diseases targeting billions of human beings and affecting a wide range of pathological conditions. However, the development of vanadium-based pharmaceuticals will probably take some time since various issues related to vanadium toxicity, speciation and multiple targeting will need to be solved before advancing to clinical trials. Despite being used for decades by researchers as an inhibitor of protein tyrosine phosphatases, it is still not totally clear which vanadium species induce or which signalling pathways transduce physiological and pharmacological effects. Vanadium chemistry is complex, and different species or complexes may induce different pathways [5], affecting different biological processes. This work intends to review what is presently known about the bone-related role of vanadium in mammals and present recent in vitro data on the mineralogenic effect of vanadate in fish, which have become promising model organisms for vertebrate bone-related studies.

The appearance of skeletal deformities is a major problem associated to intensive, large-scale culture of fish species, affecting growth, development and ultimately, survival of produced fish. Although the mechanisms working behind it are still unclear, it is known that environmental, genetic and nutritional factors play fundamental roles. Given its importance as a vertebrate model to study skeleton-related diseases, the selection of an appropriate dietary protocol is a critical step in order to diminish the nutritional effect on bone and cartilage development.

Spliced variants of insulin-like growth factor 1 (IGF-1), a small peptide with a critical role in metabolism and growth, have been identified in various vertebrate species. However, despite recent functional data in mammalian systems suggesting specific roles (e.g. in muscle formation) for their pro-peptides and/or E domains, their function remains unclear. In this study, three alternatively spliced variants of Sparus aurata proIGF-1 (1a, 1b, and 1c) were identified and their expression analyzed. In adult fish, IGF-1 gene expression was observed in various soft tissues (highest levels in liver) and calcified tissues, with IGF-1c being always the most expressed isoform. In developing larvae, each isoform presented a specific pattern of expression, characterized by different onset and extent and consistent with a possible role of IGF-1a and 1b during early post-hatching events (e.g. bone or muscle formation), while IGF-1c would be rather involved in early larvae formation but probably acts in concerted action with other isoforms at later stages. We also propose that, in adults, IGF-1a and 1b isoforms may have a local action, while isoform 1c would assume a systemic action, as its mammalian counterpart. This hypothesis was further supported by in silico analysis of isoform distribution, revealing that only IGF-1c/Ea isoform has been conserved throughout evolution and that other fish isoforms (i.e. 1a and 1b) may be associated with mechanisms of osmoregulation. We finally propose that IGF-1 variants may exhibit different modes of action (systemic or local) and may be involved in different developmental and adaptive mechanisms.

The zebrafish Danio rerio has recently emerged as an eminent vertebrate model for studyign genetics and development. Given the considerable importance of zebrafish as an experimental model, along with the significant economic costs associated with their large-scale use and maintenance of culturing facilities, it is to some extent surprising that their husbandry is poorly developed.

Matrix Gla protein (mgp), a vitamin K-dependent protein, is an important element of skeleton development as it regulates mineralization of both cartilaginous and bony structures, among others. Although mgp is found in a wide range of tissues, it is known to be expressed mostly by chondrocytes and vascular smooth muscle cells. Accordingly, inhibition of Mgp function results in over-mineralization of cartilages and growth plates and in ectopic mineralizations, particularly in the vascular system. Besides being a well characterized protein and the existence of developmental studies in zebrafish, there is still no description of monitoring mgp expression in live specimens. This work was partially funded by CCMARlplurianual funding. A.B. Brito is the recipient of a PhD fellowship from FCT.

We recently reported proliferative and anti-mineralogenic effects of vanadate on fish chondrocytes and here we investigate the signalling pathways associated with these effects. Our data show that vanadate stimulates chondrocyte proliferation through the MAPK pathway, using signalling mechanisms similar to those used by IGF-1, while it inhibits chondrocyte differentiation/mineralization through a putative PI-3K/Ras/ Erk signalling, a pathway shared with insulin. Our data also suggest that vanadate impairs ECM mineralization not only by interfering with regulatory pathways but also by inhibiting enzymatic activity of ALP. Finally, this work provides additional evidence for the conservation, throughout evolution, of mechanisms regulating chondrocyte proliferation and differentiation.

The physiological and molecular processes that regulate early cell differentiation and maturation, matrix deposition, morphogenesis and maintenance are under strict control during ontogenic skeletal development of vertebrates. A large research effort on the sequencing and genetic characterization of model fish such as zebrafish, medaka, fugu or seabream in the last two decades has provided numerous studies at various levels on the regulation of skeletal formation and demonstrated that similar developmental events occur both in mammalian and non mammalian systems.

Reliable juvenile production or recruitment requires high numbers of healthy fish larvae. Despite considerable progress in marine fish farming in the past 20 years, juvenile fish production is still fraught with problems which arise during the larval phase. In fisheries, juvenile recruitment in some populations has not recovered despite long-term moratoria on captures and protection of the broodstock. These issues highlight the growing importance of multidisciplinary fish larval research.