Metabolic bone disorders and the promise of marine osteoactive compounds.

Metabolic bone disorders and associated fragility fractures are major causes of disability and mortality worldwide and place an important financial burden on the global health systems. These disorders result from an unbalance between bone anabolic and resorptive processes and are characterized by different pathophysiological mechanisms. Drugs are available to treat bone metabolic pathologies, but they are either poorly effective or associated with undesired side effects that limit their use. The molecular mechanism underlying the most common metabolic bone disorders, and the availability, efficacy, and limitations of therapeutic options currently available are discussed here. A source for the unmet need of novel drugs to treat metabolic bone disorders is marine organisms, which produce natural osteoactive compounds of high pharmaceutical potential. In this review, we have inventoried the marine osteoactive compounds (MOCs) currently identified and spotted the groups of marine organisms with potential for MOC production. Finally, we briefly examine the availability of in vivo screening and validation tools for the study of MOCs.

The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish models.

Skeletal disorders are problematic aspects for the aquaculture industry as skeletal deformities, which affect most species of farmed fish, increase production costs and affect fish welfare. Following recent findings that show the presence of osteoactive compounds in marine organisms, we evaluated the osteogenic and mineralogenic potential of commercially available microalgae strains Skeletonema costatum and Tetraselmis striata CTP4 in several fish systems. Ethanolic extracts increased extracellular matrix mineralization in gilthead seabream (Sparus aurata) bone-derived cell cultures and promoted osteoblastic differentiation in zebrafish (Danio rerio) larvae. Long-term dietary exposure to both extracts increased bone mineralization in zebrafish and upregulated the expression of genes involved in bone formation (sp7, col1a1a, oc1, and oc2), bone remodeling (acp5a), and antioxidant defenses (cat, sod1). Extracts also improved the skeletal status of zebrafish juveniles by reducing the incidence of skeletal anomalies. Our results indicate that both strains of microalgae contain osteogenic and mineralogenic compounds, and that ethanolic extracts have the potential for an application in the aquaculture sector as dietary supplements to support fish bone health. Future studies should also identify osteoactive compounds and establish whether they can be used in human health to broaden the therapeutic options for bone erosive disorders such as osteoporosis.

mef2ca and mef2cb Double Mutant Zebrafish Show Altered Craniofacial Phenotype and Motor Behaviour.

The transcription factor MEF2C is crucial in neuronal, cardiac, bone and cartilage molecular processes, as well as for craniofacial development. MEF2C was associated with the human disease MRD20, whose patients show abnormal neuronal and craniofacial development. Zebrafish mef2ca;mef2cb double mutants were analysed for abnormalities in craniofacial and behaviour development through phenotypic analysis. Quantitative PCR was performed to investigate the expression levels of neuronal marker genes in mutant larvae. The motor behaviour was analysed by the swimming activity of 6 dpf larvae. We found that mef2ca;mef2cb double mutants display several abnormal phenotypes during early development, including those already described in zebrafish carrying mutations in each paralog, but also (i) a severe craniofacial phenotype (comprising both cartilaginous and dermal bone structures), (ii) developmental arrest due to the disruption of cardiac oedema and (iii) clear alterations in behaviour. We demonstrate that the defects observed in zebrafish mef2ca;mef2cb double mutants are similar to those previously described in MEF2C-null mice and MRD20 patients, confirming the usefulness of these mutant lines as a model for studies concerning MRD20 disease, the identification of new therapeutic targets and screening for possible rescue strategies.

Type I Diabetes in Zebrafish Reduces Sperm Quality and Increases Insulin and Glucose Transporter Transcripts.

Type I diabetes is a prominent human pathology with increasing incidence in the population; however, its cause is still unknown. This disease promotes detrimental effects on reproduction, such as lower sperm motility and DNA integrity. Hence, the investigation of the underlying mechanisms of this metabolic disturbance in reproduction and its transgenerational consequences is of the utmost importance. The zebrafish is a useful model for this research considering its high homology with human genes as well as its fast generation and regeneration abilities. Therefore, we aimed to investigate sperm quality and genes relevant to diabetes in the spermatozoa of Tg(ins:nfsb-mCherry) zebrafish, a model for type I diabetes. Diabetic Tg(ins:nfsb-mCherry) males showed significantly higher expression of transcripts for insulin a (insa) and glucose transporter (slc2a2) compared to controls. Sperm obtained from the same treatment group showed significantly lower sperm motility, plasma membrane viability, and DNA integrity compared to that from the control group. Upon sperm cryopreservation, sperm freezability was reduced, which could be a consequence of poor initial sperm quality. Altogether, the data showed similar detrimental effects related to type I diabetes in zebrafish spermatozoa at the cellular and molecular levels. Therefore, our study validates the zebrafish model for type I diabetes research in germ cells.

Distinguishing the Effects of Water Volumes versus Stocking Densities on the Skeletal Quality during the Pre-Ongrowing Phase of Gilthead Seabream (Sparus aurata).

Gilthead seabream (Sparus aurata) production is a highly valued aquaculture industry in Europe. The presence of skeletal deformities in farmed gilthead seabream represents a major bottleneck for the industry leading to economic losses, negative impacts on the consumers' perception of aquaculture, and animal welfare issues for the fish. Although past work has primarily focused on the hatchery phase to reduce the incidence of skeletal anomalies, this work targets the successive pre-ongrowing phase in which more severe anomalies affecting the external shape often arise. This work aimed to test the effects of: (i) larger and smaller tank volumes, stocked at the same density; and (ii) higher and lower stocking densities maintained in the same water volume, on the skeleton of gilthead seabream fingerlings reared for ~63 days at a pilot scale. Experimental rearing was conducted with gilthead seabream juveniles (~6.7 ± 2.5 g), which were selected as 'non-deformed' based on external inspection, stocked at three different densities (Low Density (LD): 5 kg/m3; Medium Density (MD): 10 kg/m3; High Density (HD): 20 kg/m3) in both 500 L and 1000 L tanks. Gilthead seabream were sampled for growth performance and radiographed to assess the skeletal elements at the beginning and end of the experimental trial. Results revealed that (i) LD fish were significantly longer than HD fish, although there were no differences in final weights, regardless of the water volume; (ii) an increase in the prevalence of seabream exhibiting cranial and vertebral axis anomalies was found to be associated with increased density. These results suggest that farmers can significantly reduce the presence of some cranial and axis anomalies affecting pre-ongrown gilthead seabream by reducing the stocking density.

Zebrafish Models to Study Ectopic Calcification and Calcium-Associated Pathologies.

Ectopic calcification refers to the pathological accumulation of calcium ions in soft tissues and is often the result of a dysregulated action or disrupted function of proteins involved in extracellular matrix mineralization. While the mouse has traditionally been the go-to model organism for the study of pathologies associated with abnormal calcium deposition, many mouse mutants often have exacerbated phenotypes and die prematurely, limiting the understanding of the disease and the development of effective therapies. Since the mechanisms underlying ectopic calcification share some analogy with those of bone formation, the zebrafish (Danio rerio)-a well-established model for studying osteogenesis and mineralogenesis-has recently gained momentum as a model to study ectopic calcification disorders. In this review, we outline the mechanisms of ectopic mineralization in zebrafish, provide insights into zebrafish mutants that share phenotypic similarities with human pathological mineralization disorders, list the compounds capable of rescuing mutant phenotypes, and describe current methods to induce and characterize ectopic calcification in zebrafish.

Exploring Omega-3's Impact on the Expression of Bone-Related Genes in Meagre (Argyrosomus regius).

Dietary supplementation with Omega-3 fatty acids seems to promote skeletal health. Therefore, their consumption at imbalanced or excessive levels has offered less beneficial or even prejudicial effects. Fish produced in aquaculture regimes are prone to develop abnormal skeletons. Although larval cultures are usually fed with diets supplemented with Omega-3 Long Chain Polyunsaturated fatty acids (LC-PUFAs), the lack of knowledge about the optimal requirements for fatty acids or about their impact on mechanisms that regulate skeletal development has impeded the design of diets that could improve bone formation during larval stages when the majority of skeletal anomalies appear. In this study, Argyrosomus regius larvae were fed different levels of Omega-3s (2.6% and 3.6% DW on diet) compared to a commercial diet. At 28 days after hatching (DAH), their transcriptomes were analyzed to study the modulation exerted in gene expression dynamics during larval development and identify impacted genes that can contribute to skeletal formation. Mainly, both levels of supplementation modulated bone-cell proliferation, the synthesis of bone components such as the extracellular matrix, and molecules involved in the interaction and signaling between bone components or in important cellular processes. The 2.6% level impacted several genes related to cartilage development, denoting a special impact on endochondral ossification, delaying this process. However, the 3.6% level seemed to accelerate this process by enhancing skeletal development. These results offered important insights into the impact of dietary Omega-3 LC-PUFAs on genes involved in the main molecular mechanism and cellular processes involved in skeletal development.

Fish Microbiome Modulation and Convenient Storage of Aquafeeds When Supplemented with Vitamin K1.

Vitamin K (VK) is a fat-soluble vitamin necessary for fish metabolism and health. VK stability as dietary component during aquafeed storage and its potential effect on intestinal microbiome in fish have not yet been completely elucidated. The convenient storage conditions of aquafeeds when supplemented with phylloquinone (VK1), as well as its potential effects on the gut microbiota of Senegalese sole (Solea senegalensis) juveniles, have been explored. Experimental feeds were formulated to contain 0, 250 and 1250 mg kg-1 of VK1 and were stored at different temperatures (4, -20 or -80 °C). VK stability was superior at -20 °C for short-term (7 days) storage, while storing at -80 °C was best suited for long-term storage (up to 3 months). A comparison of bacterial communities from Senegalese sole fed diets containing 0 or 1250 mg kg-1 of VK1 showed that VK1 supplementation decreased the abundance of the Vibrio, Pseudoalteromonas, and Rhodobacterace families. All these microorganisms were previously associated with poor health status in aquatic organisms. These results contribute not only to a greater understanding of the physiological effects of vitamin K, particularly through fish intestinal microbiome, but also establish practical guidelines in the industry for proper aquafeed storage when supplemented with VK1.

Resveratrol-Mediated Reversal of Doxorubicin-Induced Osteoclast Differentiation.

Secondary osteoporosis has been associated with cancer patients undertaking Doxorubicin (DOX) chemotherapy. However, the molecular mechanisms behind DOX-induced bone loss have not been elucidated. Molecules that can protect against the adverse effects of DOX are still a challenge in chemotherapeutic treatments. We investigated the effect and mechanism of DOX in osteoclast differentiation and used the Sirt 1 activator resveratrol (RES) to counteract DOX-induced effects. RAW 264.7 cells were differentiated into osteoclasts under cotreatment with DOX and RES, alone or combined. RES treatment inhibited DOX-induced osteoclast differentiation, reduced the expression of osteoclast fusion marker Oc-stamp and osteoclast differentiation markers Rank, Trap, Ctsk and Nfatc1. Conversely, RES induced the upregulation of antioxidant genes Sod 1 and Nrf 2 while DOX significantly reduced the FoxM1 expression, resulting in oxidative stress. Treatment with the antioxidant MitoTEMPO did not influence DOX-induced osteoclast differentiation. DOX-induced osteoclastogenesis was studied using the cathepsin-K zebrafish reporter line (Tg[ctsk:DsRed]). DOX significantly increased ctsk signal, while RES cotreatment resulted in a significant reduction in ctsk positive cells. RES significantly rescued DOX-induced mucositis in this model. Additionally, DOX-exposed zebrafish displayed altered locomotor behavior and locomotory patterns, while RES significantly reversed these effects. Our research shows that RES prevents DOX-induced osteoclast fusion and activation in vitro and in vivo and reduces DOX-induced mucositis, while improving locomotion parameters.

Regular Supplementation with Antioxidants Rescues Doxorubicin-Induced Bone Deformities and Mineralization Delay in Zebrafish.

Osteoporosis is characterized by an abnormal bone structure with low bone mass and degradation of microarchitecture. Oxidative stress induces imbalances in osteoblast and osteoclast activity, leading to bone degradation, a primary cause of secondary osteoporosis. Doxorubicin (DOX) is a widely used chemotherapy drug for treating cancer, known to induce secondary osteoporosis. The mechanism underlying DOX-induced bone loss is still not fully understood, but one of the relevant mechanisms is through a massive accumulation of reactive oxygen and nitrogen species (i.e., ROS and NOS) leading to oxidative stress. We investigated the effects of antioxidants Resveratrol and MitoTEMPO on DOX-induced bone impairment using the zebrafish model. DOX was shown to increase mortality, promote skeletal deformities, induce alterations on intestinal villi, impair growth and mineralization and significantly downregulate osteoblast differentiation markers osteocalcin 2 and osterix/sp7. Lipid peroxidation was significantly increased in DOX-supplemented groups as compared to control and antioxidants, suggesting ROS formation as one of the key factors for DOX-induced bone loss. Furthermore, DOX affected mineral contents, suggesting an altered mineral metabolism. However, upon supplementation with antioxidants, DOX-induced effects on mineral content were rescued. Our data show that supplementation with antioxidants effectively improves the overall growth and mineralization in zebrafish and counteracts DOX-induced bone anomalies.

Fin ray branching is defined by TRAP+ osteolytic tubules in zebrafish.

The shaping of bone structures relies on various cell types and signaling pathways. Here, we use the zebrafish bifurcating fin rays during regeneration to investigate bone patterning. We found that the regenerating fin rays form via two mineralization fronts that undergo an osteoblast-dependent fusion/stitching until the branchpoint, and that bifurcation is not simply the splitting of one unit into two. We identified tartrate-resistant acid phosphatase-positive osteolytic tubular structures at the branchpoints, hereafter named osteolytic tubules (OLTs). Chemical inhibition of their bone-resorbing activity strongly impairs ray bifurcation, indicating that OLTs counteract the stitching process. Furthermore, by testing different osteoactive compounds, we show that the position of the branchpoint depends on the balance between bone mineralization and resorption activities. Overall, these findings provide a unique perspective on fin ray formation and bifurcation, and reveal a key role for OLTs in defining the proximo-distal position of the branchpoint.

Screening of Mineralogenic and Osteogenic Compounds in Zebrafish-Tools to Improve Assay Throughput and Data Accuracy.

Bone disorders affect millions of people worldwide and treatments currently available often produce undesirable secondary effects or have limited efficacy. It is therefore of the utmost interest for patients to develop more efficient drugs with reduced off-target activities. In the long process of drug development, screening and preclinical validation have recently gained momentum with the increased use of zebrafish as a model organism to study pathological processes related to human bone disorders, and the development of zebrafish high-throughput screening assays to identify bone anabolic compounds. In this review, we provided a comprehensive overview of the literature on zebrafish bone-related assays and evaluated their performance towards an integration into screening pipelines for the discovery of mineralogenic/osteogenic compounds. Tools available to standardize fish housing and feeding procedures, synchronize embryo production, and automatize specimen sorting and image acquisition/analysis toward faster and more accurate screening outputs were also presented.

Effects of pristine or contaminated polyethylene microplastics on zebrafish development.

The presence of microplastics in the aquatic ecosystem represents a major issue for the environment and human health. The capacity of organic pollutants to adsorb onto microplastic particles raises additional concerns, as it creates a new route for toxic compounds to enter the food web. Current knowledge on the impact of pristine and/or contaminated microplastics on aquatic organisms remains insufficient, and we provide here new insights by evaluating their biological effects in zebrafish (Danio rerio). Zebrafish larvae were raised in ZEB316 stand-alone housing systems and chronically exposed throughout their development to polyethylene particles of 20-27 µm, pristine (MP) or spiked with benzo[α]pyrene (MP-BaP), supplemented at 1% w/w in the fish diet. While they had no effect at 30 days post-fertilization (dpf), MP and MP-BaP affected growth parameters at 90 and 360 dpf. Relative fecundity, egg morphology, and yolk area were also impaired in zebrafish fed MP-BaP. Zebrafish exposed to experimental diets exhibited an increased incidence of skeletal deformities at 30 dpf as well as an impaired development of caudal fin/scales, and a decreased bone quality at 90 dpf. An intergenerational bone formation impairment was also observed in the offspring of parents exposed to MP or MP-BaP through a reduction of the opercular bone in 6 dpf larvae. Beside a clear effect on bone development, histological analysis of the gut revealed a reduced number of goblet cells in zebrafish fed MP-BaP diet, a sign of intestinal inflammation. Finally, exposure of larvae to MP-BaP up-regulated the expression of genes associated with the BaP response pathway, while negatively impacting the expression of genes involved in oxidative stress. Altogether, these data suggest that long-term exposure to pristine/contaminated microplastics not only jeopardizes fish growth, reproduction performance, and skeletal health, but also causes intergenerational effects.

Zebrafish as a Model to Unveil the Pro-Osteogenic Effects of Boron-Vitamin D3 Synergism.

The micronutrient boron (B) plays a key role during the ossification process as suggested by various in vitro and in vivo studies. To deepen our understanding of the molecular mechanism involved in the osteogenicity of B and its possible interaction with vitamin D3 (VD), wild-type AB zebrafish (Danio rerio) were used for morphometric analysis and transcriptomic analysis in addition to taking advantage of the availability of specific zebrafish osteoblast reporter lines. First, osteoactive concentrations of B, VD, and their combinations were established by morphometric analysis of the opercular bone in alizarin red-stained zebrafish larvae exposed to two selected concentrations of B (10 and 100 ng/ml), one concentration of VD (10 pg/ml), and their respective combinations. Bone formation, as measured by opercular bone growth, was significantly increased in the two combination treatments than VD alone. Subsequently, a transcriptomic approach was adopted to unveil the molecular key regulators involved in the synergy. Clustering of differentially expressed genes revealed enrichment toward bone and skeletal functions in the groups co-treated with B and VD. Downstream analysis confirmed mitogen-activated protein kinase as the most regulated pathway by the synergy groups in addition to transforming growth factor-ß signaling, focal adhesion, and calcium signaling. The best-performing synergistic treatment, B at 10 ng/ml and VD at 10 pg/ml, was applied to two zebrafish transgenic lines, Tg(sp7:mCherry) and Tg(bglap:EGFP), at multiple time points to further explore the results of the transcriptomic analysis. The synergistic treatment with B and VD induced enrichment of intermediate (sp7+) osteoblast at 6 and 9 days post fertilization (dpf) and of mature (bglap +) osteoblasts at 15 dpf. The results obtained validate the role of B in VD-dependent control over bone mineralization and can help to widen the spectrum of therapeutic approaches to alleviate pathological conditions caused by VD deficiency by using low concentrations of B as a nutritional additive.

Antioxidant and Anti-inflammatory Extracts From Sea Cucumbers and Tunicates Induce a Pro-osteogenic Effect in Zebrafish Larvae.

Bone metabolic disorders such as osteoporosis are characterized by the loss of mineral from the bone tissue leading to its structural weakening and increased susceptibility to fractures. A growing body of evidence suggests that inflammation and oxidative stress play an important role in the pathophysiological processes involved in the rise of these conditions. As the currently available therapeutic strategies are often characterized by toxic effects associated with their long-term use, natural antioxidants and anti-inflammatory compounds such as polyphenols promise to be a valuable alternative for the prevention and treatment of these disorders. In this scope, the marine environment is becoming an important source of bioactive compounds with potential pharmacological applications. Here, we explored the bioactive potential of three species of holothurians (Echinodermata) and four species of tunicates (Chordata) as sources of antioxidant and anti-inflammatory compounds with a particular focus on polyphenolic substances. Hydroethanolic and aqueous extracts were obtained from animals' biomass and screened for their content of polyphenols and their antioxidant and anti-inflammatory properties. Hydroethanolic fractions of three species of tunicates displayed high polyphenolic content associated with strong antioxidant potential and anti-inflammatory activity. Extracts were thereafter tested for their capacity to promote bone formation and mineralization by applying an assay that uses the developing operculum of zebrafish (Danio rerio) to assess the osteogenic activity of compounds. The same three hydroethanolic fractions from tunicates were characterized by a strong in vivo osteogenic activity, which positively correlated with their anti-inflammatory potential as measured by COX-2 inhibition. This study highlights the therapeutic potential of polyphenol-rich hydroethanolic extracts obtained from three species of tunicates as a substrate for the development of novel drugs for the treatment of bone disorders correlated to oxidative stress and inflammatory processes.

Reversal of Doxorubicin-Induced Bone Loss and Mineralization by Supplementation of Resveratrol and MitoTEMPO in the Early Development of Sparus aurata.

Doxorubicin is a widely used chemotherapeutic drug known to induce bone loss. The mechanism behind doxorubicin-mediated bone loss is unclear, but oxidative stress has been suggested as a potential cause. Antioxidants that can counteract the toxic effect of doxorubicin on the bone would be helpful for the prevention of secondary osteoporosis. We used resveratrol, a natural antioxidant, and MitoTEMPO, a mitochondria-targeted antioxidant, to counteract doxorubicin-induced bone loss and mineralization on Sparus aurata larvae. Doxorubicin supplemented Microdiets increased bone deformities, decreased mineralization, and lipid peroxidation, whereas Resveratrol and MitoTEMPO supplemented microdiets improved mineralization, decreased bone deformities, and reversed the effects of doxorubicin in vivo and in vitro, using osteoblastic VSa13 cells. Partial Least-Squares Discriminant Analysis highlighted differences between groups on the distribution of skeletal anomalies and mineralization of skeleton elements. Calcium and Phosphorus content was negatively affected in the doxorubicin supplemented group. Doxorubicin reduced the mRNA expression of antioxidant genes, including catalase, glutathione peroxidase 1, superoxide dismutase 1, and hsp90 suggesting that ROS are central for Doxorubicin-induced bone loss. The mRNA expression of antioxidant genes was significantly increased on resveratrol alone or combined treatment. The length of intestinal villi was increased in response to antioxidants and reduced on doxorubicin. Antioxidant supplements effectively prevent bone deformities and mineralization defects, increase antioxidant response and reverse doxorubicin-induced effects on bone anomalies, mineralization, and oxidative stress. A combined treatment of doxorubicin and antioxidants was beneficial in fish larvae and showed the potential for use in preventing Doxorubicin-induced bone impairment.

New insights into benzo[⍺]pyrene osteotoxicity in zebrafish.

Persistent and ubiquitous organic pollutants, such as the polycyclic aromatic hydrocarbon benzo[⍺]pyrene (BaP), represent a major threat to aquatic organisms and human health. Beside some well-documented adverse effects on the development and reproduction of aquatic organisms, BaP was recently shown to affect fish bone formation and skeletal development through mechanisms that remain poorly understood. In this work, zebrafish bone-related in vivo assays were used to evaluate the osteotoxic effects of BaP during bone development and regeneration. Acute exposure of zebrafish larvae to BaP from 3 to 6 days post-fertilization (dpf) induced a dose-dependent reduction of the opercular bone size and a depletion of osteocalcin-positive cells, indicating an effect on osteoblast maturation. Chronic exposure of zebrafish larvae to BaP from 3 to 30 dpf affected the development of the axial skeleton and increased the incidence and severity of skeletal deformities. In young adults, BaP affected the mineralization of newly formed fin rays and scales, and impaired fin ray patterning and scale shape, through mechanisms that involve an imbalanced bone remodeling. Gene expression analyses indicated that BaP induced the activation of xenobiotic and metabolic pathways, while negatively impacting extracellular matrix formation and organization. Interestingly, BaP exposure positively regulated inflammation markers in larvae and increased the recruitment of neutrophils. A direct interaction between neutrophils and bone extracellular matrix or bone forming cells was observed in vivo, suggesting a role for neutrophils in the mechanisms underlying BaP osteotoxicity. Our work provides novel data on the cellular and molecular players involved in BaP osteotoxicity and brings new insights into a possible role for neutrophils in inflammatory bone reduction.

Fish Models of Induced Osteoporosis.

Osteopenia and osteoporosis are bone disorders characterized by reduced bone mineral density (BMD), altered bone microarchitecture and increased bone fragility. Because of global aging, their incidence is rapidly increasing worldwide and novel treatments that would be more efficient at preventing disease progression and at reducing the risk of bone fractures are needed. Preclinical studies are today a major bottleneck to the collection of new data and the discovery of new drugs, since they are commonly based on rodent in vivo systems that are time consuming and expensive, or in vitro systems that do not exactly recapitulate the complexity of low BMD disorders. In this regard, teleost fish, in particular zebrafish and medaka, have recently emerged as suitable alternatives to study bone formation and mineralization and to model human bone disorders. In addition to the many technical advantages that allow faster and larger studies, the availability of several fish models that efficiently mimic human osteopenia and osteoporosis phenotypes has stimulated the interest of the academia and industry toward a better understanding of the mechanisms of pathogenesis but also toward the discovery of new bone anabolic or antiresorptive compounds. This mini review recapitulates the in vivo teleost fish systems available to study low BMD disorders and highlights their applications and the recent advances in the field.

Effect of Dietary Manganese and Zinc Levels on Growth and Bone Status of Senegalese Sole (Solea senegalensis) Post-Larvae.

Essential dietary trace elements, such as zinc (Zn) and manganese (Mn), critically influence a wide range of physiological, metabolic, and hormonal processes in fish larvae and post-larvae. Despite their importance for normal fish growth and skeletal development, trace mineral nutrition has not been extensively studied in the early stages of development of fish. Post-larvae of an emergent aquaculture species, Senegalese sole (Solea senegalensis), were the subject of this study in order to better understand the effects of diet supplementation of trace minerals upon fish larval development and performance. Sole post-larvae were fed a combination of organic Mn (45 and 90 mg kg-1 feed) and organic Zn (100 and 130 mg kg-1 feed) and survival, growth, mineral deposition rates, and vertebral bone status were assessed. Our results showed that although no significant effect was found on the growth performance of Senegalese sole post-larvae, Mn and Zn supplementation to a commercial microdiet for marine fish larvae at higher dietary levels (Mn at 90 mg kg-1 and Zn at 130 mg kg-1) improved larval survival, decreased the severity of vertebral malformations, and increased the deposition of Mn in bone.

New Insights on Vitamin K Metabolism in Senegalese sole (Solea senegalensis) Based on Ontogenetic and Tissue-Specific Vitamin K Epoxide Reductase Molecular Data.

Vitamin K (VK) is a key nutrient for several biological processes (e.g., blood clotting and bone metabolism). To fulfill VK nutritional requirements, VK action as an activator of pregnane X receptor (Pxr) signaling pathway, and as a co-factor of γ-glutamyl carboxylase enzyme, should be considered. In this regard, VK recycling through vitamin K epoxide reductases (Vkors) is essential and should be better understood. Here, the expression patterns of vitamin K epoxide reductase complex subunit 1 (vkorc1) and vkorc1 like 1 (vkorc1l1) were determined during the larval ontogeny of Senegalese sole (Solea senegalensis), and in early juveniles cultured under different physiological conditions. Full-length transcripts for ssvkorc1 and ssvkorc1l1 were determined and peptide sequences were found to be evolutionarily conserved. During larval development, expression of ssvkorc1 showed a slight increase during absence or low feed intake. Expression of ssvkorc1l1 continuously decreased until 24 h post-fertilization, and remained constant afterwards. Both ssvkors were ubiquitously expressed in adult tissues, and highest expression was found in liver for ssvkorc1, and ovary and brain for ssvkorc1l1. Expression of ssvkorc1 and ssvkorc1l1 was differentially regulated under physiological conditions related to fasting and re-feeding, but also under VK dietary supplementation and induced deficiency. The present work provides new and basic molecular clues evidencing how VK metabolism in marine fish is sensitive to nutritional and environmental conditions.