Marine Biotechnology

, Volume 16, Issue 5, pp 522–537 | Cite as

Dietary Supplementation with Vitamin K Affects Transcriptome and Proteome of Senegalese Sole, Improving Larval Performance and Quality

  • Nadège Richard
  • Ignacio Fernández
  • Tune Wulff
  • Kristin Hamre
  • Leonor Cancela
  • Luis E. C. Conceição
  • Paulo J. GavaiaEmail author
Original Article


Nutritional factors strongly influence fish larval development and skeletogenesis, and may induce skeletal deformities. Vitamin K (VK) has been largely disregarded in aquaculture nutrition, despite its important roles in bone metabolism, in γ-carboxylation of Gla proteins, and in regulating gene expression through the pregnane X receptor (Pxr). Since the mechanisms mediating VK effects over skeletal development are poorly known, we investigated the effects of VK-supplementation on skeletal development in Senegalese sole larvae, aiming to identify molecular pathways involved. Larvae were fed live preys enriched with graded levels of phylloquinone (PK) (0, 50, and 250 mg kg−1) and survival rate, growth, VK contents, calcium content and incidence of skeletal deformities were determined, revealing an improvement of larval performance and decreasing the incidence of deformities in VK-supplemented groups. Comparative proteome analysis revealed a number of differentially expressed proteins between Control and Diet 250 associated with key biological processes including skin, muscle, and bone development. Expression analysis showed that genes encoding proteins related to the VK cycle (ggcx, vkor), VK nuclear receptor (pxr), and VK-dependent proteins (VKDPs; oc1 and grp), were differentially expressed. This study highlights the potential benefits of increasing dietary VK levels in larval diets, and brings new insights on the mechanisms mediating the positive effects observed on larval performance and skeletal development.


Vitamin K Skeletal deformities Proteomics Gene expression Solea senegalensis Flatfish larvae 



NR and IF acknowledge financial support through post-doctoral grants SFRH/BDP/65578/2009 and SFRH/BDP/82049/2011 from Portuguese Foundation for Science and Technology (FCT). This work was funded by project PDTC/MAR/105152/2008 (SPECIAL_K) from FCT.


  1. Anguis V, Cañavate JP (2005) Spawning of captive Senegal sole (Solea senegalensis) under a naturally fluctuating temperature regime. Aquaculture 243:133–145CrossRefGoogle Scholar
  2. Asaduzzaman MD, Akolkar DB, Kinoshita S, Watabe S (2013) The expression of multiple myosin heavy chain genes during skeletal muscle development of torafugu Takifugu rubripes embryos and larvae. Gene 515:144–154CrossRefPubMedGoogle Scholar
  3. Atkins GJ, Welldon KJ, Wijenayaka AR, Bonewald LF, Findlay DM (2009) Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by {gamma}-carboxylation-dependent and -independent mechanisms. Am J Physiol Cell Physiol 297:C1358–C1367CrossRefPubMedGoogle Scholar
  4. Bæverfjord G, Åsgård T, Shearer KD (1996) Development and detection of phosphorus deficiency in Atlantic salmon, Salmo salar L., parr and post-smolts. Aquac Nutr 4:1–11CrossRefGoogle Scholar
  5. Berkner KL (2005) The vitamin K-dependent carboxylase. Annu Rev Nutr 25:127–149CrossRefPubMedGoogle Scholar
  6. Berkner KL (2008) Vitamin K–dependent carboxylation. Vitam Horm 78:131–156CrossRefPubMedGoogle Scholar
  7. Boglione C, Gavaia P, Koumoundouros G, Gisbert E, Moren M, Fontagné S, Witten PE (2013a) A review on skeletal anomalies in reared European larvae and juveniles. Part 1: normal and anomalous skeletogenic processes. Rev Aquac 5:S99–S120CrossRefGoogle Scholar
  8. Boglione C, Gisbert E, Gavaia P, Witten PE, Moren M, Fontagné S, Koumoundouros G (2013b) A review on skeletal anomalies in reared European larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev Aquac 5:S121–S167CrossRefGoogle Scholar
  9. Brenner B, Kuperman A, Watzka M, Oldenburg J (2009) Vitamin K-dependent coagulation factors deficiency. Semin Thromb Hemost 35:439–446CrossRefPubMedGoogle Scholar
  10. Burns K, Duggan B, Atkinson EA, Famulski KS, Nemer M, Bleackley RC, Michalak M (1994) Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature 367:476–480CrossRefPubMedGoogle Scholar
  11. Campinho M, Silva N, Sweeney GE, Power DM (2007) Molecular, cellular and histological changes in skin from a larval to an adult phenotype during bony fish metamorphosis. Cell Tissue Res 327:267–284CrossRefPubMedGoogle Scholar
  12. Cancela ML, Conceição N, Laizé V (2012) Gla-rich protein, a new player in tissue calcification? Adv Nutr 3:174–181PubMedCentralCrossRefPubMedGoogle Scholar
  13. Chatrou ML, Winckers K, Hackeng TM, Reutelingsperger CP, Schurgers LJ (2012) Vascular calcification: the price to pay for anticoagulation therapy with vitamin K-antagonists. Blood Rev 26:155–166CrossRefPubMedGoogle Scholar
  14. Danziger J (2008) Vitamin K-dependent proteins, warfarin, and vascular calcification. Clin J Am Soc Nephrol 3:1504–1510PubMedCentralCrossRefPubMedGoogle Scholar
  15. Darias MJ, Mazurais D, Koumoundouros G, Glynatsi N, Christodoulopoulou S, Huelva C, Desbruyeres E, Le Gall MM, Quazuguel P, Cahu CL, Zambonino-Infante JL (2010) Dietary vitamin D3 affects digestive system ontogenesis and ossification in European sea bass (Dicentrachus labrax, Linnaeus, 1758). Aquaculture 298:300–307CrossRefGoogle Scholar
  16. De Boer-van den Berg MA, Verstijnen CP, Vermeer C (1986) Vitamin K-dependent carboxylase in skin. J Investig Dermatol 87:377–380CrossRefPubMedGoogle Scholar
  17. Dedhar S, Rennie PS, Shago M, Hagesteijn CY, Yang H, Filmus J, Hawley RG, Bruchovsky N, Cheng H, Matusik RJ, Giguere V (1994) Inhibition of nuclear hormone receptor activity by calreticulin. Nature 367:480–483CrossRefPubMedGoogle Scholar
  18. Derveax S, Vandesompele J, Hellemans J (2010) How to do successful gene expression analysis using real-time PCR. Methods 50:227–230CrossRefGoogle Scholar
  19. Dinis MT, Ribeiro L, Soares F, Sarasquete C (1999) A review on the cultivation potential of Solea senegalensis in Spain and in Portugal. Aquaculture 176:27–38CrossRefGoogle Scholar
  20. Engrola S, Figueira L, Conceição LEC, Gavaia PJ, Ribeiro L, Dinis MT (2009) Co-feeding in Senegalese sole larvae with inert diet from mouth opening promotes growth at weaning. Aquaculture 288:264–272CrossRefGoogle Scholar
  21. Ferland G (2012) Vitamin K and the nervous system: an overview of its actions. Adv Nutr 3:204–212PubMedCentralCrossRefPubMedGoogle Scholar
  22. Fernández I, Gisbert E (2010) Senegalese sole bone tissue originated from chondral ossification is more sensitive than dermal bone to high vitamin A content in enriched Artemia. J Appl Ichthyol 26:344–349CrossRefGoogle Scholar
  23. Fernández I, Gisbert E (2011) The effect of vitamin A on flatfish development and skeletogenesis: a review. Aquaculture 315:34–48CrossRefGoogle Scholar
  24. Fernández I, Hontoria F, Ortiz-Delgado JB, Kotzamanis Y, Estevez A, Zambonino-Infante JL, Gisbert E (2008) Larval performance and skeletal deformities in farmed gilthead sea bream (Sparus aurata) fed with graded levels of vitamin A enriched rotifers (Brachionus plicatilis). Aquaculture 283:102–115CrossRefGoogle Scholar
  25. Fernández I, Pimentel MS, Ortiz-Delgado JB, Hontoria F, Sarasquette C, Estévez A, Zambonino-Infante JL, Gisbert E (2009) Effect of dietary vitamin A on Senegalese sole (Solea senegalensis) skeletogenesis and larval quality. Aquaculture 295:250–265CrossRefGoogle Scholar
  26. Fernández-Díaz C, Kopecka J, Cañavate JP, Sarasquete C, Solé M (2006) Variations on development and stress defences in Solea senegalensis larvae fed on live and microencapsulated diets. Aquaculture 251:573–584CrossRefGoogle Scholar
  27. Figueiredo MA, Mareco EA, Silva MDP, Marins LF (2012) Muscle-specific growth hormone receptor (GHR). Transgenic Res 21:457–469CrossRefPubMedGoogle Scholar
  28. Gavaia PJ, Sarasquete MC, Cancela ML (2000) Detection of mineralized structures in very early stages of development of marine teleostei using a modified Alcian blue-Alizarin red double staining technique for bone and cartilage. Biotech Histochem 75:79–84CrossRefPubMedGoogle Scholar
  29. Gavaia PJ, Dinis MT, Cancela ML (2002) Osteological development and abnormalities of the vertebral column and caudal skeleton in larval and juvenile stages of hatchery-reared Senegal sole (Solea senegalensis). Aquaculture 211:305–323CrossRefGoogle Scholar
  30. Gavaia PJ, Simes DC, Viegas CSB, Pinto J, Ortiz-Delgado JB, Kelsh RN, Sarasquete MC, Cancela ML (2006) Osteocalcin and Matrix Gla Protein in zebrafish (Danio rerio) and Senegal sole (Solea senegalensis): comparative gene and protein expression during larval development through adulthood. Gene Expr Patterns 6:637–652CrossRefPubMedGoogle Scholar
  31. Gisbert E, Ortiz-Delgado JB, Sarasquete C (2008) Nutritional cellular biomarkers in early life stages of fish. Histol Histopathol 23:1525–1539PubMedGoogle Scholar
  32. Graff E, Waagbø R, Fivelstad S, Vermeer C, Lie Ø, Lundebye AK (2002) A multivariate study on the effects of dietary vitamin K, vitamin D3 and calcium, and dissolved carbon dioxide on growth, bone minerals, vitamin status and health performance in smolting Atlantic salmon Salmo salar L. J Fish Dis 25:599–614CrossRefGoogle Scholar
  33. Graff IE, Krossøy C, Gjerdevik K, Julshamn K (2010) Influence of dietary menadione nicotinamide bisulphite (vitamin K-3) and phylloquinone (vitamin K-1) on Atlantic salmon (Salmo salar L.) tissue levels, determined by high-performance liquid chromatography with fluorescence detection. Aquac Nutr 16:637–647CrossRefGoogle Scholar
  34. Guderley H, Leroy PH, Gagné A (2001) Thermal acclimatation, growth and burst swimming of threespine stickleback: enzymatic correlates and influence of photoperiod. Physiol Biochem Zool 74:66–74CrossRefPubMedGoogle Scholar
  35. Hanumanthaiah R, Thankavel B, Day K, Gregory M, Jagadeeswaran P (2001) Developmental expression of vitamin K-dependent gamma-carboxylase activity in zebrafish embryos: effect of warfarin. Blood Cell Mol Dis 27:992–999Google Scholar
  36. Hevrøy EM, Jordal A-EO, Hordvik I, Espe M, Hemre G-I, Olsvik PA (2006) Myosin heavy chain mRNA expression correlates higher with muscle protein accretion than growth in Atlantic salmon, Salmo salar. Aquaculture 252:453–461CrossRefGoogle Scholar
  37. Hjernø K, Højrup P (2004) PeakErazor: a windows-based program for improving peptide mass searches. In: Kamp RM, Calvete JJ, Choli-Papadopoulou T (eds) Methods in proteome and protein analysis. Springer, Berlin, pp 359–370CrossRefGoogle Scholar
  38. Ichikawa T, Horie-Inoue K, Ikeda K, Blumberg B, Inoue S (2006) Steroid and xenobiotic receptor SXR mediates vitamin K2-activated transcription of extracellular matrix-related genes and collagen accumulation in osteoblastic cells. J Biol Chem 281:16927–16934CrossRefPubMedGoogle Scholar
  39. Igarashi M, Yogiashi Y, Mihara M, Takada I, Kitagawa H, Kato S (2007) Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene. Mol Cell Biol 27(22):7947–7954PubMedCentralCrossRefPubMedGoogle Scholar
  40. Imsland AK, Foss A, Conceição LEC, Dinis MT, Delbare D, Schram E, Kamstra A, Rema P, White P (2003) A review of the culture potential of Solea solea and S. senegalensis. Rev Fish Biol Fish 13:379–407CrossRefGoogle Scholar
  41. Infante C, Manchado M, Asensio E, Cañate JP (2007) Molecular characterization, gene expression and dependence on thyroid hormones of two type I keratin genes (sseKer1 and sseKer2) in the flatfish Senegalese sole (Solea senegalensis Kaup). BMC Dev Biol 7:118–133PubMedCentralCrossRefPubMedGoogle Scholar
  42. Infante C, Matsuoka MP, Asensio E, Cañavate JP, Reith M, Manchado M (2008) Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR. BMC Mol Biol 9:28–40PubMedCentralCrossRefPubMedGoogle Scholar
  43. Infante C, Ponce M, Asensio E, Zerolo R, Manchado M (2011) Molecular characterization of a novel type II keratin gene (sseKer3) in the Senegalese sole (Solea senegalensis): differential expression of keratin genes by salinity. Comp Biochem Physiol 160B:15–23CrossRefGoogle Scholar
  44. Johnston IA, Lee H-T, Macqueen DJ, Paranthaman K, Kawashima C, Anwar A, Kinghorn JR, Dalmay T (2009) Embryonin temperature affects muscle fibre recruitment in adult zebrafish: genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes. J Exp Biol 212:1781–1793CrossRefPubMedGoogle Scholar
  45. Karsenty G, Ferron M (2012) The contribution of bone to whole-organism physiology. Nature 481:314–320CrossRefPubMedGoogle Scholar
  46. Krossøy C, Waagbø R, Fjelldal P-G, Wargelius A, Lock E-J, Graff IE, Ørnsrund R (2009) Dietary menadione nicotinamide bisulphite (vitamin K3) does not affect growth or bone health in first-feeding fry of Atlantic salmon (Salmo salar L.). Aquac Nutr 15:638–649CrossRefGoogle Scholar
  47. Krossøy C, Lock E-J, Ørnsrud R (2010) Vitamin K-dependent γ-glutamylcarboxylase in Atlantic salmon (Salmo salar L.). Fish Physiol Biochem 36:627–635CrossRefPubMedGoogle Scholar
  48. Krossøy C, Waagbø R, Ørnsrund R (2011) Vitamin K in fish nutrition. Aquac Nutr 17:585–594CrossRefGoogle Scholar
  49. Lall SP, Lewis-McCrea L (2007) Role of nutrients in skeletal metabolism and pathology in fish—an overview. Aquaculture 267:3–19CrossRefGoogle Scholar
  50. Liang C-S, Ikeda D, Kinishita S, Shimizu A, Sasaki T, Asakawa S, Shimizu N, Watabe S (2008) Myocyte enhancer factor 2 regulates expression of medaka Oryzias latipes fast skeletal myosin heavy chain genes in a temperature-dependent manner. Gene 407:42–53CrossRefPubMedGoogle Scholar
  51. Neacsu CD, Grosch M, Tejada M, Winterpacht A, Paulsson M, Wagener R, Tagariello A (2011) Ucmaa (Grp-2) is required for zebrafish skeletal development. Evidence for a functional role of its glutamate γ-carboxylation. Matrix Biol 30:369–378CrossRefPubMedGoogle Scholar
  52. Nihei Y, Kobiyama A, Ikeda D, Ono Y, Ohara S, Cole NJ, Johnston IA, Watabe S (2006) Molecular cloning and mRNA expression analysis of carp embryonic, slow and cardiac myosin heavy chain isoforms. J Exp Biol 209:188–198CrossRefPubMedGoogle Scholar
  53. Oldenburg J, Marivona M, Müller-Reible C, Waltzka M (2008) The vitamin K cycle. Vitam Horm 78:35–62CrossRefPubMedGoogle Scholar
  54. Ostermeyer U, Schmidt T (2001) Determination of vitamin K in the edible part of fish by high-performance liquid chromatography. Eur Food Res Technol 212:518–528CrossRefGoogle Scholar
  55. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: Bestkeeper-Excell-based tool using pairwise correlations. Biotechnol Lett 26:509–515CrossRefPubMedGoogle Scholar
  56. Pinto JP, Ohresser M, Cancela ML (2001) Cloning of the bone Gla protein gene from the teleost fish Sparus aurata. Evidence for overall conservation in molecular structure and pattern of expression from fish to man. Gene 270:77–91CrossRefPubMedGoogle Scholar
  57. Ribeiro L, Sarasquete C, Dinis MT (1999) Histological and histochemical development of the digestive system of Solea senegalensis (Kaup, 1858) larvae. Aquaculture 171:293–308CrossRefGoogle Scholar
  58. Roy PK, Lall SP (2007) Vitamin K deficiency inhibits mineralization and enhances deformity in vertebrae of haddock (Melanogrammus aeglefinus L.). Comp Biochem Physiol 148B:174–183CrossRefGoogle Scholar
  59. Saad FA, Hofstaetter JG (2011) Proteomic analysis of mineralising osteoblasts identifies novel genes related to bone matrix mineralisation. Int Orthop 35:447–451PubMedCentralCrossRefPubMedGoogle Scholar
  60. Shago M, Flock G, Hagesteijn C-YL, Woodside M, Grinstein S, Giguère V, Dedhar S (1997) Modulation of the retinoic acid and retinoid X receptor signaling pathways in P19 embryonal carcinoma cells by calreticulin. Exp Cell Res 230:50–60CrossRefPubMedGoogle Scholar
  61. Stafford DW (2005) The vitamin K cycle. J Thromb Hemost 3:1873–1878CrossRefGoogle Scholar
  62. Suhara Y, Wada A, Tachibana Y, Watanabe M, Nakamura K, Nakagawa K, Okano T (2010) Structure-activity relationships in the conversion of vitamin K analogues into menaquinone-4. Substrates essential to the synthesis of menaquinone-4 in cultured human cell lines. Bioorg Med Chem 18:3116–3124CrossRefPubMedGoogle Scholar
  63. Sveinsdóttir H, Vilhelmsson O, Gudmundsdóttir Á (2008) Proteome analysis of abundant proteins in two age groups of early Atlantic cod (Gadus morhua) larvae. Comp Biochem Physiol 3D:243–250Google Scholar
  64. Tabb MM, Sun A, Zhou C, Grün F, Errandi J, Romero K, Pham H, Inoue S, Mallick S, Lin M, Forman BM, Blumberg B (2003) Vitamin K2 regulation of bone homeostasis is mediated by the steroid and xenobiotic receptor SXR. J Biol Chem 278:43919–43927CrossRefPubMedGoogle Scholar
  65. Theuwissen E, Smit E, Vermeer C (2012) The role of vitamin K in soft-tissue calcification. Adv Nutr 3:166–173PubMedCentralCrossRefPubMedGoogle Scholar
  66. Udagawa M (2001) The effect of dietary vitamin K (phylloquinone and menadione) levels on the vertebral formation in mummichog Fundulus heteroclitus. Fish Sci 67:104–109CrossRefGoogle Scholar
  67. Udagawa M (2004) The effect of parental vitamin K deficiency on bone structure in mummichog Fundulus heteroclitus. J World Aquacult Soc 35:366–371CrossRefGoogle Scholar
  68. Udagawa M, Nakazoe J, Murai T (1993) Tissue distribution of phylloquinone and menaquinone-4 in sardine, Sardinops melanostictus. Comp Biochem Physiol 106B:297–301Google Scholar
  69. van Deursen JV, Ruitenbeek W, Heerschap A, Jap P, ter Laak H (1994) Creatine kinase (CK) in skeletal muscle energy metabolism: a study of mouse mutants with graded reduction in muscle CK expression. Proc Natl Acad Sci U S A 91:9091–9095PubMedCentralCrossRefPubMedGoogle Scholar
  70. Viegas CSB, Simes DC, Laizé V, Williamson MK, Price PA, Cancela ML (2008) Gla-rich protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates. J Biol Chem 283:36655–36664PubMedCentralCrossRefPubMedGoogle Scholar
  71. Yamaguchi M, Weitzmann M (2011) Vitamin K2 stimulates osteoblastogenesis and suppresses osteoclastogenesis by suppressing NF-κB activation. Int J Mol Med 27:3–14PubMedGoogle Scholar
  72. Zhang H, Lu W, Zhao Y, Rong P, Cao R, Gu W, Xiao J, Miao D, Lappe J, Recker R, Xiao GG (2011) Adipocytes derived from human bone marrow mesenchymal stem cells exert inhibitory effects on osteoblastogenesis. Curr Mol Med 11:489–502CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Nadège Richard
    • 1
  • Ignacio Fernández
    • 1
  • Tune Wulff
    • 2
  • Kristin Hamre
    • 3
  • Leonor Cancela
    • 1
    • 4
  • Luis E. C. Conceição
    • 1
    • 5
  • Paulo J. Gavaia
    • 1
    Email author
  1. 1.CCMAR-CIMAR L.A., Centro de Ciências do Mar do AlgarveUniversidade do AlgarveFaroPortugal
  2. 2.Division of Seafood ResearchDTU FoodKgs. LyngbyDenmark
  3. 3.NIFES, National Institute of Nutrition and Seafood ResearchBergenNorway
  4. 4.Department of Biomedical Sciences and MedicineUniversity of AlgarveFaroPortugal
  5. 5.SPAROS Lda, CRIA - Universidade do AlgarveFaroPortugal

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