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- Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concernsPublication . Granadeiro, Luis; Dirks, Ron P.; Ortiz-Delgado, Juan B.; J. Gavaia, Paulo; Sarasquete, Carmen; Laizé, Vincent; Leonor Cancela, M.; I, FernándezWarfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly over-represented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.
- Coordinated regulation of chromatophore differentiation and melanogenesis during the ontogeny of skin pigmentation of Solea senegalensis (Kaup, 1858)Publication . Darias, Maria J.; Andree, Karl B.; Boglino, Anais; Fernandez, Ignacio; Estevez, Alicia; Gisbert, EnricAbnormal pigmentation of Senegalese sole has been described as one problem facing the full exploitation of its commercial production. To improve our understanding of flatfish pigmentation of this commercially important species we have evaluated eleven genes related to two different processes of pigmentation: melanophore differentiation, and melanin production. The temporal distribution of gene expression peaks corresponds well with changes in pigmentation patterns and the intensity of skin melanization. Several gene ratios were also examined to put in perspective possible genetic markers for the different stages of normal pigmentation development. Further, the phenotypic changes that occur during morphogenesis correspond well with the main transitions in gene expression that occur. Given the dramatic phenotypic alterations which flatfish undergo, including the asymmetric coloration that occurs between the ocular and the blind side, and the synchrony of the two processes of morphogenesis and pigmentation ontogenesis, these species constitute an interesting model for the study of pigmentation. In this study we present a first approximation towards explaining the genetic mechanisms for regulating pigmentation ontogeny in Senegalese sole, Solea senegalensis.
- The role of calcium concentration in the invasive capacity of Corbicula fluminea in crystalline basinsPublication . Ferreira-Rodriguez, Noe; I, Fernández; Varandas, Simone; Cortes, Rui; Cancela, M. Leonor; Pardo, IsabelThe natural variation of environmental factors in freshwater basins determines their biodiversity. Among them, calcium is a key physiological compound for freshwater invertebrates. It is required for shell formation, muscle contraction, it mediates gene expression and allows counteracting acidosis during stress periods, among other functions. Although the distribution of different freshwater species has been suggested to be linked with the environmental calcium concentration, as yet no research studies have confirmed this. Identifying whether environmental calcium concentrations might determine the invasion success of alien species would be critical in developing and implementing effective management strategies to control them. Here, a multidisciplinary approach integrating field surveys, analytical chemistry techniques, molecular biology analyses and a lab-scale experiment was taken to decipher whether the environmental calcium Concentration might hamper the establishment of Corbicula fluminea in northwestern Iberian rivers. A Principal Component Analysis on water chemistry variables from 13 water bodies identified environmental calcium concentration, among others, as one key factor that best characterized the distribution area of C. fluminea. The calcium content in animals' bodies from two representative rivers was dependent on the environmental calcium concentration of freshwater basins; the lower the concentration, the lower the body's content. The expression of stress- and calcium homeostasis-related genes was higher in C fluminea from low calcium concentration environments than in those from calcium-rich freshwater basins. Finally, under experimental conditions, lower water calcium concentrations decreased C fluminea growth rates. The present data suggest, for the first time, that environmental calcium concentration may act as a determinant factor on the invasion success of C. fluminea in freshwater environments. Our results provide new clues for the identification of basins with increased risk of potential invasion by C fluminea based on environmental calcium levels. (C) 2016 Elsevier B.V. All rights reserved.
- Circulating small non-coding RNAs provide new insights into vitamin K nutrition and reproductive physiology in teleost fishPublication . I, Fernández; Fernandes, Jorge M. O.; Roberto, Vânia; Kopp, Martina; Oliveira, Catarina; Riesco, Marta F.; Dias, Jorge; Cox, Cymon J.; Leonor Cancela, M.; Cabrita, Elsa; Gavaia, PauloBackground: Vitamin K (VK) is a fat-soluble vitamin known for its essential role in blood coagulation, but also on other biological processes (e.g. reproduction, brain and bone development) have been recently suggested. Nevertheless, the molecular mechanisms behind its particular function on reproduction are not yet fully understood. Methods: The potential role of VK on reproduction through nutritional supplementation in Senegalese sole (Solea senegalensis) was assessed by gonadal maturation and 11-ketosterone, testosterone and estriol plasma levels when fed with control or VK supplemented (1250 mg kg(-1) of VK,) diets along a six month trial. At the end, sperm production and quality (viability and DNA fragmentation) were evaluated. Circulating small non-coding RNAs (sncRNAs) in blood plasma from males were also studied through RNA-Seq. Results: Fish fed with dietary VK supplementation had increased testosterone levels and lower sperm DNA fragmentation. SncRNAs from blood plasma were found differentially expressed when nutritional and sperm quality conditions were compared. PiR-675//676//4794//5462 and piR-74614 were found up-regulated in males fed with dietary VK supplementation. Let-7g, let-7e(18nt), let-7a-1, let-7a-3//7a-2//7a-1, let-7e(23nt) and piR-675//676//4794//5462 were found to be up-regulated and miR-146a and miR-146a-1//146a-2//146a-3 down-regulated when fish with low and high sperm DNA fragmentation were compared. Bioinformatic analyses of predicted mRNAs targeted by sncRNAs revealed the potential underlying pathways. Conclusions: VK supplementation improves fish gonad maturation and sperm quality, suggesting an unexpected and complex regulation of the nutritional status and reproductive performance through circulating sncRNAs. General significance: The use of circulating sncRNAs as reliable and less-invasive physiological biomarkers in fish nutrition and reproduction has been unveiled.
- Vitamin A affects flatfish development in a thyroid hormone signaling and metamorphic stage dependent mannerPublication . Fernandez, Ignacio; Ortiz-Delgado, Juan B.; Darias, Maria J.; Hontoria, Francisco; Andree, Karl B.; Manchado, Manuel; Sarasquete, Carmen; Gisbert, EnricVitamin A (VA) and retinoid derivatives are known morphogens controlling vertebrate development. Despite the research effort conducted during the last decade, the precise mechanism of how VA induces post-natal bone changes, and particularly those operating through crosstalk with the thyroid hormones (THs) remain to be fully understood. Since effects and mechanisms seem to be dose and time-dependent, flatfish are an interesting study model as they undergo a characteristic process of metamorphosis driven by THs that can be followed by external appearance. Here, we studied the effects of VA imbalance that might determine Senegalese sole (Solea senegalensis) skeletogenetic phenotype through development of thyroid follicles, THs homeostasis and signaling when a dietary VA excess was specifically provided during pre-, pro-or post-metamorphic stages using enriched rotifers and Artemia as carriers. The increased VA content in enriched live prey was associated to a higher VA content in fish at all developmental stages. Dietary VA content clearly affected thyroid follicle development, T3 and T4 immunoreactive staining, skeletogenesis and mineralization in a dose and time-dependent fashion. Gene expression analysis showed that VA levels modified the mRNA abundance of VA- and TH-specific nuclear receptors at specific developmental stages. Present results provide new and key knowledge to better understand how VA and TH pathways interact at tissue, cellular and nuclear level at different developmental periods in Senegalese sole, unveiling how dietary modulation might determine juvenile phenotype and physiology.
- Normal and histopathological organization of the opercular bone and vertebrae in gilthead sea bream Sparus aurataPublication . Ortiz-Delgado, Juan B.; Fernández Monzón, Ignacio Valentín; Sarasquete, Carmen; Gisbert, EnricThis study provides a comprehensive description of the tissue organization of non-deformed and deformed opercula and vertebrae from gilthead sea bream Sparus aurata juveniles by means of histological, histochemical and immunohistochemical approaches. Two types of opercular anomalies are described: the folding of the opercle and subopercle into the gill chamber, starting at the upper corner of the branchial cleft and extending down to its lower third; and the partial lack of the operculum (opercle, subopercle, interopercle and preopercle underdeveloped) with a regression of the loose edge extending down to its lower third. Histological observations revealed a rare type of bone remodelling process in the opercular structure, which consisted of the coalescence of contacting bone tissues (presumably from the preopercle and opercle), resulting in skeletal tissue with a trabecular aspect filled by a single-cell epithelium of cubic osteoblastic-like cells. Differences in collagen fiber thickness and its 3-dimensional arrangement between normal and deformed opercula were also found. Lordotic vertebrae were characterized by the formation of fibrous cartilage in the haemal and/or neural sides, indicating that a metaplastic shift occurred during the process of lordosis. Another major histomorphological change found in lordotic vertebrae was the complete loss of notochordal sheath integrity. Histological alterations were coupled with an imbalance of cell death and cell proliferation processes in lordotic vertebrae as well as that of bone formation/resorption, and extracellular matrix deposition activity differences which might have resulted from the remodelling process occurring in lordotic vertebrae. Altogether, these results provide an increase in our basic knowledge of bone disorders that contribute to our understanding of the mechanisms by which these skeletal anomalies appear in this fish species and which hamper its production efficiency.