Regulation of stanniocalcin secretion by calcium and PTHrP in Gilthead Seabream (Sparus aurata)

Calcium balance is of paramount importance for vertebrates. In fish, the endocrine modulators of calcium homeostasis include the stanniocalcin (STC), and some members of the parathyroid hormone (PTH) family, such as the PTH-related protein (PTHrP), acting as antagonists. STC is ubiquitously expressed in higher vertebrates. In turn, bony fish exhibit specific STC-producing glands named the corpuscles of Stannius (CS). Previous studies pointed to a calcium-sensing receptor (CaSR) involvement in the secretion of STC, but little is known of the involvement of other putative regulators. The CS provides a unique model to deepen the study of STC secretion. We developed an ex vivo assay to culture CS of fish and a competitive ELISA method to measure STC concentrations. As expected, STC released from the CS responds to CaSR stimulation by calcium, calcimimetics, and calcilytic drugs. Moreover, we uncover the presence (by PCR) of two PTHrP receptors in the CS, e.g., PTH1R and PTH3R. Thus, ex vivo incubations revealed a dose-response inhibition of STC secretion in response to PTHrP at basal Ca2+ concentrations. This inhibition is achieved through specific and reversible second messenger pathways (transmembrane adenylyl cyclases and phospholipase C), as the use of specific inhibitors highlights. Together, these results provide evidence for endocrine modulation between two antagonist hormones, STC and PTHrP.info:eu-repo/semantics/publishedVersio

Biometry and plasmatic stress-related parameters in brill (Scophthalmus rhombus)cultured at different stocking densities.

The effects of the stocking density on the physiological stress and biometric features of the brill were studied. Fish (491&plusmn;20 g) were cultured at three different stocking densities: 1; 5 and 15 Kg m-2 (LSD, MSD and HSD) during 5 weeks. Survival and several biometric, feeding and plasmatic parameters were assessed. Although final weight and specific growth rate decreased in higher densities, there were not significant differences between MSD and HSD. Differences for survival rate, feed efficiency, conversion index and feed intake were not detected among treatments. The minimum HSI was found in the HSD treatment, and condition factor varied inversely regards to stocking density. Plasma cortisol and osmolality were directly related to stocking density though the former was not significantly different among treatments. Plasma lactate and glucose significantly increased while stocking density rose. Nevertheless, free fatty acids did not vary among treatments, and triglycerides only decreased in LSD. This work was supported by Interreg Project 0251_ECOAQUA_5_E, financed by the EDRF (European Regional Development Fund).www.juntadeandalucia.es/agriculturaypesca/ifapa/ecoaqua. &nbsp;Se estudiaron los efectos de la densidad de cultivo sobre el estr&eacute;s fisiol&oacute;gico y par&aacute;metros biom&eacute;tricos en el parracho. Los peces (491&plusmn;20 g) fueron cultivados a tres densidades diferentes: 1; 5 y 15 Kg m-2 (LSD, MSD y HSD) durante 5 semanas. El peso final y crecimiento decrecieron con la densidad, pero no hubo diferencias significativas entre MSD y HSD. No se detectaron diferencias significativas entre tratamientos para supervivencia, eficiencia alimentaria, &iacute;ndice de conversi&oacute;n y tasa de ingesti&oacute;n. El HIS m&iacute;nimo fue para la HSD y el factor de condici&oacute;n vari&oacute; inversamente a la densidad. La osmolalidad y el cortisol, glucosa y lactato plasm&aacute;ticos estuvieron directamente relacionados con la densidad. Sin embargo, los &aacute;cidos grasos libres no variaron entre tratamientos. Este trabajo fue apoyado por el proyecto Interreg 0251_ECOAQUA_5_E , financiado por el FEDER (Fondo Europeo de Desarrollo Regional).www.juntadeandalucia.es/agriculturaypesca/ifapa/ecoaqua.</p

SURVIVAL OF ATLANTIC BLUEFIN TUNA (THUNNUS THYNNUS) LARVAE HATCHED AT DIFFERENT PH AND SALINITY CONDITIONS

In this study, we assessed the effect of pH and salinity as independent factors on larval survival (LS) of Atlantic bluefin tuna (ABFT –Thunnus thynnus) together with their Na+/K+-ATPase and V-type H+-ATPase activities. Fertilized eggs of ABFT were obtained on 25 June 2016 from a spontaneous spawning of broodstock in the farming facilities at El Gorguel (Cartagena, SE Spain) of Caladeros del Mediterráneo Company. The fertilized eggs were transferred to facilities of the Spanish Institute of Oceanography (IEO) in Mazarrón (SE Spain). In a first experiment, eggs (n = 150 per treatment, in 3 replicates) were exposed to sea water salinity (SW: 38 ppt) and four pH treatments until hatch was completed (44 hours at 23 ºC): 8.0 (control), 7.7 (near future), 7.5 (far future) and 7.3 (lower). In a second experiment eggs (n = 150 per treatment, in 3 replicates) were exposed to eleven salinities treatments and constant pH 8.0 (control) until hatch was completed (44 hours at 23 ºC): 27 , 30 , 33 , 36 , 37 , 38 (control), 39 , 40 , 43 , 46 and 49 ppt. No significant differences in LS were observed with pH treatment, but lower H+-ATPase activity was detected at control environmental pH (pH 8.0). A ‘‘U-shaped’’ relationship was observed between hatching salinity and both Na+/K+-ATPase and H+-ATPase activities in whole larvae hatched, increasing both activities in groups exposed to extreme salinities. However, LS showed an inverse “U shape” curve respect to environmental salinity with higher values at intermediate salinities and lower LS at extreme salinities. These results suggest higher survival rates with lower active pumps activities. Survival results are discussed in terms of osmoregulatory cost adapting to a pH and salinity predicted for the near future scenarios. This work was funding by the European Union’s Horizon 2020research and innovation programme under Grant Agreement No. 678193

Characterization of the peripheral thyroid system of gilthead seabream acclimated to different ambient salinities

Thyroid hormones are involved in many developmental and physiological processes, including osmoregulation. The regulation of the thyroid system by environmental salinity in the euryhaline gilthead seabream (Sparus aurata) is still poorly characterized. To this end seabreams were exposed to four different environmental salinities (5, 15, 40 and 55 ppt) for 14 days, and plasma free thyroid hormones (fT3, ff4), outer ring deiodination and Na+/K+ -ATPase activities in gills and kidney, as well as other osmoregulatory and metabolic parameters were measured. Low salinity conditions (5 ppt) elicited a significant increase in fT3 (29%) and ff4 (184%) plasma concentrations compared to control animals (acclimated to 40 ppt, natural salinity conditions in the Bay of Cadiz, Spain), while the amount of pituitary thyroid stimulating hormone subunit 13 (tshb) transcript abundance remained unchanged. In addition, plasma fT4 levels were positively correlated to renal and branchial deiodinase type 2 (dio2) mRNA expression. Gill and kidney T4-outer ring deiodination activities correlated positively with dio2 mRNA expression and the highest values were observed in fish acclimated to low salinities (5 and 15 ppt). The high salinity (55 ppt) exposure caused a significant increase in tshb expression (65%), but deiodinase gene expression (diol and dio2) and activity did not change and were similar to controls (40 ppt). In conclusion, acclimation to different salinities led to changes in the peripheral regulation of thyroid hormone metabolism in seabream. Therefore, thyroid hormones are involved in the regulation of ion transport and osmoregulatory physiology in this species. The conclusions derived from this study may also allow aquaculturists to modulate thyroid metabolism in seabream by adjusting culture salinity. (C) 2016 Elsevier Inc. All rights reserved.Socrates/Erasmus Grant from the European UnionUniversity of Cadiz [UCA 2009-074-FPI]Ministerio de Education y Ciencia, Spain [AGL2007-61211/ACU]FEDER, Spain [AGL2007-61211/ACU]Proyecto de Excelencia (Junta de Andalucia) [PO7-RNM-02843]Science Foundation (FCT) of Portugal [SFRH/BPD/89889/2012, SFRH/BPD/84033/2012]info:eu-repo/semantics/acceptedVersio

Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds

In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg2+ and Mn2+, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis

Does Mutational Robustness Inhibit Extinction by Lethal Mutagenesis in Viral Populations?

Lethal mutagenesis is a promising new antiviral therapy that kills a virus by raising its mutation rate. One potential shortcoming of lethal mutagenesis is that viruses may resist the treatment by evolving genomes with increased robustness to mutations. Here, we investigate to what extent mutational robustness can inhibit extinction by lethal mutagenesis in viruses, using both simple toy models and more biophysically realistic models based on RNA secondary-structure folding. We show that although the evolution of greater robustness may be promoted by increasing the mutation rate of a viral population, such evolution is unlikely to greatly increase the mutation rate required for certain extinction. Using an analytic multi-type branching process model, we investigate whether the evolution of robustness can be relevant on the time scales on which extinction takes place. We find that the evolution of robustness matters only when initial viral population sizes are small and deleterious mutation rates are only slightly above the level at which extinction can occur. The stochastic calculations are in good agreement with simulations of self-replicating RNA sequences that have to fold into a specific secondary structure to reproduce. We conclude that the evolution of mutational robustness is in most cases unlikely to prevent the extinction of viruses by lethal mutagenesis

A Multi-Step Process of Viral Adaptation to a Mutagenic Nucleoside Analogue by Modulation of Transition Types Leads to Extinction-Escape

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin —by avoiding the biased repertoire of transition mutations produced by this purine analogue—and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure

What can we learn from glucocorticoid administration in fish? Effects of cortisol and dexamethasone on intermediary metabolism of gilthead seabream (Sparus aurata L.)

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