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  • Effect of acute copper sulfate exposure on olfactory responses to amino acids and pheromones in goldfish (Carassius auratus)
    Publication . Kolmakov, Nikolai N.; Hubbard, Peter; Lopes, Orlando; Canario, Adelino V. M.
    Exposure of olfactory epithelium to environmentally relevant concentrations of copper disrupts olfaction in fish. To examine the dynamics of recovery at both functional and morphological levels after acute copper exposure, unilateral exposure of goldfish olfactory epithelia to 100 μM CuSO4 (10 min) was followed by electro-olfactogram (EOG) recording and scanning electron microscopy. Sensitivity to amino acids (L-arginine and L-serine), generally considered food-related odorants, recovered most rapidly (three days), followed by that to catecholamines(3-O-methoxytyramine),bileacids(taurolithocholic acid) and the steroid pheromone, 17,20 -dihydroxy-4-pregnen- 3-one 20-sulfate, which took 28 days to reach full recovery. Sensitivity to the postovulatory pheromone prostaglandin F2R had not fully recovered even at 28 days. These changes in sensitivity were correlated with changes in the recovery of ciliated and microvillous receptor cell types. Microvillous cells appeared largely unaffected by CuSO4 treatment. Cilia in ciliated receptor neurones, however, appeared damaged one day post-treatment and were virtually absent after three days but had begun to recover after 14 days. Together, these results support the hypothesis that microvillous receptor neurones detect amino acids whereas ciliated receptor neurones were not functional and are responsible for detection of social stimuli (bile acidsandpheromones).Furthermore, differences in sensitivity to copper may be due to different transduction pathways in the different cell types.
  • Water cortisol and testosterone in Nile tilapia (Oreochromis niloticus) recirculating aquaculture systems
    Publication . Mota, Vasco; Martins, Catarina I. M.; Eding, Ep H.; Canario, Adelino; Verreth, Johan A. J.
    The accumulation of steroids released by fish in recirculating aquaculture systems (RAS) may potentially influence their physiology and behavior. The present study examined the release rate of cortisol and testosterone by Nile tilapia, Oreochromis niloticus, and their accumulation in six identical lab scale RAS operated at different water exchange rates (150 L/kg feed/day, (LowRAS) and 1500 L/kg feed/day, (HighRAS)) and how steroid accumulation is affected by grading and weighing induced stress. Water cortisol and testosterone concentrations during the experimental period ranged between 1.0 and 5.1 ng/L and between 1.4 and 9.4 ng/L, respectively. Water cortisol concentration was 34% and 43% significantly higher in LowRAS when compared with HighRAS at week 3 and week 4, whereas for water testosterone concentration the two treatments did not differ. Overall steroid release rates were 0.02 +/- 0.02 and 0.04 +/- 0.02 ng/g body weight/h for cortisol and testosterone, respectively. Two hours after grading and weighing water cortisol concentration increased 30% in the LowRAS whereas water testosterone concentration remained unchanged. Calculated cortisol and testosterone discharge from the system were, respectively, 87% and 89% lower in LowRAS than in HighRAS. These findings show that reduced water usage and acute stressors can induce significant accumulation of cortisol in the rearing water at levels close to olfactory detection. Accumulation of metabolites, in particular steroids, should be taken in consideration when designing and managing RAS to prevent exceeding allowable concentrations. Statement of relevance: RAS containing Nile tilapia, cortisol and testosterone are released to the culture water. Water cortisol concentration depends on the water exchange rate that is used and higher concentrations are expected in lower water exchange rates. Fish grading and weighing has a short-term effect on the water cortisol concentrations, after which concentrations return to the basal levels. These findings show that reduction in water usage and fish handling may lead to a significant increase of hormones in the rearing water, which emphasizes the importance of accounting for steroids in the design and management of RAS. (C) 2016 Elsevier B.V. All rights reserved.
  • Evidence that olfactory sensitivities to calcium and sodium are mediated by different mechanisms in the goldfish Carassius auratus
    Publication . Hubbard, Peter; Canario, Adelino V. M.
    The current study investigated whether the olfactory sensitivity to Ca2+ and Na+ is mediated by the same mechanism in the goldfish, a freshwater teleost. The olfactory responses, as assessed by recording the electro-encephalogram (EEG) from the olfactory bulb, to changes in external [Ca2+] and to [Na+] were recorded in the absence and presence of increasing concentrations of the other ion. Low concentrations of Na+ (0–1.0 mM) had no significant effects on the olfactory response to changes in [Ca2+] in terms of EC50, Imax or Hill co-efficient (n = 8). A relatively high concentration of Na+ (10 mM) significantly reduced the Imax and increased the EC50. One hundred millimolars Na+reduced the olfactory response to Ca2+ to undetectable levels. Conversely, low concentrations of Ca2+ (0.1 and 1.0 mM) significantly attenuated the olfactory response to changes in environmental [Na+], reducing the amplitude of response and increasing the threshold of detection (n = 7). However, a high concentration of Ca2+ (10 mM) failed to attenuate the olfactory response to Na+ completely. Taken together, these results suggest that, at normal environmental concentrations of these ions, Ca2+ and Na+ are detected by distinct and separate cellular mechanisms. However, there seems to be a degree of overlap between the two mechanisms. The exact mechanisms involved, and their biological roles, remain to be established.
  • Toll-like receptor evolution: does temperature matter?
    Publication . Sousa, Carmen; Fernandes, Stefan A.; Cardoso, João; Wang, Ying; Zhai, Wanying; Guerreiro, Pedro; Chen, Liangbiao; Canario, A.V.M.; Power, Deborah
    Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns (PAMPs) and are an ancient and well-conserved group of pattern recognition receptors (PRRs). The isolation of the Antarctic continent and its unique teleost fish and microbiota prompted the present investigation into Tlr evolution. Gene homologues of tlr members in teleosts from temperate regions were present in the genome of Antarctic Nototheniidae and the non-Antarctic sister lineage Bovichtidae. Overall, in Nototheniidae apart from D. mawsoni, no major tlr gene family expansion or contraction occurred. Instead, lineage and species-specific changes in the ectodomain and LRR of Tlrs occurred, particularly in the Tlr11 superfamily that is well represented in fish. Positive selective pressure and associated sequence modifications in the TLR ectodomain and within the leucine-rich repeats (LRR), important for pathogen recognition, occurred in Tlr5, Tlr8, Tlr13, Tlr21, Tlr22, and Tlr23 presumably associated with the unique Antarctic microbiota. Exposure to lipopolysaccharide (Escherichia coli O111:B4) Gram negative bacteria did not modify tlr gene expression in N. rossii head-kidney or anterior intestine, although increased water temperature (+4 degrees C) had a significant effect.
  • In vitro metabolism of pregnenolone to 7 alpha-hydroxypregnenolone by rainbow trout embryos
    Publication . Petkam, R.; Renaud, R. L.; Freitas, A. M. M. S.; Canario, Adelino V. M.; Raeside, J. I.; Kime, D. E.; Leatherland, J. F.
    Tissues taken from rainbow trout embryos at several developmental stages, were incubated in the presence of radioactively-labelled pregnenolone in order to determine the capability of salmonid embryos to metabolize steroids, such as pregnenolone, that are incorporated into the oocyte during gonadal growth and maturation. High performance liquid chromatography was used to separate the steroid products, and gas chromatography–mass spectrometry was applied for the chemical identification of the product. 7α-Hydroxypregnenolone, previously known to be produced only by ovarian tissues, was found to be the sole metabolite of pregnenolone metabolism by rainbow trout embryos. Sulfate and glucuronide conjugated forms of 7α-hydroxypregnenolone were also produced. We hypothesize that this metabolite provides a pathway for excretion of pregnenolone, enabling the embryo to maintain its own steroid milieu, although the possibility of 7α-hydroxypregnenolone also playing a physiological role cannot be excluded.
  • Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships
    Publication . Moncaut, Natalia P.; Somoza, G. M.; Power, Deborah; Canario, Adelino V. M.
    Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50–55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the ‘‘classical’’ role of pituitary function regulation.
  • Time for change in Portuguese science
    Publication . Canario, Adelino V. M.
    There is an almost perfect positive relationship between scientific output and gross domestic product (GDP) in Western Europe (Fig. 1). But two countries — Ireland and Portugal — spoil the relationship.
  • Brain aromatase activity and mRNA expression in a fish with male alternative reproductive tactics.
    Publication . Gonçalves, D.; Domingues, A.; Alpedrinha, J.; Teodósio, H. R.; Canario, Adelino V. M.; Oliveira, R. F.
    In southern Portugal, a population of the peacock blenny Salaria pavo inhabits a mudflat area where there is a scarcity of hard-substrate necessary for nesting. Males compete strongly for the access to the few available nest sites and only large males are able to acquire nests.
  • Cortisol and parathyroid hormone-related peptide are reciprocally modulated by negative feedback
    Publication . Guerreiro, P. M.; Rotllant, J.; Fuentes, J.; Power, Deborah; Canario, Adelino V. M.
    In previous in vitro studies, we have shown that the N-terminal region of parathyroid hormone-related protein (PTHrP) can stimulate cortisol production in sea bream, Sparus auratus, interrenal tissue, possibly through a paracrine action. In the current study, the systemic interaction between cortisol and PTHrP was studied in vivo. Sustained elevated blood cortisol levels, induced either by cortisol injection or conWnement stress, suppressed circulating PTHrP 6 and 24-fold,respectively, by comparison to control Wsh.reduced cortisol levels, prevented the decrease of plasma PTHrP observed in conWned Wsh and raised plasma PTHrPrespectively, by comparison to control fish.