Transgenic models in endocrinology

Major advances in the field of endocrinology have frequently been driven by the application of new technologies, and transgenic technology is no exception

European sea bass (Dicentrarchus labrax) skin and scale transcriptomes

Fish skin and their appendages, the mineralized scales, are important organs for protection and homeostasis, but little is known about their specific transcript or protein repertoire. This study used RNA-seq to generate transcriptome data for skin and scales in the European sea bass (Dicentrarchus labrax), an important species for fisheries and aquaculture. RNA was extracted from the pectoral skin and from scales collected above the midline of immature one-year old sea bass. More than 20 x 10(6) reads were obtained for each tissue, using RNA-seq Illumina technology. De novo assembly resulted in 31,842 transcripts (of 500 base pairs or greater) for skin and 20,423 transcripts for scale. This dataset provides a useful resource for both aquaculture and fish conservation studies and for research into the physiology and molecular biology of fish skin and scales. (C) 2017 Elsevier B.V. All rights reserved.Foundation for Science and Technology of Portugal (FCT) [PTDC/AAG-GLO/4003/2012, CCMAR/Multi/04326/2013, SFRH/BPD/84033/2012

A noninvasive monitoring device for anesthetics in fish

A noninvasive device capable of recording both gill and lateral fin movements was assembled and used to analyze initial and post-treatment activity frequency (Hz) in fish exposed to anesthetics. Exposure of platy fish (Xiphosphorus maculatus) to saponins from quillaja bark (0.185 mM and 0.555 mM) initially caused hyperactivity, but within five minutes all activity ceased and the fish failed to recover. In contrast, clove oil (67 μg/L) added to water at 22oC reduced activity by 22.8% ± 8.9% (P = 0.038) after 125 ± 19 sec, a sedative effect that was totally reversible. Cinnamon oil compared with clove oil had a significantly longer time to sedation (125 ± 19 versus 235 ± 24 sec, P = 0.02), although no significant difference in the decline in activity was noted.DMP acknowledges the support of an STMS exchange grant funded in the context of COST 925 action

Water calcium concentration modifies whole-body calcium uptake in sea bream larvae during short-term adaptation to altered salinities

Whole-body calcium uptake was studied in gilthead sea bream larvae (9–83·mg) in response to changing environmental salinity and [Ca2+]. Calcium uptake increased with increased fish size and salinity. Fish exposed to calcium-enriched, diluted seawater showed increased calcium uptake compared with fish in diluted seawater alone. Calcium uptake was unchanged in Na+- enriched, diluted seawater. Overall, [Ca2+], and not salinity/osmolarity per se, appears to be the main factor contributing to calcium uptake. By contrast, drinking was reduced by a decrease in salinity/osmolarity but was little affected by external [Ca2+]. Calculations of the maximum contribution from drinking-associated calcium uptake showed that it became almost insignificant (less than 10%) through a strong decrease in drinking rate at low salinities (0–8‰). Diluted seawater enriched in calcium to the concentration present in full-strength seawater (i.e. constant calcium, decreasing salinity) restored intestinal calcium uptake to normal. Extra-intestinal calcium uptake also benefited from calcium addition but to a lesser extent

Duplicated membrane estrogen receptors in the European sea bass (Dicentrarchus labrax): Phylogeny, expression and regulation throughout the reproductive cycle

The numerous estrogen functions reported across vertebrates have been classically explained by their binding to specific transcription factors, the nuclear estrogen receptors (ERs). Rapid non-genomic estrogenic responses have also been recently identified in vertebrates including fish, which can be mediated by membrane receptors such as the G protein-coupled estrogen receptor (Gper). In this study, two genes for Gper, namely gpera and gperb, were identified in the genome of a teleost fish, the European sea bass. Phylogenetic analysis indicated they were most likely retained after the 3R teleost-specific whole genome duplication and raises questions about their function in male and female sea bass. Gpera expression was mainly restricted to brain and pituitary in both sexes while gperb had a widespread tissue distribution with higher expression levels in gill filaments, kidney and head kidney. Both receptors were detected in the hypothalamus and pituitary of both sexes and significant changes in gpers expression were observed throughout the annual reproductive season. In female pituitaries, gpera showed an overall increase in expression throughout the reproductive season while gperb levels remained constant. In the hypothalamus, gpera had a higher expression during vitellogenesis and decreased in fish entering the ovary maturation and ovulation stage, while gperb expression increased at the final atresia stage. In males, gpers expression was constant in the hypothalamus and pituitary throughout the reproductive cycle apart from the mid- to late testicular development stage transition when a significant up-regulation of gpera occurred in the pituitary. The differential sex, seasonal and subtype-specific expression patterns detected for the two novel gper genes in sea bass suggests they may have acquired different and/or complementary roles in mediating estrogens actions in fish, namely on the neuroendocrine control of reproduction.info:eu-repo/semantics/publishedVersio

Thermal imprinting modifies adult stress and innate immune responsiveness in the teleost sea bream

The impact of thermal imprinting on the plasticity of the hypothalamic-pituitary-interrenal (HPI) axis and stress response in an adult ectotherm, the gilthead sea bream (Sparus aurata, L.), during its development was assessed. Fish were reared under 4 thermal regimes, and the resulting adults exposed to acute confinement stress and plasma cortisol levels and genes of the HPI axis were monitored. Changes in immune function, a common result of stress, were also evaluated using histomorphometric measurements of melanomacrophages centers (MMCs) in the head kidney and by monitoring macrophage-related transcripts. Thermal history significantly modified the HPI responsiveness in adult sea bream when eggs and larvae were reared at a higher than optimal temperature (HT, 22 degrees C), and they had a reduced amplitude in their cortisol response and significantly upregulated pituitary pomc and head kidney star transcripts. Additionally, after an acute stress challenge, immune function was modified and the head kidney of adult fish reared during development at high temperatures (HT and LHT, 18-22 degrees C) had a decreased number of MMCs and a significant downregulation of dopachrome tautomerase. Thermal imprinting during development influenced adult sea bream physiology and increased plasma levels of glucose and sodium even in the absence of an acute stress in fish reared under a high-low thermal regime (HLT, 22-18 degrees C). Overall, the results demonstrate that temperature during early development influences the adult HPI axis and immune function in a teleost fish.project Lifecycle EU-FP7 [222719]FCT- Foundation for Science and Technology [CCMAR/Multi/04326/2013]info:eu-repo/semantics/publishedVersio

Phylogeny, expression patterns and regulation of DNA Methyltransferases in early development of the flatfish, Solea senegalensis

Background: The identification of DNA methyltransferases (Dnmt) expression patterns during development and their regulation is important to understand the epigenetic mechanisms that modulate larval plasticity in marine fish. In this study, dnmt1 and dnmt3 paralogs were identified in the flatfish Solea senegalensis and expression patterns in early developmental stages and juveniles were determined. Additionally, the regulation of Dnmt transcription by a specific inhibitor (5-aza-2 '-deoxycytidine) and temperature was evaluated. Results: Five paralog genes of dnmt3, namely dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb. 1 and dnmt3bb. 2 and one gene for dnmt1 were identified. Phylogenetic analysis revealed that the dnmt gene family was highly conserved in teleosts and three fish-specific genes, dnmt3aa, dnmt3ba and dnmt3bb. 2 have evolved. The spatio-temporal expression patterns of four dnmts (dnmt1, dnmt3aa, dnmt3ab and dnmt3bb. 1) were different in early larval stages although all of them reduced expression with the age and were detected in neural organs and dnmt3aa appeared specific to somites. In juveniles, the four dnmt genes were expressed in brain and hematopoietic tissues such as kidney, spleen and gills. Treatment of sole embryos with 5-aza-2 '-deoxycytidine down-regulated dntm1 and up-regulated dntm3aa. Moreover, in lecithotrophic larval stages, dnmt3aa and dnmt3ab were temperature sensitive and their expression was higher in larvae incubated at 16 degrees C relative to 20 degrees C. Conclusion: Five dnmt3 and one dnmt1 paralog were identified in sole and their distinct developmental and tissue-specific expression patterns indicate that they may have different roles during development. The inhibitor 5-aza-2 '-deoxycytidine modified the transcript abundance of dntm1 and dntm3aa in embryos, which suggests that a regulatory feedback mechanism exists for these genes. The impact of thermal regime on expression levels of dnmt3aa and dnmt3ab in lecithotrophic larval stages suggests that these paralogs might be involved in thermal programing.INIA and EU through FEDER [RTA2013-00023-C0201

Expression of the myosin light chains 1 and 2 in the developing fast muscle of gilthead sea bream (Sparus aurata)

Myosin, the major component of striated muscle, is a complex molecule of heavy and light chains, which undergo continuous replacement to meet developmental and environmental demands. A range of myosin isoforms are expressed in early developmental stages and are of special interest as they offer information about muscle formation and function early in life. In addition, they can act as markers for the study of prenatal events with an effect on postnatal growth performance. In this study, the spatial and temporal expression of embryonic myosin light chains 1 (MLC1) and 2 (MLC2) was studied in sea bream larvae post-hatch by in situ hybridization using riboprobes

Gill transcriptome response to changes in environmental calcium in the green spotted puffer fish

Abstract Background Calcium ion is tightly regulated in body fluids and for euryhaline fish, which are exposed to rapid changes in environmental [Ca2+], homeostasis is especially challenging. The gill is the main organ of active calcium uptake and therefore plays a crucial role in the maintenance of calcium ion homeostasis. To study the molecular basis of the short-term responses to changing calcium availability, the whole gill transcriptome obtained by Super Serial Analysis of Gene Expression (SuperSAGE) of the euryhaline teleost green spotted puffer fish, Tetraodon nigroviridis, exposed to water with altered [Ca2+] was analysed. Results Transfer of T. nigroviridis from 10 ppt water salinity containing 2.9 mM Ca2+ to high (10 mM Ca2+ ) and low (0.01 mM Ca2+) calcium water of similar salinity for 2-12 h resulted in 1,339 differentially expressed SuperSAGE tags (26-bp transcript identifiers) in gills. Of these 869 tags (65%) were mapped to T. nigroviridis cDNAs or genomic DNA and 497 (57%) were assigned to known proteins. Thirteen percent of the genes matched multiple tags indicating alternative RNA transcripts. The main enriched gene ontology groups belong to Ca2+ signaling/homeostasis but also muscle contraction, cytoskeleton, energy production/homeostasis and tissue remodeling. K-means clustering identified co-expressed transcripts with distinct patterns in response to water [Ca2+] and exposure time. Conclusions The generated transcript expression patterns provide a framework of novel water calcium-responsive genes in the gill during the initial response after transfer to different [Ca2+]. This molecular response entails initial perception of alterations, activation of signaling networks and effectors and suggests active remodeling of cytoskeletal proteins during the initial acclimation process. Genes related to energy production and energy homeostasis are also up-regulated, probably reflecting the increased energetic needs of the acclimation response. This study is the first genome-wide transcriptome analysis of fish gills and is an important resource for future research on the short-term mechanisms involved in the gill acclimation responses to environmental Ca2+ changes and osmoregulation.Peer Reviewe

Temperature responsiveness of gilthead sea bream bone; an in vitro and in vivo approach

This study aimed to characterize the molecules involved in osteogenesis in seabream and establish using in vitro/in vivo approaches the responsiveness of selected key genes to temperature. The impact of a temperature drop from 23 to 13 degrees C was evaluated in juvenile fish thermally imprinted during embryogenesis. Both, in vitro/in vivo, Fib1a, appeared important in the first stages of bone formation, and Col1A1, ON and OP, in regulating matrix production and mineralization. OCN mRNA levels were up-regulated in the final larval stages when mineralization was more intense. Moreover, temperature-dependent differential gene expression was observed, with lower transcript levels in the larvae at 18 degrees C relative to those at 22 degrees C, suggesting bone formation was enhanced in the latter group. Results revealed that thermal imprinting affected the long-term regulation of osteogenesis. Specifically, juveniles under the low and low-to-high-temperature regimes had reduced levels of OCN when challenged, indicative of impaired bone development. In contrast, gene expression in fish from the high and high-to-low-temperature treatments was unchanged, suggesting imprinting may have a protective effect. Overall, the present study revealed that thermal imprinting modulates bone development in seabream larvae, and demonstrated the utility of the in vitro MSC culture as a reliable tool to investigate fish osteogenesis."Ministerio de Economia y Competitividad" (MINECO) [BES-2015-074654]; Portuguese Science Foundation (FCT) [SFRH/BPD/111512/2015, SFRH/BD/81625/2011]; MINECO, Spain [AGL2010-17324, AGL2014-57974-R]; "Generalitat de Catalunya" (XRAq); Generalitat de Catalunya [2014SGR-01371]; FCT, Portugal [CCMAR/Multi/04326/2013]; European Union [LIFECYCLE EU-FP7 222719]info:eu-repo/semantics/publishedVersio