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

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

Expression patterns of MLC isoforms during halibut (Hippoglossus hippoglossus L.) metamorphosis

Atlantic halibut is an important commercial fish in the countries of the North Atlantic and is emerging as a promising species for marine cold-water aquaculture. The axial musculature of the developing larvae is the largest and most rapidly growing tissue and during the transition from larval to adult muscle fibre types significant changes in fibre morphology and gene transcription occur. In fact the change in myotome height correlates well with different larval halibut stages. In the present study the spatial and temporal expression of myosin light chain 1 (MLC1), 2 (MLC2) and 3 (MLC3) was studied in metamorphosing halibut by in situ hybridization. As a first step to establishing a role for the thyroid axis in halibut muscle development whole body thyroid hormone (TH) concentrations were also determined. In first feeding larvae MLC1, MLC2A and MLC3 transcripts had a similar distribution and were confined to the muscle fibres of the germinal zones. In pre-metamorphic larvae transcripts were highly expressed throughout the epaxial and hypaxial musculature and expression levels reached a maximum in larvae starting metamorphosis, this change coincided with a significant increase in the concentration of thyroid hormones. By the time larvae reached the metamorphic climax, MLC1, MLC2A and MLC3 expression was still high throughout the musculature but expression was confined to fibres adjacent to the myosepts and to small cells scattered in the musculature, possibly satellite cells. MLC2A was also expressed in the red muscle fibres; no transition between larval and adult MLC isoforms was detected.This work was funded by European Union project CT 96-1442, ARRDE-Q5Rs-2002-01192 and Portuguese Ministry of Science and Technology – Pluriannual funding to CCMAR. We thank Heiddis Smáradóttir, (Fiskey, Iceland) for providing the animal samples

Editorial

In a globally changing climate and with the alarming loss of biodiversity Comparative Endocrinology becomes ever more relevant as a scientific discipline. The comparative endocrinologist continues to be at the frontline of scientific endeavour trying to understand the myriad complexities of life forms on earth by looking at the regulatory factors that underpin key events in an organisms’ life and ensure maintenance of homeostasis and species survival

Characterization of fish scale regeneration: environmental and endocrine control

Fish scales are calcified structures present in skin which have a protective function but which are also implicated in calcium homeostasis. Information about the molecular and cellular organisation of scales and the endocrine factors which regulate their turnover is scarce. In the present study as a first step to studying the influence of hormones on scale metabolism we aimed to characterize elasmoid scales from the sea bream (Sparus auratus), the dynamics of scale regeneration and the possible regulation of this process by calcium availability. Regenerating scales were evident 7 days after their removal in fish maintained in seawater (SW) and brackish water (BW). In BW, the activity of enzymes involved in scale turnover, tartrate resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP), increased indicating active remodelling. The higher activity of TRAP compared to ALP suggests that the reduced calcium availability in BW led to calcium mobilization from the scales. The results suggest that the scales can be used as a regulated reservoir of calcium in this fish.info:eu-repo/semantics/publishedVersio

Distribution of giardia duodenalis assemblages A and B among children living in a remote Indigenous Community of the Northern Territory, Australia

Giardiasis is a communicable gastrointestinal disease caused by Giardia duodenalis and two genetic assemblages, A and B, cause human infection. In remote Indigenous communities of Australia, giardiasis is highly prevalent among children but disease transmission is poorly understood. This study investigated the prevalence of Giardia and genetic subtypes contributing to human disease in a remote Indigenous community, in the Northern Territory of Australia. Eighty-seven faecal samples were collected from 74 children (<15 years) over an 18 month period, and the distribution of positive cases relative to participant age and gender were examined. Screening by microscopy and 18S rRNA PCR amplification showed 66.7% (58/87) of faecal samples were positive for Giardia. Both males and females were equally affected and high detection rates were obtained for participants aged 0–<5 years and 5–<10 years (66.0 and 60.0% respectively). For 58.6% of the positive samples, Giardia was only detected by 18S rRNA PCR. Approximately 75% of cases were assemblage B, and subassemblage analyses using terminal restriction fragment length polymorphism of the glutamate dehydrogenase gene demonstrated that a variety of genetic variants were present. The high proportion of positive cases that were not detectable by microscopy, and dominance of assemblage B cases highlights the need for further research in this community, to assess the contribution of Giardia to chronic gastrointestinal disease among children, and to understand conditions conductive to assemblage B transmission

Calcium balance in sea bream (Sparus aurata): the effect of oestradiol-17 beta

In all teleost fishes vitellogenesis is triggered and maintained by oestradiol-17 (E2) and is accompanied by an increase of blood plasma calcium and phosphate. The action of this hormone on calcium metabolism was investigated by treating fast-growing immature juvenile sea bream (Sparus aurata) with coconut butter implants alone (control) or implants containing 10 μg/g E2. Treatment with E2 induced the production of circulating vitellogenin, a 2·5-fold increase in plasma ionic Ca2+ and a 10-fold increase in plasma total calcium, largely bound to protein. In contrast to freshwater species, which obtain most of their calcium from the environment directly through the gills, the intestinal component of calcium uptake of the salt water-living sea bream represented up to 60–70% of the total uptake. The whole body calcium uptake, expressed as the sum of calcium obtained via intestinal and extra-intestinal (likely branchial) routes increased significantly in response to E2. Combined influx and unchanged efflux rates resulted in a significant 31% increase in net calcium uptake. There was no evidence for an effect of E2 on the calcium and phosphate content of the scales or the tartrate-resistant acid phosphatase activity (an index for bone/scale osteoclast activity). While most freshwater fish appear to rely on internal stores of calcium, i.e. bone and/or scales to increase calcium availability, the marine sea bream accommodates calcium-transporting mechanisms to obtain calcium from the environment and preserve internal stores. These observations suggest that a fundamental difference may exist in the E2-dependent calcium regulation between freshwater and marine teleosts

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

Molecular cloning and sequence of Sparus aurata skeletal myosin light chains expressed in white muscle: developmental expression and thyroid regulation

Two full-length cDNA clones encoding the skeletal myosin light chain 2 (MLC2; 1452 bp) and myosin light chain 3 (MLC3; 972 bp) were isolated from a cDNA library prepared from gilthead sea bream Sparus aurata larvae. The MLC2 cDNA encoded a predicted protein of 170 residues that was 79 % identical to rabbit MLC2 over the entire length and 87 % identical within the Ca2+- binding region. The deduced amino acid sequence of MLC3 was 153 residues in length and was 91 % and 69% identical to the zebrafish and rabbit MLC3, respectively. Northern blot analysis revealed that in adults both transcripts were expressed in fast white muscle only. MLC2 appeared earlier in development: MLC2 transcripts were detectable from the beginning of segmentation, whereas MLC3 transcripts did not appear until 27 h post-fertilisation. At this developmental stage, a second MLC2 transcript of 0.89 kilobase-pairs was present. MLCs exhibited a different age-related pattern of response to varied thyroidal states, which were experimentally induced by the administration of 1 mgg-1 body mass of thyroxine (T4) or triiodothyronine (T3), or 5 ng g-1 body mass of the hypothyroidal compound thiourea; MLC3 expression was not significantly affected, whereas levels of MLC2 transcripts were significantly elevated in the white muscle only of juvenile sea bream after administration of T4. Although the mechanism of thyroidal regulation of MLC expression remains unknown, the present results suggest that different regulatory mechanisms exist for different MLCs

In vitro screening for estrogenic endocrine disrupting compounds using Mozambique tilapia and sea bass scales

A wide range of estrogenic endocrine disruptors (EDCs) are accumulating in the environment and may disrupt the physiology of aquatic organisms. The effects of EDCs on fish have mainly been assessed using reproductive endpoints and in vivo animal experiments. We used a simple non-invasive assay to evaluate the impact of estrogens and EDCs on sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) scales. These were exposed to estradiol (E2), two phytoestrogens and six anthropogenic estrogenic/anti-estrogenic EDCs and activities of enzymes related to mineralized tissue turnover (TRAP, tartrate-resistant acid phosphatase and ALP, alkaline phosphatase) were measured. Semi-quantitative RT-PCR detected the expression of both membrane and nuclear estrogen receptors in the scales of both species, confirming scales as a target for E2 and EDCs through different mechanisms. Changes in TRAP or ALP activities after 30 minute and 24 h exposure were detected in sea bass and tilapia scales treated with E2 and three EDCs, although compound-, time- and dose-specific responses were observed for the two species. These results support again that the mineralized tissue turnover of fish is regulated by estrogens and reveals that the scales are a mineralized estrogen-responsive tissue that may be affected by some EDCs. The significance of these effects for whole animal physiology needs to be further explored. The in vitro fish scale bioassay is a promising non-invasive screening tool for E2 and EDCs effects, although the low sensitivity of TRAP/ALP quantification limits their utility and indicates that alternative endpoints are required