Browsing by Author "Thorne, Michael A. S."
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- Comparative analysis of a teleost skeleton transcriptome provides insight into its regulationPublication . Vieira, Florbela A.; Thorne, Michael A. S.; Stueber, K.; Darias, M.; Reinhardt, Richard; Clark, M. S.; Gisbert, Enric; Power, DeborahAn articulated endoskeleton that is calcified is a unifying innovation of the vertebrates, however the molecular basis of the structural divergence between terrestrial and aquatic vertebrates, such as teleost fish, has not been determined. In the present study long-read next generation sequencing (NGS, Roche 454 platform) was used to characterize acellular perichondral bone (vertebrae) and chondroid bone (gill arch) in the gilthead sea bream (Sparus auratus). A total of 15.97 and 14.53 Mb were produced, respectively from vertebrae and gill arch cDNA libraries and yielded 32,374 and 28,371 contigs (consensus sequences) respectively. 10,455 contigs from vertebrae and 10,625 contigs from gill arches were annotated with gene ontology terms. Comparative analysis of the global transcriptome revealed 4249 unique transcripts in vertebrae, 4201 unique transcripts in the gill arches and 3700 common transcripts. Several core gene networks were conserved between the gilthead sea bream and mammalian skeleton. Transcripts for putative endocrine factors were identified in acellular gilthead sea bream bone suggesting that in common with mammalian bone it can act as an endocrine tissue. The acellular bone of the vertebra, in contrast to current opinion based on histological analysis, was responsive to a short fast and significant (p < 0.05) down-regulation of several transcripts identified by NGS, osteonectin, osteocalcin, cathepsin K and IGFI occurred. In gill arches fasting caused a significant (p < 0.05) down-regulation of osteocalcin and up-regulation of MMP9.
- European sea bass (Dicentrarchus labrax) skin and scale transcriptomesPublication . Pinto, Patricia IS; Thorne, Michael A. S.; Power, DeborahFish 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.
- Gill transcriptome response to changes in environmental calcium in the green spotted puffer fishPublication . Pinto, Patricia IS; Matsumura, H.; Thorne, Michael A. S.; Power, Deborah; Terauchi, Ryohei; Reinhardt, Richard; Canario, Adelino V. M.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.
- Identification of molecular and physiological responses to chronic environmental challenge in an invasive species The Pacific oyster, Crassostrea gigasPublication . Clark, M. S.; Thorne, Michael A. S.; Araújo Amaral, Ana Margarida; Vieira, Florbela A.; Batista, Frederico; Reis, João; Power, DeborahUnderstanding the environmental responses of an invasive species is critical in predicting how ecosystem composition may be transformed in the future, especially under climate change. In this study, Crassostrea gigas, a species well adapted to the highly variable intertidal environment, was exposed to the chronic environmental challenges of temperature (19 and 24°C) and pH (ambient seawater and a reduction of 0.4 pH units) in an extended 3-month laboratory-based study. Physiological parameters were measured (condition index, shell growth, respiration, excretion rates, O:N ratios, and ability to repair shell damage) alongside molecular analyses. Temperature was by far the most important stressor, as demonstrated by reduced condition indexes and shell growth at 24°C, with relatively little effect detected for pH. Transcriptional profiling using candidate genes and SOLiD sequencing of mantle tissue revealed that classical “stress” genes, previously reported to be upregulated under acute temperature challenges, were not significantly expressed in any of the treatments, emphasizing the different response between acute and longer term chronic stress. The transcriptional profiling also elaborated on the cellular responses underpinning the physiological results, including the identification of the PI3K/AKT/mTOR pathway as a potentially novel marker for chronic environmental challenge. This study represents a first attempt to understand the energetic consequences of cumulative thermal stress on the intertidal C. gigas which could significantly impact on coastal ecosystem biodiversity and function in the future.
- Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyroseqeuencingPublication . Clark, M. S.; Thorne, Michael A. S.; Vieira, Florbela A.; Cardoso, João CR; Power, Deborah; Peck, Lloyd S.Abstract Background The Antarctic clam, Laternula elliptica, is an infaunal stenothermal bivalve mollusc with a circumpolar distribution. It plays a significant role in bentho-pelagic coupling and hence has been proposed as a sentinel species for climate change monitoring. Previous studies have shown that this mollusc displays a high level of plasticity with regard to shell deposition and damage repair against a background of genetic homogeneity. The Southern Ocean has amongst the lowest present-day CaCO3 saturation rate of any ocean region, and is predicted to be among the first to become undersaturated under current ocean acidification scenarios. Hence, this species presents as an ideal candidate for studies into the processes of calcium regulation and shell deposition in our changing ocean environments. Results 454 sequencing of L. elliptica mantle tissue generated 18,290 contigs with an average size of 535 bp (ranging between 142 bp-5.591 kb). BLAST sequence similarity searching assigned putative function to 17% of the data set, with a significant proportion of these transcripts being involved in binding and potentially of a secretory nature, as defined by GO molecular function and biological process classifications. These results indicated that the mantle is a transcriptionally active tissue which is actively proliferating. All transcripts were screened against an in-house database of genes shown to be involved in extracellular matrix formation and calcium homeostasis in metazoans. Putative identifications were made for a number of classical shell deposition genes, such as tyrosinase, carbonic anhydrase and metalloprotease 1, along with novel members of the family 2 G-Protein Coupled Receptors (GPCRs). A membrane transport protein (SEC61) was also characterised and this demonstrated the utility of the clam sequence data as a resource for examining cold adapted amino acid substitutions. The sequence data contained 46,235 microsatellites and 13,084 Single Nucleotide Polymorphisms(SNPs/INDELS), providing a resource for population and also gene function studies. Conclusions This is the first 454 data from an Antarctic marine invertebrate. Sequencing of mantle tissue from this non-model species has considerably increased resources for the investigation of the processes of shell deposition and repair in molluscs in a changing environment. A number of promising candidate genes were identified for functional analyses, which will be the subject of further investigation in this species and also used in model-hopping experiments in more tractable and economically important model aquaculture species, such as Crassostrea gigas and Mytilus edulis.
- Skin healing and scale regeneration in fed and unfed sea bream, Sparus auratusPublication . Vieira, Florbela A.; Gregorio, Silvia; Ferraresso, Serena; Thorne, Michael A. S.; Costa, Rita; Milan, Massimo; Bargelloni, Luca; Clark, M. S.; Canario, Adelino V. M.; Power, DeborahAbstract Background Fish scales are an important reservoir of calcium and phosphorus and together with the skin function as an integrated barrier against environmental changes and external aggressors. Histological studies have revealed that the skin and scales regenerate rapidly in fish when they are lost or damaged. In the present manuscript the histological and molecular changes underlying skin and scale regeneration in fed and fasted sea bream (Sparus auratus) were studied using a microarray 3 and 7 days after scale removal to provide a comprehensive molecular understanding of the early stages of these processes. Results Histological analysis of skin/scales revealed 3 days after scale removal re-epithelisation and formation of the scale pocket had occurred and 53 and 109 genes showed significant up or down-regulation, respectively. Genes significantly up-regulated were involved in cell cycle regulation, cell proliferation and adhesion, immune response and antioxidant activities. 7 days after scale removal a thin regenerated scale was visible and only minor changes in gene expression occurred. In animals that were fasted to deplete mineral availability the expression profiles centred on maintaining energy homeostasis. The utilisation of fasting as a treatment emphasised the competing whole animal physiological requirements with regard to barrier repair, infection control and energy homeostasis. Conclusions The identification of numerous genes involved in the mitotic checkpoint and cell proliferation indicate that the experimental procedure may be useful for understanding cell proliferation and control in vertebrates within the context of the whole animal physiology. In response to skin damage genes of immune surveillance were up-regulated along with others involved in tissue regeneration required to rapidly re-establish barrier function. Additionally, candidate fish genes were identified that may be involved in cytoskeletal re-modelling, mineralization and stem cells, which are of potential use in aquaculture and fish husbandry, as they may impact on the ability of the fish to produce structural proteins, such as muscle, efficiently.
- Transcriptome profiling of the gills of a euryhaline teleost fish, Tetraodon nigroviridis, in response to altered calcium concentrations in waterPublication . Pinto, Patricia IS; Power, Deborah; Canario, Adelino V. M.; Thorne, Michael A. S.; Reinhardt, Richard; Matsumura, H.; Terauchi, R.Fish are able to maintain their extracellular [Ca2+] relatively constant through regulatory mechanisms that control the influx or efflux of Ca2+ in osmoregulatory organs. To understand in depth the molecular basis of these adjustments, the whole gill transcriptome of the euryhaline teleost Tetraodon nigroviridis exposed to high and low [Ca2+] was obtained using Super Serial Analysis of Gene Expression (SuperSAGE).