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Abstract Background Transcriptomes are powerful resources, providing a window on the expressed portion of the genome that can be generated rapidly and at low cost for virtually any organism. However, because many genes have tissue-specific expression patterns, developing a complete transcriptome usually requires a 'discovery pool' of individuals to be sacrificed in order to harvest mRNA from as many different types of tissue as possible. This hinders transcriptome development in large, charismatic and endangered species, many of which stand the most to gain from such approaches. To circumvent this problem in a model pinniped species, we 454 sequenced cDNA from testis, heart, spleen, intestine, kidney and lung tissues obtained from nine adult male Antarctic fur seals (Arctocephalus gazella) that died of natural causes at Bird Island, South Georgia. Results After applying stringent quality control criteria based on length and annotation, we obtained 12,397 contigs which, in combination with 454 data previously obtained from skin, gave a total of 23,096 unique contigs. Homology was found to 77.0% of dog (Canis lupus familiaris) transcripts, suggesting that the combined assembly represents a substantial proportion of this species' transcriptome. Moreover, only 0.5% of transcripts revealed sequence similarity to bacteria, implying minimal contamination, and the percentage of transcripts involved in cell death was low at 2.6%. Transcripts with immune-related annotations were almost five-fold enriched relative to skin and represented 13.2% of all spleen-specific contigs. By reference to the dog, we also identified transcripts revealing homology to five class I, ten class II and three class III genes of the Major Histocompatibility Complex and derived the putative genomic distribution of 17,121 contigs, 2,119 in silico mined microsatellites and 9,382 single nucleotide polymorphisms. Conclusions Our findings suggest that transcriptome development based on samples collected post mortem may greatly facilitate genomic studies, not only of marine mammals but also more generally of species that are of conservation concern.

Abstract 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.

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.

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.

Abstract Background Insects provide tractable models for enhancing our understanding of the physiological and cellular processes that enable survival at extreme low temperatures. They possess three main strategies to survive the cold: freeze tolerance, freeze avoidance or cryoprotective dehydration, of which the latter method is exploited by our model species, the Arctic springtail Megaphorura arctica, formerly Onychiurus arcticus (Tullberg 1876). The physiological mechanisms underlying cryoprotective dehydration have been well characterised in M. arctica and to date this process has been described in only a few other species: the Antarctic nematode Panagrolaimus davidi, an enchytraied worm, the larvae of the Antarctic midge Belgica antarctica and the cocoons of the earthworm Dendrobaena octaedra. There are no in-depth molecular studies on the underlying cold survival mechanisms in any species. Results A cDNA microarray was generated using 6,912 M. arctica clones printed in duplicate. Analysis of clones up-regulated during dehydration procedures (using both cold- and salt-induced dehydration) has identified a number of significant cellular processes, namely the production and mobilisation of trehalose, protection of cellular systems via small heat shock proteins and tissue/cellular remodelling during the dehydration process. Energy production, initiation of protein translation and cell division, plus potential tissue repair processes dominate genes identified during recovery. Heat map analysis identified a duplication of the trehalose-6-phosphate synthase (TPS) gene in M. arctica and also 53 clones co-regulated with TPS, including a number of membrane associated and cell signalling proteins. Q-PCR on selected candidate genes has also contributed to our understanding with glutathione-S-transferase identified as the major antioxdidant enzyme protecting the cells during these stressful procedures, and a number of protein kinase signalling molecules involved in recovery. Conclusion Microarray analysis has proved to be a powerful technique for understanding the processes and genes involved in cryoprotective dehydration, beyond the few candidate genes identified in the current literature. Dehydration is associated with the mobilisation of trehalose, cell protection and tissue remodelling. Energy production, leading to protein production, and cell division characterise the recovery process. Novel membrane proteins, along with aquaporins and desaturases, have been identified as promising candidates for future functional analyses to better understand membrane remodelling during cellular dehydration.

Abstract Background Microscopic monogonont rotifers, including the euryhaline species Brachionus plicatilis, are typically found in water bodies where environmental factors restrict population growth to short periods lasting days or months. The survival of the population is ensured via the production of resting eggs that show a remarkable tolerance to unfavorable conditions and remain viable for decades. The aim of this study was to generate Expressed Sequence Tags (ESTs) for molecular characterisation of processes associated with the formation of resting eggs, their survival during dormancy and hatching. Results Four normalized and four subtractive libraries were constructed to provide a resource for rotifer transcriptomics associated with resting-egg formation, storage and hatching. A total of 47,926 sequences were assembled into 18,000 putative transcripts and analyzed using both Blast and GO annotation. About 28–55% (depending on the library) of the clones produced significant matches against the Swissprot and Trembl databases. Genes known to be associated with desiccation tolerance during dormancy in other organisms were identified in the EST libraries. These included genes associated with antioxidant activity, low molecular weight heat shock proteins and Late Embryonic Abundant (LEA) proteins. Real-time PCR confirmed that LEA transcripts, small heat-shock proteins and some antioxidant genes were upregulated in resting eggs, therefore suggesting that desiccation tolerance is a characteristic feature of resting eggs even though they do not necessarily fully desiccate during dormancy. The role of trehalose in resting-egg formation and survival remains unclear since there was no significant difference between resting-egg producing females and amictic females in the expression of the tps-1 gene. In view of the absence of vitellogenin transcripts, matches to lipoprotein lipase proteins suggest that, similar to the situation in dipterans, these proteins may serve as the yolk proteins in rotifers. Conclusion The 47,926 ESTs expand significantly the current sequence resource of B. plicatilis. It describes, for the first time, genes putatively associated with resting eggs and will serve as a database for future global expression experiments, particularly for the further identification of dormancy related genes.

Abstract Background Ice, snow and temperatures of -14°C are conditions which most animals would find difficult, if not impossible, to survive in. However this exactly describes the Arctic winter, and the Arctic springtail Onychiurus arcticus regularly survives these extreme conditions and re-emerges in the spring. It is able to do this by reducing the amount of water in its body to almost zero: a process that is called "protective dehydration". The aim of this project was to generate clones and sequence data in the form of ESTs to provide a platform for the future molecular characterisation of the processes involved in protective dehydration. Results Five normalised libraries were produced from both desiccating and rehydrating populations of O. arcticus from stages that had previously been defined as potentially informative for molecular analyses. A total of 16,379 EST clones were generated and analysed using Blast and GO annotation. 40% of the clones produced significant matches against the Swissprot and trembl databases and these were further analysed using GO annotation. Extraction and analysis of GO annotations proved an extremely effective method for identifying generic processes associated with biochemical pathways, proving more efficient than solely analysing Blast data output. A number of genes were identified, which have previously been shown to be involved in water transport and desiccation such as members of the aquaporin family. Identification of these clones in specific libraries associated with desiccation validates the computational analysis by library rather than producing a global overview of all libraries combined. Conclusion This paper describes for the first time EST data from the arctic springtail (O. arcticus). This significantly enhances the number of Collembolan ESTs in the public databases, providing useful comparative data within this phylum. The use of GO annotation for analysis has facilitated the identification of a wide variety of ESTs associated with a number of different biochemical pathways involved in the dehydration and recovery process in O. arcticus.

Within the last decade, thin ultra-low velocity zone (ULVZ) layering, sitting directly on top of the core–mantle boundary (CMB), has begun to be investigated using the flip-reverse-stack (FRS) method. In this method, pre- and post-cursor arrivals that are symmetrical in time about the ScS arrival, but with opposite polarities, are stacked. This same methodology has also been applied to high velocity layering, with indications that ultra-high velocity zones (UHVZs) may also exist. Thus far, studies using the FRS technique have relied on 1-D synthetic predictions to infer material properties of ULVZs. 1-D ULVZ models predominantly show a SdS precursor that reflects off the top of the ULVZ and an ScscS reverberation within the ULVZ that arrives as a post-cursor. 1-D UHVZ models are more complex and have a different number of arrivals depending on a variety of factors including UHVZ thickness, velocity contrast, and lateral extent. 1-D modelling approaches assume that lower mantle heterogeneity is constant and continuous everywhere across the lower mantle. However, lower mantle features display lateral heterogeneity and are either finite in extent or display local thickness variations. We examine the interaction of the ScS wavefield with ULVZs and UHVZs in 2.5-D geometries of finite extent. We show that multiple additional arrivals exist that are not present in 1-D predictions. In particular, multipath ScS arrivals as well as additional post-cursor arrivals are generated. Subsequent processing by the FRS method generates complicated FRS traces with multiple peaks. Furthermore, post-cursor arrivals can be generated even when the ScS ray path does not directly strike the heterogeneity from above. Analysing these predictions for 2.5-D models using 1-D modelling techniques demonstrates that a cautious approach must be adopted in utilization and interpretation of FRS traces to determine if the ScS wavefield is interacting with a ULVZ or UHVZ through a direct strike on the top of the feature. In particular, traveltime delays or advances of the ScS arrival should be documented and symmetrical opposite polarity arrivals should be demonstrated to exist around ScS. The latter can be quantified by calculation of a time domain multiplication trace. Because multiple post-cursor arrivals are generated by finite length heterogeneities, interpretation should be confined to single layer models rather than to interpret the additional peaks as internal layering. Furthermore, strong trade-offs exist between S -wave velocity perturbation and thickness making estimations of ULVZ or UHVZ elastic parameters highly uncertain. We test our analysis methods using data from an event occurring in the Fiji-Tonga region recorded in North America. The ScS bounce points for this event sample the CMB region to the southeast of Hawaii, in a region where ULVZs have been identified in several recent studies. We see additional evidence for a ULVZ in this region centred at 14°N and 153°W with a lateral scale of at least 250 km × 360 km. Assuming a constant S -wave velocity decrease of −10 or −20 per cent with respect to the PREM model implies a ULVZ thickness of up to 16 or 9 km, respectively. [ABSTRACT FROM AUTHOR]

Ultra-low velocity zones (ULVZs) have been identified as regions of extremely low velocity anomalies in the Earth's lowermost mantle using seismic observations from reflected, refracted and diffracted arrivals along the mantle side of the core–mantle boundary. Estimation of ULVZ geometrical (i.e. shape and size) and elastic (i.e. velocity and density) parameters with uncertainties is crucial in understanding the role of ULVZs in the ongoing dynamic processes within the Earth's mantle; however, these parameters are still poorly known due to uncertainties and tradeoffs of the seismically resolved ULVZ geometries and elastic parameters. Computation of synthetic waveforms for 2-D and 3-D ULVZs shapes is currently computationally feasible, but past studies utilize higher dimensional waveform modelling of mostly only low-frequency diffracted waves. Most studies focusing on high-frequency core-reflected waveforms (e.g. ScP) still use 1-D modelling approaches to determine ULVZ properties. This approach might lead to wrong results if the imaged structures have inherently 3-D geometries. This study investigates high-frequency synthetic ScP waveforms for various 2.5-D ULVZ geometries showing that additional seismic arrivals are generated even when the ScP geometrical ray path does not directly strike the location of the ULVZ. The largest amplitude additional phases in the 2.5-D models are post-cursor arrivals that are generated at the edges of the finite-length ULVZs. These newly identified ScP post-cursors can arrive within the ScsP post-cursor time window traditionally analysed in 1-D ULVZ studies. These post-cursors might then be misidentified or constructively/destructively interfere with the ScsP post-cursor, leading to incorrect estimation of ULVZ parameters. In this study we investigate the bias introduced by the 2.5-D morphologies on the 1-D estimated ULVZ elastic properties in a Bayesian waveform inversion scheme. We further expand the Bayesian method by including the data noise covariance matrix in the inversion and compare it to an autoregressive noise model that was utilized in previous studies. From the application to the observed ScP data, we find that the new approach converges faster, particularly for the inversion of data from multiple events, and the new algorithm retrieves ULVZ parameters with more realistic uncertainties. The inversion of 2.5-D synthetic ScP waveforms suggests that the retrieved ULVZ parameters can be misleading with unrealistically high confidence if we do not consider the data noise covariance matrix in the inversion. Our new approach can also retrieve the shape of a multidimensional Gaussian ULVZ if its length is 12° or longer in the great circle arc direction. However, 2.5-D synthetic waveforms show additional waveform complexity which can constructively interfere with the ScP wavefield. Hence, in many cases the estimation of ULVZ properties using 1-D forward modelling can provide incorrect ULVZ parameters. Hence previous ULVZ modelling efforts using 1-D parameter estimation methods may be incorrect. [ABSTRACT FROM AUTHOR]