The transcriptome of metamorphosing flatfish.

BACKGROUND: Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. Metamorphosis in vertebrates is driven by thyroid hormones (THs), but how they orchestrate the cellular, morphological and functional modifications associated with maturation to juvenile/adult states in flatfish is an enigma. Since THs act via thyroid receptors that are ligand activated transcription factors, we hypothesized that the maturation of tissues during metamorphosis should be preceded by significant modifications in the transcriptome. Targeting the unique metamorphosis of flatfish and taking advantage of the large size of Atlantic halibut (Hippoglossus hippoglossus) larvae, we determined the molecular basis of TH action using RNA sequencing. RESULTS: De novo assembly of sequences for larval head, skin and gastrointestinal tract (GI-tract) yielded 90,676, 65,530 and 38,426 contigs, respectively. More than 57 % of the assembled sequences were successfully annotated using a multi-step Blast approach. A unique set of biological processes and candidate genes were identified specifically associated with changes in morphology and function of the head, skin and GI-tract. Transcriptome dynamics during metamorphosis were mapped with SOLiD sequencing of whole larvae and revealed greater than 8,000 differentially expressed (DE) genes significantly (p < 0.05) up- or down-regulated in comparison with the juvenile stage. Candidate transcripts quantified by SOLiD and qPCR analysis were significantly (r = 0.843; p < 0.05) correlated. The majority (98 %) of DE genes during metamorphosis were not TH-responsive. TH-responsive transcripts clustered into 6 groups based on their expression pattern during metamorphosis and the majority of the 145 DE TH-responsive genes were down-regulated. CONCLUSIONS: A transcriptome resource has been generated for metamorphosing Atlantic halibut and over 8,000 DE transcripts per stage were identified. Unique sets of biological processes and candidate genes were associated with changes in the head, skin and GI-tract during metamorphosis. A small proportion of DE transcripts were TH-responsive, suggesting that they trigger gene networks, signalling cascades and transcription factors, leading to the overt changes in tissue occurring during metamorphosis.

Comparative analysis of a teleost skeleton transcriptome provides insight into its regulation.

An 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 14.53Mb 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.

Proteomics of cryoprotective dehydration in Megaphorura arctica Tullberg 1876 (Onychiuridae: Collembola).

The Arctic springtail, Megaphorura arctica Tullberg 1876 (Onychiuridae: Collembola), is one of the few organisms known to survive the extreme stresses of its environment by using cryoprotective dehydration. We have undertaken a proteomics study comparing M. arctica, acclimated at -2°C, the temperature known to induce the production of the anhydroprotectant trehalose in this species, and -6°C, the temperature at which trehalose expression plateaus, against control animals acclimated at +5°C. Using difference gel electrophoresis, and liquid chromatography tandem mass spectrometry, we identified three categories of differentially expressed proteins with specific functions, up-regulated in both the -2°C and -6°C animals, that were involved in metabolism, membrane transport and protein folding. Proteins involved in cytoskeleton organisation were only up-regulated in the -6°C animals.

Gene expression associated with changes in cold tolerance levels of the Antarctic springtail, Cryptopygus antarcticus.

The ability of the Antarctic microarthropod Cryptopygus antarcticus (Collembola, Isotomidae) to survive low temperatures has been well studied at the physiological level, with recent investigations indicating the importance of the moulting process in conferring this ability. This study investigated gene expression in groups of C. antarcticus that have distinct differences in their ability to survive low temperatures. A microarray containing c. 5400 C. antarcticus expressed sequence tags was used to investigate gene expression differences between groups of animals with different supercooling points (SCP), and to low temperatures close to their SCP. By demonstrating the involvement of moult-related genes in the differential survival of two groups of C. antarcticus with distinct SCP profiles, the results of this investigation add support to the suggestion that moulting plays a role in conferring cold tolerance in C. antarcticus.

Age-related thermal response: the cellular resilience of juveniles.

Understanding species' responses to environmental challenges is key to predicting future biodiversity. However, there is currently little data on how developmental stages affect responses and also whether universal gene biomarkers to environmental stress can be identified both within and between species. Using the Antarctic clam, Laternula elliptica, as a model species, we examined both the tissue-specific and age-related (juvenile versus mature adult) gene expression response to acute non-lethal warming (12 h at 3 °C). In general, there was a relatively muted response to this sub-lethal thermal challenge when the expression profiles of treated animals, of either age, were compared with those of 0 °C controls, with none of the "classical" stress response genes up-regulated. The expression profiles were very variable between the tissues of all animals, irrespective of age with no single transcript emerging as a universal biomarker of thermal stress. However, when the expression profiles of treated animals of the different age groups were directly compared, a very different pattern emerged. The profiles of the younger animals showed significant up-regulation of chaperone and antioxidant transcripts when compared with those of the older animals. Thus, the younger animals showed evidence of a more robust cellular response to warming. These data substantiate previous physiological analyses showing a more resilient juvenile population.

A draft fur seal genome provides insights into factors affecting SNP validation and how to mitigate them.

Custom genotyping arrays provide a flexible and accurate means of genotyping single nucleotide polymorphisms (SNPs) in a large number of individuals of essentially any organism. However, validation rates, defined as the proportion of putative SNPs that are verified to be polymorphic in a population, are often very low. A number of potential causes of assay failure have been identified, but none have been explored systematically. In particular, as SNPs are often developed from transcriptomes, parameters relating to the genomic context are rarely taken into account. Here, we assembled a draft Antarctic fur seal (Arctocephalus gazella) genome (assembly size: 2.41 Gb; scaffold/contig N50 : 3.1 Mb/27.5 kb). We then used this resource to map the probe sequences of 144 putative SNPs genotyped in 480 individuals. The number of probe-to-genome mappings and alignment length together explained almost a third of the variation in validation success, indicating that sequence uniqueness and proximity to intron-exon boundaries play an important role. The same pattern was found after mapping the probe sequences to the Walrus and Weddell seal genomes, suggesting that the genomes of species divergent by as much as 23 million years can hold information relevant to SNP validation outcomes. Additionally, reanalysis of genotyping data from seven previous studies found the same two variables to be significantly associated with SNP validation success across a variety of taxa. Finally, our study reveals considerable scope for validation rates to be improved, either by simply filtering for SNPs whose flanking sequences align uniquely and completely to a reference genome, or through predictive modelling.

Transcriptome of the Atlantic halibut (Hippoglossus hippoglossus).

Although the Atlantic halibut (Hippoglossus hippoglossus) is an important commercial species, there is still a deficit with regard to the number of transcripts in the databases, which can be accessed and exploited for targeted candidate gene and pathway studies. In this study, the RNAs from head, skin and GI tract from different developmental stages were sequenced to generate 22,272 contigs of 500 base pairs or greater as a molecular resource for this species.

Transcription profiling of acute temperature stress in the Antarctic plunderfish Harpagifer antarcticus.

Harpagifer antarcticus (the Antarctic plunderfish), a shallow-water benthic fish distributed around the Antarctic Peninsula, is a member of the notothenioid family, one of whose adaptations to the cold waters of Antarctica has been the loss of the classic heat shock response. In order to gain a more comprehensive understanding of the effects of temperature stress on H. antarcticus, we constructed a liver cDNA library and a 10,371 feature microarray. This was hybridized with material from a time course series of animals held at 6°C for 48h. The resulting expression profiles show that this fish displays the classical vertebrate acute inflammatory response. There was also a pronounced signal for increased energy requirements via up-regulation of genes involved in the ß oxidation of fatty acids and also a strong signature of response to oxidative stress. Genes in the latter category did not include the "classic" antioxidants such as glutathione S-transferase, but genes involved in the production of reducing potential in the form of NADPH, peroxisome proliferation via peroxisomal acyl co-enzyme A oxidase 1 and genes known to be up-regulated by hypoxia-inducible factor 1 (HIF1). These identifications provide clear support for oxygen being the whole animal limiting factor at least in acute short-term temperature challenges. The classical heat shock proteins were not up-regulated during this trial, although numerous clones for each were present on the gene chip, confirming the lack of this response in this species. These data significantly increase our knowledge of the cellular stress response from animals in this unique environment.