A comparative study of tissue protein synthesis rates in an Antarctic, Harpagifer antarcticus and a temperate, Lipophrys pholis teleost.

The affect of temperature on tissue protein synthesis rates has been reported in temperate and tropical, but not Antarctic fishes. Previous studies have generally demonstrated low growth rates in Antarctic fish species in comparison to temperate relatives and elevated levels of protein turnover. This study investigates how low temperatures effect tissue protein synthesis and hence tissue growth in a polar fish species. Groups of Antarctic, Harpagifer antarcticus and temperate, Lipophrys pholis, were acclimated to a range of overlapping water temperatures and protein synthesis was measure in white muscle (WM), liver and gastrointestinal tract (GIT). WM protein synthesis rates increased linearly with temperature in both species (H. antarcticus 0.16-0.23%.d-1, L. pholis, 0.31-0.76%.d-1), while liver (H. antarcticus 0.24-0.27%.d-1, L. pholis, 0.44-1.03%.d-1) and GIT were unaffected by temperature in H. antarcticus but increased non-linearly in L.pholis (H. antarcticus 0.22-0.26%.d-1, L. pholis, 0.40-0.86%.d-1). RNA to protein ratios were unaffected by temperature in H. antarcticus but increased weakly, in L.pholis WM and liver. In L.pholis, RNA translational efficiency increased significantly with temperature in all tissues, but only in liver in H. antarcticus. At the overlapping temperature of 3 °C, protein synthesis (WM 26%, Liver, 39%, GIT, 35%) and RNA translational efficiency (WM 273%, Liver, 271%, GIT, 300%) were significantly lower in H. antarcticus than L.pholis, while RNA to protein ratios were significantly higher (WM 270%, Liver 170%, GIT 186%). Tissue specific effects of temperature are detectable in both species. This study provides the first evidence, that tissue protein synthesis rates are constrained in Antarctic fishes.

Testing the Resilience, Physiological Plasticity and Mechanisms Underlying Upper Temperature Limits of Antarctic Marine Ectotherms.

Antarctic marine ectotherms live in the constant cold and are characterised by limited resilience to elevated temperature. Here we tested three of the central paradigms underlying this resilience. Firstly, we assessed the ability of eight species, from seven classes representing a range of functional groups, to survive, for 100 to 303 days, at temperatures 0 to 4 °C above previously calculated long-term temperature limits. Survivors were then tested for acclimation responses to acute warming and acclimatisation, in the field, was tested in the seastar Odontaster validus collected in different years, seasons and locations within Antarctica. Finally, we tested the importance of oxygen limitation in controlling upper thermal limits. We found that four of 11 species studied were able to survive for more than 245 days (245-303 days) at higher than previously recorded temperatures, between 6 and 10 °C. Only survivors of the anemone Urticinopsis antarctica did not acclimate CTmax and there was no evidence of acclimatisation in O. validus. We found species-specific effects of mild hyperoxia (30% oxygen) on survival duration, which was extended (two species), not changed (four species) or reduced (one species), re-enforcing that oxygen limitation is not universal in dictating thermal survival thresholds. Thermal sensitivity is clearly the product of multiple ecological and physiological capacities, and this diversity of response needs further investigation and interpretation to improve our ability to predict future patterns of biodiversity.

Genomics of cold adaptations in the Antarctic notothenioid fish radiation.

Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes.

The environmental cellular stress response: the intertidal as a multistressor model.

The wild poses a multifaceted challenge to the maintenance of cellular function. Therefore, a multistressor approach is essential to predict the cellular mechanisms which promote homeostasis and underpin whole-organism tolerance. The intertidal zone is particularly dynamic, and thus, its inhabitants provide excellent models to assess mechanisms underpinning multistressor tolerance. Here, we critically review our current understanding of the regulation of the cellular stress response (CSR) under multiple abiotic stressors in intertidal organisms and consider to what extent a multistressor approach brings us closer to understanding responses in the wild. The function of the CSR has been well documented in laboratory and field exposures with a view to understanding single-stressor thermal effects. Multistressor studies still remain relatively limited in comparison but have applied three main approaches: (i) laboratory application of multiple stressors in isolation, (ii) multiple stressors applied in combination, and (iii) field-based correlation of multiple stressors against the CSR. The application of multiple stressors in isolation has allowed the identification of putative, shared stress pathways but overlooks non-additive stressor interactions on the CSR. Combined stressor studies are relatively limited in number but already highlight variable effects on the CSR dependent upon stressor type, timing, and magnitude. Field studies have allowed the identification of responsive components of the CSR to various stressors in situ but are correlative, not causative. A combined approach involving laboratory multistressor studies linking the CSR to whole-organism tolerance as well as field studies is required if we are to understand the role of the CSR in the natural environment.

Evolutionary conservation and divergence of the transcriptional regulation of bivalve shell secretion across life-history stages.

Adult molluscs produce shells with diverse morphologies and ornamentations, different colour patterns and microstructures. The larval shell, however, is a phenotypically more conserved structure. How do developmental and evolutionary processes generate varying diversity at different life-history stages within a species? Using live imaging, histology, scanning electron microscopy and transcriptomic profiling, we have described shell development in a heteroconchian bivalve, the Antarctic clam, Laternula elliptica, and compared it to adult shell secretion processes in the same species. Adult downstream shell genes, such as those encoding extracellular matrix proteins and biomineralization enzymes, were largely not expressed during shell development. Instead, a development-specific downstream gene repertoire was expressed. Upstream regulatory genes such as transcription factors and signalling molecules were largely conserved between developmental and adult shell secretion. Comparing heteroconchian data with recently reported pteriomorphian larval shell development data suggests that, despite being phenotypically more conserved, the downstream effectors constituting the larval shell 'tool-kit' may be as diverse as that of adults. Overall, our new data suggest that a larval shell formed using development-specific downstream effector genes is a conserved and ancestral feature of the bivalve lineage, and possibly more broadly across the molluscs.

Life in the freezer: protein metabolism in Antarctic fish.

Whole-animal, in vivo protein metabolism rates have been reported in temperate and tropical, but not Antarctic fish. Growth in Antarctic species is generally slower than lower latitude species. Protein metabolism data for Antarctic invertebrates show low rates of protein synthesis and unusually high rates of protein degradation. Additionally, in Antarctic fish, increasing evidence suggests a lower frequency of successful folding of nascent proteins and reduced protein stability. This study reports the first whole-animal protein metabolism data for an Antarctic fish. Groups of Antarctic, Harpagifer antarcticus, and temperate, Lipophrys pholis, fish were acclimatized to a range of overlapping water temperatures and food consumption, whole-animal growth and protein metabolism measured. The rates of protein synthesis and growth in Antarctic, but not temperate fish, were relatively insensitive to temperature and were significantly lower in H. antarcticus at 3°C than in L. pholis. Protein degradation was independent of temperature in H. antarcticus and not significantly different to L. pholis at 3°C, while protein synthesis retention efficiency was significantly higher in L. pholis than H. antarcticus at 3°C. These results suggest Antarctic fish degrade a significantly larger proportion of synthesized protein than temperate fish, with fundamental energetic implications for growth at low temperatures.

Molecular Responses to Thermal and Osmotic Stress in Arctic Intertidal Mussels (Mytilus edulis): The Limits of Resilience.

Increases in Arctic temperatures have accelerated melting of the Greenland icesheet, exposing intertidal organisms, such as the blue mussel Mytilus edulis, to high air temperatures and low salinities in summer. However, the interaction of these combined stressors is poorly described at the transcriptional level. Comparing expression profiles of M. edulis from experimentally warmed (30 °C and 33 °C) animals kept at control (23‱) and low salinities (15‱) revealed a significant lack of enrichment for Gene Ontology terms (GO), indicating that similar processes were active under all conditions. However, there was a progressive increase in the abundance of upregulated genes as each stressor was applied, with synergistic increases at 33 °C and 15‱, suggesting combined stressors push the animal towards their tolerance thresholds. Further analyses comparing the effects of salinity alone (23‱, 15‱ and 5‱) showed high expression of stress and osmoregulatory marker genes at the lowest salinity, implying that the cell is carrying out intracellular osmoregulation to maintain the cytosol as hyperosmotic. Identification of aquaporins and vacuolar-type ATPase transcripts suggested the cell may use fluid-filled cavities to excrete excess intracellular water, as previously identified in embryonic freshwater mussels. These results indicate that M. edulis has considerable resilience to heat stress and highly efficient mechanisms to acclimatise to lowered salinity in a changing world.

Variable heat shock response in Antarctic biofouling serpulid worms.

The classical heat shock response (HSR) with up-regulation of hsp70 in response to warming is often absent in Antarctic marine species. Whilst in Antarctic fish, this is due to a mutation in the gene promoter region resulting in permanent constitutive expression of the inducible form of hsp70; there are further questions as to whether evolution to life below 0 °C has resulted in a generalised alteration to the HSR in Antarctic marine invertebrates. However, the number of species investigated to date is limited. In the first evaluation of the HSR in two spirorbid polychaetes Romanchella perrieri and Protolaeospira stalagmia, we show highly variable results of HSR induction depending on warming regimes. These animals were subjected to in situ warming (+ 1 °C and + 2 °C above ambient conditions) using heated settlement panels for 18 months, and then the HSR was tested in R. perrieri using acute and chronic temperature elevation trials. The classic HSR was not induced in response to acute thermal challenge in this species (2 h at 15 °C) and significant down-regulation of hsp90 occurred during chronic warming at 4 °C for 30 days. Analysis of heat shock protein (HSP) genes in a transcriptome study of P. stalagmia, which had been warmed in situ for 18 months, showed up-regulation of HSP70 and HSP90 family members, thus further emphasising the complexity of the response in Antarctic marine species. It is increasingly apparent that the Antarctic HSR has evolved in a species-specific manner to life in the cold.

Sweepstake reproductive success and collective dispersal produce chaotic genetic patchiness in a broadcast spawner.

A long-standing paradox of marine populations is chaotic genetic patchiness (CGP), temporally unstable patterns of genetic differentiation that occur below the geographic scale of effective dispersal. Several mechanisms are hypothesized to explain CGP including natural selection, spatiotemporal fluctuations in larval source populations, self-recruitment, and sweepstake reproduction. Discriminating among them is extremely difficult but is fundamental to understanding how marine organisms reproduce and disperse. Here, we report a notable example of CGP in the Antarctic limpet, an unusually tractable system where multiple confounding explanations can be discounted. Using population genomics, temporally replicated sampling, surface drifters, and forward genetic simulations, we show that CGP likely arises from an extreme sweepstake event together with collective larval dispersal, while selection appears to be unimportant. Our results illustrate the importance of neutral demographic forces in natural populations and have important implications for understanding the recruitment dynamics, population connectivity, local adaptation, and resilience of marine populations.

Large within, and between, species differences in marine cellular responses: Unpredictability in a changing environment.

Predicting the impacts of altered environments on future biodiversity requires a detailed understanding of organism responses to change. To date, studies evaluating mechanisms underlying marine organism stress responses have largely concentrated on oxygen limitation and the use of heat shock proteins as biomarkers. However, whether these biomarkers represent responses that are consistent across species and different environmental stressors remains open to question. Here we show that responses to four different thermal stresses (three rates of thermal ramping (1 °C h-1, 1 °C day-1 or 1 °C 3 day-1) and a three-month acclimation to warming of 2 °C) applied to three species of Antarctic marine invertebrate produced highly individual responses in gene expression profiles, both within and between species. Mapping the gene expression profiles from each treatment for each of the three species, identified considerable difference in numbers of differentially regulated transcripts ranging from 10 to 3011. When these data were correlated across the different temperature treatments, there was no evidence for a common response with only 0-2 transcripts shared between all four treatments within any one species. There were also no shared differentially expressed genes across species, even at the same thermal ramping rates. The classical cellular stress response (CSR) i.e. up-regulation of heat shock proteins, was only strongly present in two species at the fastest ramping rate of 1 °C h-1, albeit with different sets of stress genes expressed in each species. These data demonstrate the wide variability in response to warming at the molecular level in marine species. Therefore, identification of biodiversity stress responses engendered by changing conditions will require evaluation at the species level using targeted key members of the ecosystem, strongly correlated to the local biotic and abiotic factors.

Transcriptomic analysis of shell repair and biomineralization in the blue mussel, Mytilus edulis.

BACKGROUND: Biomineralization by molluscs involves regulated deposition of calcium carbonate crystals within a protein framework to produce complex biocomposite structures. Effective biomineralization is a key trait for aquaculture, and animal resilience under future climate change. While many enzymes and structural proteins have been identified from the shell and in mantle tissue, understanding biomieralization is impeded by a lack of fundamental knowledge of the genes and pathways involved. In adult bivalves, shells are secreted by the mantle tissue during growth, maintenance and repair, with the repair process, in particular, amenable to experimental dissection at the transcriptomic level in individual animals. RESULTS: Gene expression dynamics were explored in the adult blue mussel, Mytilus edulis, during experimentally induced shell repair, using the two valves of each animal as a matched treatment-control pair. Gene expression was assessed using high-resolution RNA-Seq against a de novo assembled database of functionally annotated transcripts. A large number of differentially expressed transcripts were identified in the repair process. Analysis focused on genes encoding proteins and domains identified in shell biology, using a new database of proteins and domains previously implicated in biomineralization in mussels and other molluscs. The genes implicated in repair included many otherwise novel transcripts that encoded proteins with domains found in other shell matrix proteins, as well as genes previously associated with primary shell formation in larvae. Genes with roles in intracellular signalling and maintenance of membrane resting potential were among the loci implicated in the repair process. While haemocytes have been proposed to be actively involved in repair, no evidence was found for this in the M. edulis data. CONCLUSIONS: The shell repair experimental model and a newly developed shell protein domain database efficiently identified transcripts involved in M. edulis shell production. In particular, the matched pair analysis allowed factoring out of much of the inherent high level of variability between individual mussels. This snapshot of the damage repair process identified a large number of genes putatively involved in biomineralization from initial signalling, through calcium mobilization to shell construction, providing many novel transcripts for future in-depth functional analyses.

A Bivalve Biomineralization Toolbox.

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization SMPs derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by the analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis, and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid-base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.

Transcriptional frontloading contributes to cross-tolerance between stressors.

The adaptive value of phenotypic plasticity for performance under single stressors is well documented. However, plasticity may only truly be adaptive in the natural multifactorial environment if it confers resilience to stressors of a different nature, a phenomenon known as cross-tolerance. An understanding of the mechanistic basis of cross-tolerance is essential to aid prediction of species resilience to future environmental change. Here, we identified mechanisms underpinning cross-tolerance between two stressors predicted to increasingly challenge aquatic ecosystems under climate change, chronic warming and hypoxia, in an ecologically-important aquatic invertebrate. Warm acclimation improved hypoxic performance through an adaptive hypometabolic strategy and changes in the expression of hundreds of genes that are important in the response to hypoxia. These 'frontloaded' genes showed a reduced reaction to hypoxia in the warm acclimated compared to the cold acclimated group. Frontloaded genes included stress indicators, immune response and protein synthesis genes that are protective at the cellular level. We conclude that increased constitutive gene expression as a result of warm acclimation reduced the requirement for inducible stress responses to hypoxia. We propose that transcriptional frontloading contributes to cross-tolerance between stressors and may promote fitness of organisms in environments increasingly challenged by multiple anthropogenic threats.

Resilience in Greenland intertidal Mytilus: The hidden stress defense.

The Arctic is experiencing particularly rapid rates of warming, consequently invasive boreal species are now able to survive the less extreme Arctic winter temperatures. Whilst persistence of intertidal and terrestrial species in the Arctic is primarily determined by their ability to tolerate the freezing winters, air temperatures in the Arctic summer can reach 36 °C in the intertidal, which is beyond the upper thermal limits of many marine species. This is normally lethal for the conspicuous ecosystem engineer Mytilus edulis. Transcriptomic analyses were undertaken on both in situ collected and experimentally warmed animals to understand whether M. edulis is able to tolerate these very high summer temperatures. Surprisingly there was no significant enrichment for Gene Ontology terms (GO) when comparing the inner and outer fjord intertidal animals with outer fjord subtidal (control) animals, representing animals collected at 27 °C, 19 °C and 3 °C respectively. This lack of differentiation indicated a wide acclimation ability in this species. Conversely, significant enrichment for processes such as signal transduction, cytoskeleton and cellular protein modification was identified in the expression profiles of the 22 °C and 32 °C experimentally heated animals. This difference in gene expression between in situ collected and experimentally warmed animals was almost certainly due to the former being acclimated to a fluctuating, but predictable, temperature regime, which has increased their thermal tolerances. Interestingly, there was no evidence for enrichment of the classical cellular stress response in any of the animals sampled. Identification of a massive expansion of the HSPA12 heat shock protein 70 kDa gene family presented the possibility of these genes acting as intertidal regulators underpinning thermal resilience. This expansion has resulted in a modified cellular stress response, as an evolutionary adaptation to the rigour of the invasive intertidal life style. Thus, M. edulis appear to have considerable capacity to withstand the current rates of Arctic warming, and the very large attendant thermal variation.

Deciphering mollusc shell production: the roles of genetic mechanisms through to ecology, aquaculture and biomimetics.

Most molluscs possess shells, constructed from a vast array of microstructures and architectures. The fully formed shell is composed of calcite or aragonite. These CaCO3 crystals form complex biocomposites with proteins, which although typically less than 5% of total shell mass, play significant roles in determining shell microstructure. Despite much research effort, large knowledge gaps remain in how molluscs construct and maintain their shells, and how they produce such a great diversity of forms. Here we synthesize results on how shell shape, microstructure, composition and organic content vary among, and within, species in response to numerous biotic and abiotic factors. At the local level, temperature, food supply and predation cues significantly affect shell morphology, whilst salinity has a much stronger influence across latitudes. Moreover, we emphasize how advances in genomic technologies [e.g. restriction site-associated DNA sequencing (RAD-Seq) and epigenetics] allow detailed examinations of whether morphological changes result from phenotypic plasticity or genetic adaptation, or a combination of these. RAD-Seq has already identified single nucleotide polymorphisms associated with temperature and aquaculture practices, whilst epigenetic processes have been shown significantly to modify shell construction to local conditions in, for example, Antarctica and New Zealand. We also synthesize results on the costs of shell construction and explore how these affect energetic trade-offs in animal metabolism. The cellular costs are still debated, with CaCO3 precipitation estimates ranging from 1-2 J/mg to 17-55 J/mg depending on experimental and environmental conditions. However, organic components are more expensive (~29 J/mg) and recent data indicate transmembrane calcium ion transporters can involve considerable costs. This review emphasizes the role that molecular analyses have played in demonstrating multiple evolutionary origins of biomineralization genes. Although these are characterized by lineage-specific proteins and unique combinations of co-opted genes, a small set of protein domains have been identified as a conserved biomineralization tool box. We further highlight the use of sequence data sets in providing candidate genes for in situ localization and protein function studies. The former has elucidated gene expression modularity in mantle tissue, improving understanding of the diversity of shell morphology synthesis. RNA interference (RNAi) and clustered regularly interspersed short palindromic repeats - CRISPR-associated protein 9 (CRISPR-Cas9) experiments have provided proof of concept for use in the functional investigation of mollusc gene sequences, showing for example that Pif (aragonite-binding) protein plays a significant role in structured nacre crystal growth and that the Lsdia1 gene sets shell chirality in Lymnaea stagnalis. Much research has focused on the impacts of ocean acidification on molluscs. Initial studies were predominantly pessimistic for future molluscan biodiversity. However, more sophisticated experiments incorporating selective breeding and multiple generations are identifying subtle effects and that variability within mollusc genomes has potential for adaption to future conditions. Furthermore, we highlight recent historical studies based on museum collections that demonstrate a greater resilience of molluscs to climate change compared with experimental data. The future of mollusc research lies not solely with ecological investigations into biodiversity, and this review synthesizes knowledge across disciplines to understand biomineralization. It spans research ranging from evolution and development, through predictions of biodiversity prospects and future-proofing of aquaculture to identifying new biomimetic opportunities and societal benefits from recycling shell products.

Gene network analyses support subfunctionalization hypothesis for duplicated hsp70 genes in the Antarctic clam.

A computationally predicted gene regulatory network (GRN), generated from mantle-specific gene expression profiles in the Antarctic clam Laternula elliptica, was interrogated to test the regulation and interaction of duplicated inducible hsp70 paralogues. hsp70A and hsp70B were identified in the GRN with each paralogue falling into unique submodules that were linked together by a single shared second neighbour. Annotations associated with the clusters in each submodule suggested that hsp70A primarily shares regulatory relationships with genes encoding ribosomal proteins, where it may have a role in protecting the ribosome under stress. hsp70B, on the other hand, interacted with a suite of genes involved in signalling pathways, including four transcription factors, cellular response to stress and the cytoskeleton. Given the contrasting submodules and associated annotations of the two hsp70 paralogues, the GRN analysis suggests that each gene is carrying out additional separate functions, as well as being involved in the traditional chaperone heat stress response, and therefore supports the hypothesis that subfunctionalization has occurred after gene duplication. The GRN was specifically produced from experiments investigating biomineralization; however, this study shows the utility of such data for investigating multiple questions concerning gene duplications, interactions and putative functions in a non-model species.

Molecular mechanisms of biomineralization in marine invertebrates.

Much recent marine research has been directed towards understanding the effects of anthropogenic-induced environmental change on marine biodiversity, particularly for those animals with heavily calcified exoskeletons, such as corals, molluscs and urchins. This is because life in our oceans is becoming more challenging for these animals with changes in temperature, pH and salinity. In the future, it will be more energetically expensive to make marine skeletons and the increasingly corrosive conditions in seawater are expected to result in the dissolution of these external skeletons. However, initial predictions of wide-scale sensitivity are changing as we understand more about the mechanisms underpinning skeletal production (biomineralization). These studies demonstrate the complexity of calcification pathways and the cellular responses of animals to these altered conditions. Factors including parental conditioning, phenotypic plasticity and epigenetics can significantly impact the production of skeletons and thus future population success. This understanding is paralleled by an increase in our knowledge of the genes and proteins involved in biomineralization, particularly in some phyla, such as urchins, molluscs and corals. This Review will provide a broad overview of our current understanding of the factors affecting skeletal production in marine invertebrates. It will focus on the molecular mechanisms underpinning biomineralization and how knowledge of these processes affects experimental design and our ability to predict responses to climate change. Understanding marine biomineralization has many tangible benefits in our changing world, including improvements in conservation and aquaculture and exploitation of natural calcified structure design using biomimicry approaches that are aimed at producing novel biocomposites.