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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.

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.

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.

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.

Background Marine soft sediments are some of the most widespread habitats in the ocean, playing a vital role in global carbon cycling, but are amongst the least studied with regard to species composition and ecosystem functioning. This is particularly true of the Polar Regions, which are currently undergoing rapid climate change, the impacts of which are poorly understood. Compared to other latitudes, Polar sediment habitats also experience additional environmental drivers of strong seasonality and intense disturbance from iceberg scouring, which are major structural forces for hard substratum communities. This study compared sediment assemblages from two coves, near Rothera Point, Antarctic Peninsula, 67°S in order to understand the principal drivers of community structure, for the first time, evaluating composition across all size classes from mega- to micro-fauna. Results Morpho-taxonomy identified 77 macrofaunal species with densities of 464–16,084 individuals m−2. eDNA metabarcoding of microfauna, in summer only, identified a higher diversity, 189 metazoan amplicon sequence variants (ASVs) using the 18S ribosomal RNA and 249 metazoan ASVs using the mitochondrial COI gene. Both techniques recorded a greater taxonomic diversity in South Cove than Hangar Cove, with differences in communities between the coves, although the main taxonomic drivers varied between techniques. Morphotaxonomy identified the main differences between coves as the mollusc, Altenaeum charcoti, the cnidarian Edwardsia sp. and the polychaetes from the family cirratulidae. Metabarcoding identified greater numbers of species of nematodes, crustaceans and Platyhelminthes in South Cove, but more bivalve species in Hangar Cove. There were no detectable differences in community composition, measured through morphotaxonomy, between seasons, years or due to iceberg disturbance. Conclusions This study found that unlike hard substratum communities the diversity of Antarctic soft sediment communities is correlated with the same factors as other latitudes. Diversity was significantly correlated with grain size and organic content, not iceberg scour. The increase in glacial sediment input as glaciers melt, may therefore be more important than increased iceberg disturbance. Electronic supplementary material The online version of this article (10.1186/s12898-019-0244-x) contains supplementary material, which is available to authorized users.

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12192-021-01235-z.

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.

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.

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.

We present a new software tool in R, the PhylSim package, which allows the user to simulate trait evolution on a phylogeny, while varying the speciation rate and evolutionary model over the course of the simulation. In particular, the user can specify any number of regimes, with a different speciation rate for each regime, or set the speciation rate to vary when the value of a trait exceeds a certain threshold. The evolutionary model can also be varied as many times as necessary over the course of the simulation, to account for traits tending toward different optima at different periods in evolutionary history. We illustrate the use of the package with an example, a simulation of the adaptive radiation of Antarctic notothenioids in the Southern Ocean.

Motivation The molecular processes regulating molluscan shell production remain relatively uncharacterized, despite the clear evolutionary and societal importance of biomineralization. Results Here we built the first computationally predicted gene regulatory network (GRN) for molluscan biomineralization using Antarctic clam (Laternula elliptica) mantle gene expression data produced over an age-categorized shell damage-repair time-course. We used previously published in vivo in situ hybridization expression data to ground truth gene interactions predicted by the GRN and show that candidate biomineralization genes from different shell layers, and hence microstructures, were connected in unique modules. We characterized two biomineralization modules of the GRN and hypothesize that one module is responsible for translating the extracellular proteins required for growing, repairing or remodelling the nacreous shell layer, whereas the second module orchestrates the transport of both ions and proteins to the shell secretion site, which are required during normal shell growth, and repair. Our findings demonstrate that unbiased computational methods are particularly valuable for studying fundamental biological processes and gene interactions in non-model species where rich sources of gene expression data exist, but annotation rates are poor and the ability to carry out true functional tests are still lacking. Availability and implementation The raw RNA-Seq data is freely available for download from NCBI SRA (Accession: PRJNA398984), the assembled and annotated transcriptome can be viewed and downloaded from molluscDB (ensembl.molluscdb.org) and in addition, the assembled transcripts, reconstructed GRN, modules and detailed annotations are all available as Supplementary Files. Supplementary information Supplementary data are available at Bioinformatics online.

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.

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. FCT: UID/Multi/04326/2019; European Marine Biological Research Infrastructure Cluster-EMBRIC (EU H2020 research and innovation program) 654008; European Union Seventh Framework Programme [FP7] ITN project 'CACHE: Calcium in a Changing Environment' under REA 60505; NERC Natural Environment Research Council NE/J500173/1 info:eu-repo/semantics/publishedVersion

The evolution of antifreeze glycoproteins has enabled notothenioid fish to flourish in the freezing waters of the Southern Ocean. Whilst successful at the biodiversity level to life in the cold, paradoxically at the cellular level these stenothermal animals have problems producing, folding and degrading proteins at their ambient temperatures of down to −1.86°C. In this first multi-species transcriptome comparison of the amino acid composition of notothenioid proteins with temperate teleost proteins, we show that, unlike psychrophilic bacteria, Antarctic fish provide little evidence for the mass alteration of protein amino acid composition to enhance protein folding and reduce protein denaturation in the cold. The exception was the significant over-representation of positions where leucine in temperate fish proteins was replaced by methionine in the notothenioid orthologues. Although methionine may increase stability in critical proteins, we hypothesise that a more likely explanation for the extra methionines is that they have been preferentially assimilated into the genome because they act as redox sensors. This redox hypothesis is supported by the enrichment of duplicated genes within the notothenioid transcriptomes which centre around Mapk signalling, a major pathway in the cellular cascades associated with responses to environmental stress. Whilst notothenioid fish show cold-associated problems with protein homeostasis, they may have modified only a selected number of biochemical pathways to work efficiently below 0°C. Even a slight warming of the Southern Ocean might disrupt the critical functions of this handful of key pathways with considerable impacts for the functioning of this ecosystem in the future.

Morphologies of animals can vary significantly, and variation in characters in relation to taxonomic discrimination is often either underestimated or not evaluated. It can, however, make the identification and separation of species on morphological criteria less sound. Here, we investigate variation in morphological characters used to separate species of the Antarctic starfish genus Odontaster. Three closely related species in this genus have recently been identified using molecular barcoding (Odontaster validus, Odontaster roseus and Odontaster pearsei), and their morphologies have also been reported to be distinct, allowing them to be identified as separate species and not cryptic on morphological criteria. Our study shows that variation in overall shape, colour and for the number of spinelets on the abactinal plates of individuals in a single population of O. validus collected from one locality covers the whole range of that reported for O. roseus and overlaps that of O. pearsei. These may be separate species at the molecular level, but care needs to be used when using morphological criteria to separate them, and more care is needed to include assessment of morphological variation when constructing taxonomic keys for these species.

VAS was funded by a NERC DTG studentship (Project Reference: NE/J500173/1) to the British Antarctic Survey. MSC and LSP were financed by NERC core funding to the British Antarctic Survey. Acclimation, via phenotypic flexibility, is a potential means for a fast response to climate change. Understanding the molecular mechanisms underpinning phenotypic flexibility can provide a fine-scale cellular understanding of how organisms acclimate. In the last 30 years, Mya truncata populations around the UK have faced an average increase in sea surface temperature of 0.7 °C and further warming of between 1.5 and 4 °C, in all marine regions adjacent to the UK, is predicted by the end of the century. Hence, data are required on the ability of M. truncata to acclimate to physiological stresses, and most notably, chronic increases in temperature. Animals in the present study were exposed to chronic heat-stress for 2 months prior to shell damage and subsequently, only 3, out of 20 damaged individuals, were able to repair their shells within 2 weeks. Differentially expressed genes (between control and damaged animals) were functionally enriched with processes relating to cellular stress, the immune response and biomineralisation. Comparative transcriptomics highlighted genes, and more broadly molecular mechanisms, that are likely to be pivotal in this lack of acclimation. This study demonstrates that discovery-led transcriptomic profiling of animals during stress-response experiments can shed light on the complexity of biological processes and changes within organisms that can be more difficult to detect at higher levels of biological organisation. Publisher PDF

Marine encrusting communities play vital roles in benthic ecosystems and have major economic implications with regards to biofouling. However, their ability to persist under projected warming scenarios remains poorly understood and is difficult to study under realistic conditions. Here, using heated settlement panel technologies, we show that after 18 months Antarctic encrusting communities do not acclimate to either +1 °C or +2 °C above ambient temperatures. There is significant up-regulation of the cellular stress response in warmed animals, their upper lethal temperatures decline with increasing ambient temperature and population genetic analyses show little evidence of differential survival of genotypes with treatment. By contrast, biofilm bacterial communities show no significant differences in community structure with temperature. Thus, metazoan and bacterial responses differ dramatically, suggesting that ecosystem responses to future climate change are likely to be far more complex than previously anticipated., Genetic adaptation and physiological acclimation can potentially buffer species against climate change. Here, the authors perform a long-term warming experiment of Antarctic encrusting communities and show that focal animal species failed to acclimate and lacked genetic variation in tolerance to warming.

The pre-conditioning of adult marine invertebrates to altered conditions, such as low pH, can significantly impact offspring outcomes, a process which is often referred to as transgenerational plasticity (TGP). This study describes for the first time, the gene expression profiles associated with TGP in the green sea urchin Psammechinus miliaris and evaluates the transcriptional contribution to larval resilience. RNA-Seq was used to determine how the expression profiles of larvae spawned into low pH from pre-acclimated adults differed to those of larvae produced from adults cultured under ambient pH. The main findings demonstrated that adult conditioning to low pH critically pre-loads the embryonic transcriptional pool with antioxidants to prepare the larvae for the “new” conditions. In addition, the classic cellular stress response, measured via the production of heat shock proteins (the heat shock response (HSR)), was separately evaluated. None of the early stage larvae either spawned in low pH (produced from both ambient and pre-acclimated adults) or subjected to a separate heat shock experiment were able to activate the full HSR as measured in adults, but the capacity to mount an HSR increased as development proceeded. This compromised ability clearly contributes to the vulnerability of early stage larvae to acute environmental challenge.

Bivalves have evolved a range of complex shell forming mechanisms that are reflected by their incredible diversity in shell mineralogy and microstructures. A suite of proteins exported to the shell matrix space plays a significant role in controlling these features, in addition to underpinning some of the physical properties of the shell itself. Although, there is a general consensus that a minimum basic protein tool kit is required for shell construction, to date, this remains undefined. In this study, the shell matrix proteins (SMPs) of four highly divergent bivalves (The Pacific oyster, Crassostrea gigas; the blue mussel, Mytilus edulis; the clam, Mya truncata, and the king scallop, Pecten maximus) were analyzed in an identical fashion using proteomics pipeline. This enabled us to identify the critical elements of a “basic tool kit” for calcification processes, which were conserved across the taxa irrespective of the shell morphology and arrangement of the crystal surfaces. In addition, protein domains controlling the crystal layers specific to aragonite and calcite were also identified. Intriguingly, a significant number of the identified SMPs contained domains related to immune functions. These were often are unique to each species implying their involvement not only in immunity, but also environmental adaptation. This suggests that the SMPs are selectively exported in a complex mix to endow the shell with both mechanical protection and biochemical defense.

Members of the Myidae family are ecologically and economically important, but there is currently very little molecular data on these species. The present study sequenced and assembled the mantle transcriptome of Mya truncata from the North West coast of Scotland and identified candidate biomineralisation genes. RNA-Seq reads were assembled to create 20,106 contigs in a de novo transciptome, 18.81% of which were assigned putative functions using BLAST sequence similarity searching (cuttoff E-value 1E − 10). The most highly expressed genes were compared to the Antarctic clam (Laternula elliptica) and showed that many of the dominant biological functions (muscle contraction, energy production, biomineralisation) in the mantle were conserved. There were however, differences in the constitutive expression of heat shock proteins, which were possibly due to the M. truncata sampling location being at a relatively low latitude, and hence relatively warm, in terms of the global distribution of the species. Phylogenetic analyses of the Tyrosinase proteins from M. truncata showed a gene expansion which was absent in L. elliptica. The tissue distribution expression patterns of putative biomineralisation genes were investigated using quantitative PCR, all genes showed a mantle specific expression pattern supporting their hypothesised role in shell secretion. The present study provides some preliminary insights into how clams from different environments – temperate versus polar – build their shells. In addition, the transcriptome data provides a valuable resource for future comparative studies investigating biomineralisation.

The calcareous shells secreted by bivalve molluscs display diverse and species specific structural compositions, which indicates possible divergent biomineralization processes. Thus, studying multiple mollusc species will provide a more comprehensive understanding of shell formation. Here, the transcriptomes of the mantle tissues responsible for shell deposition were characterized in three commercially relevant bivalve species. Using high-throughput sequencing and bioinformatics tools, de novo transcriptome assemblies of mantle tissues were generated for the mussel Mytilus edulis, the oyster Crassostrea gigas and the scallop Pecten maximus. These transcriptomes were annotated, and contigs with similarity to proteins known to have shell formation roles in other species were identified. Comparison of the shell formation specific proteins in the three bivalves indicates the possibility of species specific shell proteins.

Cold polar marine species have very slow embryonic and larval development rates. Antarctic echinoids, bivalve molluscs and brooding gastropods develop up to 12 times slower than temperate and tropical species, departing from Arrhenius relationships and outside the normal Q 10 of 2–3 associated with 10 °C reductions in biochemical reaction rates. The slowing of development at temperatures around 0 °C has been reported previously to be much greater than for other parts of the global marine temperature range. Here we spawned and reared embryos and larvae of the Antarctic limpet Nacella polaris at 0.6 °C to the post-torsional veliger stage. Spawned eggs were 221 µm in diameter. Development rates were three times slower than any previously reported for patellogastropod limpets, with first division at 2.5 h post-fertilisation, the gastrula stage being reached after 55 h, hatching occurring after 70–75 h and the trochophore stage being reached after around 100 h. The marked slowing of development around 0 °C matches that previously reported for other polar taxa. This supports the hypothesis that there is a cold marine physiological transition to markedly slower physiological rates at temperatures near 0 °C. The transition is especially apparent here for development, but has also been reported for growth, both of which involve significant protein synthesis.

The physiological processes driving the rapid rates of calcification in larval bivalves are poorly understood. Here, we use a calcification substrate‐limited approach (low dissolved inorganic carbon, C T) and mRNA sequencing to identify proteins involved in bicarbonate acquisition during shell formation. As a secondary approach, we examined expression of ion transport and shell matrix proteins (SMPs) over the course of larval development and shell formation. We reared four families of Mytilus edulis under ambient (ca. 1865 µmol/kg) and low C T (ca. 941 µmol/kg) conditions and compared expression patterns at six developmental time points. Larvae reared under low C T exhibited a developmental delay, and a small subset of contigs was differentially regulated between ambient and low C T conditions. Of particular note was the identification of one contig encoding an anion transporter (SLC26) which was strongly upregulated (2.3–2.9 fold) under low C T conditions. By analyzing gene expression profiles over the course of larval development, we are able to isolate sequences encoding ion transport and SMPs to enhance our understanding of cellular pathways underlying larval calcification processes. In particular, we observe the differential expression of contigs encoding SLC4 family members (sodium bicarbonate cotransporters, anion exchangers), calcium‐transporting ATPases, sodium/calcium exchangers, and SMPs such as nacrein, tyrosinase, and transcripts related to chitin production. With a range of candidate genes, this work identifies ion transport pathways in bivalve larvae and by applying comparative genomics to investigate temporal expression patterns, provides a foundation for further studies to functionally characterize the proteins involved in larval calcification.

1. Phenotypic plasticity is essential for the persistence of organisms under changing environmental conditions but the control of the relevant cellular mechanisms including which genes are involved and the regulation of those genes remains unclear. One way to address this issue is to evaluate links between gene expression, methylation and phenotype using transplantation and common garden experiments within genetically homogeneous populations. 2. This approach was taken using the Antarctic limpet Nacella concinna. In this species, two distinct phenotypes are associated with the intertidal and subtidal zones. The in situ gene expression and methylation profiles of intertidal and subtidal cohorts were directly compared before and after reciprocal transplantation as well as after a common garden acclimation to aquarium conditions for 9 months. 3. Expression profiles showed significant modulation of cellular metabolism to habitat zone with the intertidal profile characterised by transcription modules for antioxidant production, DNA repair and the cytoskeleton reflecting the need to cope with continually fluctuating and stressful conditions including wave action, UV irradiation and desiccation. 4. Transplantation had an effect on gene expression. The subtidal animals transplanted to the intertidal zone modified their gene expression patterns towards that of an intertidal profile. In contrast, many of the antioxidant genes were still differentially expressed in the intertidal animals several weeks after transplantation into the relatively benign subtidal zone. 5. Furthermore, a core of genes involved in antioxidation was still preferentially expressed in intertidal animals at the end of the common garden experiment. Thus, acclimation in an aquarium tank for 9 months did not completely erase the intertidal gene expression profile. 6. Significant methylation differences were measured between intertidal and subtidal animals from the wild and after transplantation, which were reduced on common garden acclimation. This suggests that epigenetic factors play an important role in physiological flexibility associated with environmental niche.

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.

Summary Forecasting assemblage-level responses to climate change remains one of the greatest challenges in global ecology [1, 2]. Data from the marine realm are limited because they largely come from experiments using limited numbers of species [3], mesocosms whose interior conditions are unnatural [4], and long-term correlation studies based on historical collections [5]. We describe the first ever experiment to warm benthic assemblages to ecologically relevant levels in situ. Heated settlement panels were used to create three test conditions: ambient and 1°C and 2°C above ambient (predicted in the next 50 and 100 years, respectively [6]). We observed massive impacts on a marine assemblage, with near doubling of growth rates of Antarctic seabed life. Growth increases far exceed those expected from biological temperature relationships established more than 100 years ago by Arrhenius. These increases in growth resulted in a single " r -strategist" pioneer species (the bryozoan Fenestrulina rugula ) dominating seabed spatial cover and drove a reduction in overall diversity and evenness. In contrast, a 2°C rise produced divergent responses across species growth, resulting in higher variability in the assemblage. These data extend our ability to expand, integrate, and apply our knowledge of the impact of temperature on biological processes to predict organism, species, and ecosystem level ecological responses to regional warming.

An increasing number of studies are showing that Antarctic mega- and macrofauna are highly diverse, however, little is known about meiofaunal biodiversity in sediment communities, which are a vital part of a healthy and functional ecosystem. This is the first study to analyse community DNA (targeting meiofauna) using metabarcoding to investigate biodiversity levels in sediment communities of the Antarctic Peninsula. The results show that almost all of the meiofaunal biodiversity in the benthic habitat has yet to be characterised, levels of biodiversity were higher than expected and similar to temperate regions, albeit with the existence of potentially new and locally adapted species never described before at the molecular level. The Rothera meiofaunal sample sites showed four dominant eukaryotic groups, the nematodes, arthropods, platyhelminthes, and the annelids; some of which could comprise species complexes. Comparisons with deep-sea data from the same region suggest little exchange of Operational Taxonomic Units (OTUs) between depths with the nematodes prevalent at all depths, but sharing the shallow water benthos with the copepods. This study provides a preliminary analysis of benthic Antarctic Peninsula meiofauna using high throughput sequencing which substantiates how little is known on the biodiversity of one of the most diverse, yet underexplored communities of the Antarctic: the benthos. NERC within the Polar Sciences for Planet Earth Programme; NERC Antarctic Funding Initiative Collaborative Gearing Scheme [57]; CCMAR from the Portuguese Science Foundation [FCT.UID/Multi/04326/2013]; Portuguese Foundation for Science and Technology (FCT) [SFRH/BPD/80447/2014]; European Community Marie Curie International Incoming Fellowship project MARMEDIV [253251]

Understanding species' responses to environmental change underpins our abilities to make predictions on future biodiversity under any range of scenarios. In spite of the huge biodiversity in most ecosystems, a model species approach is often taken in environmental studies. To date, we still do not know how many species we need to study to input into models and inform on ecosystem‐level responses to change. In this study, we tested current paradigms on factors setting thermal limits by investigating the acute warming response of six Antarctic marine invertebrates: a crustacean Paraceradocus miersi, a brachiopod Liothyrella uva, two bivalve molluscs, Laternula elliptica, Aequiyoldia eightsii, a gastropod mollusc Marseniopsis mollis and an echinoderm Cucumaria georgiana. Each species was warmed at the rate of 1 °C h−1 and taken to the same physiological end point (just prior to heat coma). Their molecular responses were evaluated using complementary metabolomics and transcriptomics approaches with the aim of discovering the underlying mechanisms of their resilience or sensitivity to warming. The responses were species‐specific; only two showed accumulation of anaerobic end products and three exhibited the classical heat shock response with expression of HSP70 transcripts. These diverse cellular measures did not directly correlate with resilience to heat stress and suggested that each species may have a different critical point of failure. Thus, one unifying molecular mechanism underpinning response to warming could not be assigned, and no overarching paradigm was supported. This biodiversity in response makes future ecosystems predictions extremely challenging, as we clearly need to develop a macrophysiology‐type approach to cellular evaluations of the environmental stress response, studying a range of well‐rationalized members from different community levels and of different phylogenetic origins rather than extrapolating from one or two arbitrary model species.

Brittle stars are included within a whole range of species, which contribute to knowledge in the medically important area of tissue regeneration. All brittle stars regenerate lose limbs, but the rate at which this occurs is highly variable and species-specific. One of the slowest rates of arm regeneration reported so far is that of the Antarctic Ophionotus victoriae. Additionally, O. victoriae also has an unusual delay in the onset of regeneration of about 5months. Both processes are of interest for the areas of regeneration biology and adaptation to cold environments. One method of understanding the details of regeneration events in brittle stars is to characterise the genes involved. In the largest transcriptome study of any ophiuroid to date, we describe the results of mRNA pyrosequencing from pooled samples of regenerating arms of O. victoriae. The sequencing reads resulted in 18,000 assembled contiguous sequences of which 19% were putatively annotated by blast sequence similarity searching. We focus on the identification of major gene families and pathways with potential relevance to the regenerative processes including the Wnt/β-catenin pathway, Hox genes, the SOX gene family and the TGF beta signalling pathways. These data significantly increase the amount of ophiuroid sequences publicly available and provide candidate transcripts for the further investigation of the unusual regenerative process in this Antarctic ophiuroid.

The Antarctic clam Laternula elliptica lives almost permanently below 0 °C and therefore is a valuable and tractable model to study the mechanisms of biomineralisation in cold water. The present study employed a multidisciplinary approach using histology, immunohistochemistry, electron microscopy, proteomics and gene expression to investigate this process. Thirty seven proteins were identified via proteomic extraction of the nacreous shell layer, including two not previously found in nacre; a novel T-rich Mucin-like protein and a Zinc-dependent metalloprotease. In situ hybridisation of seven candidate biomineralisation genes revealed discrete spatial expression patterns within the mantle tissue, hinting at modular organisation, which is also observed in the mantle tissues of other molluscs. All seven of these biomineralisation candidates displayed evidence of multifunctionality and strong association with vesicles, which are potentially involved in shell secretion in this species.

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

In our changing world there is a critical need for scientists to provide evidence-based analyses to underpin future predictions of ecosystem functioning, particularly with regard to food security and ecosystem services. Climate change is increasingly impacting on our daily lives and nowhere is this more prescient than the marine environment with the increasing acidification of the World's oceans, commonly known as Ocean Acidification (OA).

This study investigated the effects of long-term incubation to near-future combined warming (+2 °C) and ocean acidification (−0.3 and −0.5 pH units) stressors, relative to current conditions (−0.3 °C and pH 8.0), on the energetics of food processing in the Antarctic sea urchin, Sterechinus neumayeri. After an extended incubation of 40 months, energy absorbed, energy lost through respiration and lost as waste were monitored through two feeding cycles. Growth parameters (mass of somatic and gonad tissues and the CHN content of gonad) were also measured. There were no significant effects of combined ocean acidification (OA) and temperature stressors on the growth of somatic or reproductive tissue. Despite more food being consumed in the low temperature control, once food processing and maintenance costs were subtracted, there were no significant effects of treatment on the scope for growth. The biggest significant differences were between amounts of food consumed during the two feeding cycles. More food was consumed by the low temperature (0 °C) control animals, indicating a potential effect of the changed conditions on digestive efficiency. Also, in November, more food was consumed, with a higher absorption efficiency, which resulted in a higher scope for growth in November than September and may reflect increased energetic needs associated with a switch to summer physiology. The effect of endogenous seasonal cycles and environmental variability on organism capacity is discussed.

The Arctic springtail, Megaphorura arctica, survives sub-zero temperatures in a dehydrated state via trehalose-dependent cryoprotective dehydration. Regulation of trehalose biosynthesis is complex; based in part on studies in yeast and fungi, its connection with oxidative stress caused by exposure of cells to oxidants, such as hydrogen peroxide (H2O2), or dehydration, is well documented. In this respect, we measured the amount of H2O2 and antioxidant enzyme activities (superoxide dismutases: copper, zinc—CuZnSOD and manganese containing–MnSOD, and catalase—CAT), as the regulatory components determining H2O2 concentrations, in Arctic springtails incubated at 5 °C (control) versus −2 °C (threshold temperature for trehalose biosynthesis). Because ecdysone also stimulates trehalose production in insects and regulates the expression of genes involved in redox homeostasis and antioxidant protection in Drosophila, we measured the levels of the active physiological form of ecdysone—20-hydroxyecdysone (20-HE). Significantly elevated H2O2 and 20-HE levels were observed in M. arctica incubated at −2 °C, supporting a link between ecdysone, H2O2, and trehalose levels during cryoprotective dehydration. CAT activity was found to be significantly lower in M. arctica incubated at −2 °C versus 5 °C, suggesting reduced H2O2 breakdown. Furthermore, measurement of the free radical composition in Arctic springtails incubated at 5 °C (controls) versus −2 °C by Electron Paramagnetic Resonance spectroscopy revealed melanin-derived free radicals at −2 °C, perhaps an additional source of H2O2. Our results suggest that H2O2 and ecdysone play important roles in the cryoprotective dehydration process in M. arctica, linked with the regulation of trehalose biosynthesis.

Regeneration of arms in brittle stars is thought to proceed slowly in low temperature environments. Here a survey of natural arm damage and arm regeneration rates is documented in the Antarctic brittle star Ophiura crassa. This relatively small ophiuroid, a detritivore found amongst red macroalgae, displays high levels of natural arm damage and repair. This is largely thought to be due to ice damage in the shallow waters it inhabits. The time scale of arm regener- ation was measured in an aquarium-based 10 mo experiment. There was a delayed regeneration phase of 7 mo before arm growth was detectable in this species. This is 2 mo longer than the longest time previously described, which was in another Antarctic ophiuroid, Ophionotus victo- riae. The subsequent regeneration of arms in O. crassa occurred at a rate of approximately 0.16 mm mo �1 . To date, this is the slowest regeneration rate known of any ophiuroid. The confir- mation that such a long delay before arm regeneration occurs in a second Antarctic species pro- vides strong evidence that this phenomenon is yet another characteristic feature of Southern Ocean species, along with deferred maturity, slowed growth and development rates. It is unclear whether delayed initial regeneration phases are adaptations to, or limitations of, low temperature environments.