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Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins—including gene expression, chromatin occupancy and three-dimensional conformation—we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral patterning mechanisms within the developing mandibular, hyoid and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralising BMP and endothelin signalling pathways and their effectors, the joint markers bapx1 and gdf5 and pro-chondrogenic transcription factors barx1 and gsc, and the dorsalising transcription factor pou3f3. Additionally, we find that mesenchymal expression ofeya1/six1is an ancestral feature of the mandibular arch of jawed vertebrates, while differences in notch signalling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the dorsoventral axis of the pharyngeal arches, includingscamp5as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.

The gill skeleton of cartilaginous fishes (sharks, skates, rays and holocephalans) exhibits a striking anterior–posterior polarity, with a series of fine appendages called branchial rays projecting from the posterior margin of the gill arch cartilages. We previously demonstrated in the skate (Leucoraja erinacea) that branchial rays derive from a posterior domain of pharyngeal arch mesenchyme that is responsive to Shh signalling from a distal gill arch epithelial ridge (GAER) signalling centre. However, how branchial ray progenitors are specified exclusively within posterior gill arch mesenchyme is not known. Here we show that genes encoding several Wnt ligands are expressed in the ectoderm immediately adjacent to the skate GAER, and that these Wnt signals are transduced largely in the anterior arch environment. Using pharmacological manipulation, we show that inhibition of Wnt signalling results in an anterior expansion of Shh signal transduction in developing skate gill arches, and in the formation of ectopic anterior branchial ray cartilages. Our findings demonstrate that ectodermal Wnt signalling contributes to gill arch skeletal polarity in skate by restricting Shh signal transduction and chondrogenesis to the posterior arch environment and highlights the importance of signalling interactions at embryonic tissue boundaries for cell fate determination in vertebrate pharyngeal arches.

The vast majority of extant vertebrate diversity lies within the bony and cartilaginous fish lineages of jawed vertebrates. There is a long history of elegant experimental investigation of development in bony vertebrate model systems (e.g., mouse, chick, frog and zebrafish). However, studies on the development of cartilaginous fishes (sharks, skates and rays) have, until recently, been largely descriptive, owing to the challenges of embryonic manipulation and culture in this group. This, in turn, has hindered understanding of the evolution of developmental mechanisms within cartilaginous fishes and, more broadly, within jawed vertebrates. The little skate (Leucoraja erinacea) is an oviparous cartilaginous fish and has emerged as a powerful and experimentally tractable developmental model system. Here, we discuss the collection, husbandry and management of little skate brood stock and eggs, and we present an overview of key stages of skate embryonic development. We also discuss methods for the manipulation and culture of skate embryos and illustrate the range of tools and approaches available for studying this system. Finally, we summarize a selection of recent studies on skate development that highlight the utility of this system for inferring ancestral anatomical and developmental conditions for jawed vertebrates, as well as unique aspects of cartilaginous fish biology.

Skates are cartilaginous fish whose novel body plan features remarkably enlarged wing-like pectoral fins that allow them to thrive in benthic environments. The molecular underpinnings of this unique trait, however, remain elusive. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins (gene expression, chromatin occupancy and three-dimensional (3D) conformation) we find skate-specific genomic rearrangements that alter the 3D regulatory landscape of genes involved in the planar cell polarity (PCP) pathway. Functional inhibition of PCP signaling resulted in marked reduction of anterior fin size, confirming this pathway as a major contributor of batoid fin morphology. We also identified a fin-specific enhancer that interacts with 3' HOX genes, consistent with the redeployment of Hox gene expression in anterior pectoral fins, and confirmed the potential of this element to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganizations and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

Single-cell sequencing technologies are revolutionizing biology, but they are limited by the need to dissociate live samples. Here, we present ACME (ACetic-MEthanol), a dissociation approach for single-cell transcriptomics that simultaneously fixes cells. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, and are sortable and permeable. As a proof of principle, we provide single-cell transcriptomic data of different species, using both droplet-based and combinatorial barcoding single-cell methods. ACME uses affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life. This work was supported by an MRC grant (MR/S007849/1) and a Royal Society Grant (RGS\R1\191278) to JS. HG-C was supported by a Nigel Groome studentship from Oxford Brookes University. PA-C was supported by an EMBO Long Term Fellowship (ALTF-217-2018). JN was supported by funding from a BBSRC grant (BB/M011224/1) and the Osk. Huttunen Foundation (Doctoral grant)

Single-cell sequencing technologies are revolutionizing biology, but are limited by the need to dissociate fresh samples that can only be fixed at later stages. We present ACME (ACetic-MEthanol) dissociation, a cell dissociation approach that fixes cells as they are being dissociated. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, can be sorted by Fluorescence-Activated Cell Sorting (FACS) and are permeable, enabling combinatorial single-cell transcriptomic approaches. As a proof of principle, we have performed SPLiT-seq with ACME cells to obtain around ∼34K single cell transcriptomes from two planarian species and identified all previously described cell types in similar proportions. ACME is based on affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.

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

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

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.

Microplastics (MPs) are prevalent in marine ecosystems. Because toxicants (termed here “co-contaminants”) can sorb to MPs, there is potential for MPs to alter co-contaminant bioavailability. Our objective was to demonstrate sorption of two co-contaminants with different physicochemistries [phenanthrene (Phe), log10Kow = 4.57; and 17α-ethinylestradiol (EE2), log10Kow = 3.67] to MPs; and assess whether co-contaminant bioavailability was increased after MP settlement. Bioavailability was indicated by gene expression in larval zebrafish. Both Phe and EE2 sorbed to MPs, which reduced bioavailability by a maximum of 33% and 48% respectively. Sorption occurred, but was not consistent with predictions based on co-contaminant physicochemistry (Phe having higher log10Kow was expected to have higher sorption). Contaminated MPs settled to the bottom of the exposures did not lead to increased bioavailability of Phe or EE2. Phe was 48% more bioavailable than predicted by a linear sorption model, organism-based measurements therefore contribute unique insight into MP co-contaminant bioavailability.

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.

Mya truncata, a soft shell clam, is presented as a new model to study biomineralization through a proteomics approach. In this study, the shell and mantle tissue were analysed in order to retrieve knowledge about the secretion of shell matrix proteins (SMPs). Out of 67 and 127 shell and mantle proteins respectively, 16 were found in both shell and mantle. Bioinformatic analysis of SMP sequences for domain prediction revealed the presence of several new domains such as fucolectin tachylectin-4 pentraxin-1 (FTP), scavenger receptor, alpha-2-macroglobulin (α2 M), lipocalin and myosin tail along with previously reported SMP domains such as chitinase, carbonic anhydrase, tyrosinase, sushi, and chitin binding. Interestingly, these newly predicted domains are attributed with molecular functions other than biomineralization. These findings suggest that shells may not only act as protective armour from predatory action, but could also actively be related to other functions such as immunity. In this context, the roles of SMPs in biomineralization need to be looked in a new perspective.

Marine debris, mostly consisting of plastic, is a global problem, negatively impacting wildlife, tourism and shipping. However, despite the durability of plastic, and the exponential increase in its production, monitoring data show limited evidence of concomitant increasing concentrations in marine habitats. There appears to be a considerable proportion of the manufactured plastic that is unaccounted for in surveys tracking the fate of environmental plastics. Even the discovery of widespread accumulation of microscopic fragments (microplastics) in oceanic gyres and shallow water sediments is unable to explain the missing fraction. Here, we show that deep-sea sediments are a likely sink for microplastics. Microplastic, in the form of fibres, was up to four orders of magnitude more abundant (per unit volume) in deep-sea sediments from the Atlantic Ocean, Mediterranean Sea and Indian Ocean than in contaminated sea-surface waters. Our results show evidence for a large and hitherto unknown repository of microplastics. The dominance of microfibres points to a previously underreported and unsampled plastic fraction. Given the vastness of the deep sea and the prevalence of microplastics at all sites we investigated, the deep-sea floor appears to provide an answer to the question— where is all the plastic?

A broad range of chemical contaminants and pollutants have been measured within the Chagos Archipelago. Contamination is amongst the lowest in the world. Whilst much data is in the open literature, the chapter also includes details of extensive pollution monitoring for the atoll Diego Garcia which hosts a military facility. Hydrocarbons present are primarily of a natural origin with negligible evidence of contamination from petroleum or combustion origins. Tar balls, however, have been reported on several beaches in the Archipelago. Analyses of faecal steroids provide negligible evidence of sewage contamination. ‘Persistent organic pollutants’ (POPs), including PCBs and pesticides, were generally below analytical detection limits, as were polyfluorinated compounds, brominated, chlorinated and organo-phosphorous flame retardants, fluorinated tensides, and surfactants (PFOS). Antifouling biocides and herbicides in Diego Garcia show negligible contamination. Metal concentrations are very low. Levels of most contaminants are typically comparable to those recorded in environments perceived to be pristine, for example, the Antarctic. In Diego Garcia, extensive monitoring includes regular analyses in accredited US laboratories of over one hundred metals and organic contaminants. Results generally reveal concentrations below detection limits. This is in agreement with the open literature surveys. These legislated assessments are designed to ensure both environmental and human health preservation. Whilst many detection limits are higher than those of the independent surveys, they generally confirm the pristine nature of the Archipelago. Beach surveys, however, revealed a surprisingly high number of pieces of debris throughout the Archipelago, mainly plastics of South East Asian origin. The number of litter pieces in Diego Garcia was less than in the other atolls, reductions being attributed to beach clean-up events. Microplastic contamination is shown to be both widespread and relatively high compared to other locations on a global scale, and there were significantly more microplastics at uninhabited atolls compared to the Diego Garcia, showing the potential for microplastics to accumulate in remote locations. Holothurian (sea cucumber) poaching has been another significant environmental pressure on the coral reefs of Chagos and is included in this review, in view of the reported ecological benefits of the group to reef health and resilience.