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Abstract Molluscan mitochondrial genomes are unusual because they show wide variation in size, radical genome rearrangements and frequently show high variation (> 10%) within species. As progress in understanding this variation has been limited, we used whole genome sequencing of a six-generation matriline of the terrestrial snail Cepaea nemoralis, as well as whole genome sequences from wild-collected C. nemoralis, the sister species C. hortensis, and multiple other snail species to explore the origins of mitochondrial DNA (mtDNA) variation. The main finding is that a high rate of SNP heteroplasmy in somatic tissue was negatively correlated with mtDNA copy number in both Cepaea species. In individuals with under ten mtDNA copies per nuclear genome, more than 10% of all positions were heteroplasmic, with evidence for transmission of this heteroplasmy through the germline. Further analyses showed evidence for purifying selection acting on non-synonymous mutations, even at low frequency of the rare allele, especially in cytochrome oxidase subunit 1 and cytochrome b. The mtDNA of some individuals of Cepaea nemoralis contained a length heteroplasmy, including up to 12 direct repeat copies of tRNA-Val, with 24 copies in another snail, Candidula rugosiuscula, and repeats of tRNA-Thr in C. hortensis. These repeats likely arise due to error prone replication but are not correlated with mitochondrial copy number in C. nemoralis. Overall, the findings provide key insights into mechanisms of replication, mutation and evolution in molluscan mtDNA, and so will inform wider studies on the biology and evolution of mtDNA across animal phyla.

Molluscan mitochondrial genomes are unusual because they show wide variation in size, radical genome rearrangements and frequently show high variation (> 10%) within species. As progress in understanding this variation has been limited, we used whole genome sequencing of a six-generation matriline of the terrestrial snail Cepaea nemoralis, as well as whole genome sequences from wild-collected C. nemoralis, the sister species C. hortensis, and multiple other snail species to explore the origins of mitochondrial DNA (mtDNA) variation. The main finding is that a high rate of SNP heteroplasmy in somatic tissue was negatively correlated with mtDNA copy number in both Cepaea species. In individuals with under ten mtDNA copies per nuclear genome, more than 10% of all positions were heteroplasmic, with evidence for transmission of this heteroplasmy through the germline. Further analyses showed evidence for purifying selection acting on non-synonymous mutations, even at low frequency of the rare allele, especially in cytochrome oxidase subunit 1 and cytochrome b. The mtDNA of some individuals of Cepaea nemoralis contained a length heteroplasmy, including up to 12 direct repeat copies of tRNA-Val, with 24 copies in another snail, Candidula rugosiuscula, and repeats of tRNA-Thr in C. hortensis. These repeats likely arise due to error prone replication but are not correlated with mitochondrial copy number in C. nemoralis. Overall, the findings provide key insights into mechanisms of replication, mutation and evolution in molluscan mtDNA, and so will inform wider studies on the biology and evolution of mtDNA across animal phyla. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract Background Host immune function can contribute to numerous ecological/evolutionary processes. Ecoimmunological studies, however, typically use one/few phenotypic immune assays and thus do not consider the complexity of the immune system. Therefore, “omics” resources that allow quantifying immune activity across multiple pathways are needed for ecoimmunological models. We applied short-read based RNAseq (Illumina NextSeq 500, PE-81) to characterise transcriptome profiles of Lymnaea stagnalis (Gastropoda), a multipurpose model snail species. We used a genetically diverse snail stock and exposed individuals to immune elicitors (injury, bacterial/trematode pathogens) and changes in environmental conditions that can alter immune activity (temperature, food availability). Results Immune defence factors identified in the de novo assembly covered elements broadly described in other gastropods. For instance, pathogen-recognition receptors (PRR) and lectins activate Toll-like receptor (TLR) pathway and cytokines that regulate cellular and humoral defences. Surprisingly, only modest diversity of antimicrobial peptides and fibrinogen related proteins were detected when compared with other taxa. Additionally, multiple defence factors that may contribute to the phenotypic immune assays used to quantify antibacterial activity and phenoloxidase (PO)/melanisation-type reaction in this species were found. Experimental treatments revealed factors from non-self recognition (lectins) and signalling (TLR pathway, cytokines) to effectors (e.g., antibacterial proteins, PO enzymes) whose transcription depended on immune stimuli and environmental conditions, as well as components of snail physiology/metabolism that may drive these effects. Interestingly, the transcription of many factors (e.g., PRR, lectins, cytokines, PO enzymes, antibacterial proteins) showed high among-individual variation. Conclusions Our results indicate several uniform aspects of gastropod immunity, but also apparent differences between L. stagnalis and some previously examined taxa. Interestingly, in addition to immune defence factors that responded to immune elicitors and changes in environmental conditions, many factors showed high among-individual variation across experimental snails. We propose that such factors are highly important to be included in future ecoimmunological studies because they may be the key determinants of differences in parasite resistance among individuals both within and between natural snail populations.

Background: Host immune function can contribute to numerous ecological/evolutionary processes. Ecoimmunological studies, however, typically use one/few phenotypic immune assays and thus do not consider the complexity of the immune system. Therefore, "omics" resources that allow quantifying immune activity across multiple pathways are needed for ecoimmunological models. We applied short-read based RNAseq (Illumina NextSeq 500, PE-81) to characterise transcriptome profiles of Lymnaea stagnalis (Gastropoda), a multipurpose model snail species. We used a genetically diverse snail stock and exposed individuals to immune elicitors (injury, bacterial/trematode pathogens) and changes in environmental conditions that can alter immune activity (temperature, food availability). Results: Immune defence factors identified in the de novo assembly covered elements broadly described in other gastropods. For instance, pathogen-recognition receptors (PRR) and lectins activate Toll-like receptor (TLR) pathway and cytokines that regulate cellular and humoral defences. Surprisingly, only modest diversity of antimicrobial peptides and fibrinogen related proteins were detected when compared with other taxa. Additionally, multiple defence factors that may contribute to the phenotypic immune assays used to quantify antibacterial activity and phenoloxidase (PO)/melanisation-type reaction in this species were found. Experimental treatments revealed factors from non-self recognition (lectins) and signalling (TLR pathway, cytokines) to effectors (e.g., antibacterial proteins, PO enzymes) whose transcription depended on immune stimuli and environmental conditions, as well as components of snail physiology/metabolism that may drive these effects. Interestingly, the transcription of many factors (e.g., PRR, lectins, cytokines, PO enzymes, antibacterial proteins) showed high among-individual variation. Conclusions: Our results indicate several uniform aspects of gastropod immunity, but also apparent differences between L. stagnalis and some previously examined taxa. Interestingly, in addition to immune defence factors that responded to immune elicitors and changes in environmental conditions, many factors showed high among-individual variation across experimental snails. We propose that such factors are highly important to be included in future ecoimmunological studies because they may be the key determinants of differences in parasite resistance among individuals both within and between natural snail populations. [ABSTRACT FROM AUTHOR]

Abstract Background Protein expression patterns underlie physiological processes and phenotypic differences including those occurring during early development. The Pacific oyster (Crassostrea gigas) undergoes a major phenotypic change in early development from free-swimming larval form to sessile benthic dweller while proliferating in environments with broad temperature ranges. Despite the economic and ecological importance of the species, physiological processes occurring throughout metamorphosis and the impact of temperature on these processes have not yet been mapped out. Results Towards this, we comprehensively characterized protein abundance patterns for 7978 proteins throughout metamorphosis in the Pacific oyster at different temperature regimes. We used a multi-statistical approach including principal component analysis, ANOVA-simultaneous component analysis, and hierarchical clustering coupled with functional enrichment analysis to characterize these data. We identified distinct sets of proteins with time-dependent abundances generally not affected by temperature. Over 12 days, adhesion and calcification related proteins acutely decreased, organogenesis and extracellular matrix related proteins gradually decreased, proteins related to signaling showed sinusoidal abundance patterns, and proteins related to metabolic and growth processes gradually increased. Contrastingly, different sets of proteins showed temperature-dependent abundance patterns with proteins related to immune response showing lower abundance and catabolic pro-growth processes showing higher abundance in animals reared at 29 °C relative to 23 °C. Conclusion Although time was a stronger driver than temperature of metamorphic proteome changes, temperature-induced proteome differences led to pro-growth physiology corresponding to larger oyster size at 29 °C, and to altered specific metamorphic processes and possible pathogen presence at 23 °C. These findings offer high resolution insight into why oysters may experience high mortality rates during this life transition in both field and culture settings. The proteome resource generated by this study provides data-driven guidance for future work on developmental changes in molluscs. Furthermore, the analytical approach taken here provides a foundation for effective shotgun proteomic analyses across a variety of taxa.

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. [ABSTRACT FROM AUTHOR]

Background: Protein expression patterns underlie physiological processes and phenotypic differences including those occurring during early development. The Pacific oyster (Crassostrea gigas) undergoes a major phenotypic change in early development from free-swimming larval form to sessile benthic dweller while proliferating in environments with broad temperature ranges. Despite the economic and ecological importance of the species, physiological processes occurring throughout metamorphosis and the impact of temperature on these processes have not yet been mapped out. Results: Towards this, we comprehensively characterized protein abundance patterns for 7978 proteins throughout metamorphosis in the Pacific oyster at different temperature regimes. We used a multi-statistical approach including principal component analysis, ANOVA-simultaneous component analysis, and hierarchical clustering coupled with functional enrichment analysis to characterize these data. We identified distinct sets of proteins with time-dependent abundances generally not affected by temperature. Over 12 days, adhesion and calcification related proteins acutely decreased, organogenesis and extracellular matrix related proteins gradually decreased, proteins related to signaling showed sinusoidal abundance patterns, and proteins related to metabolic and growth processes gradually increased. Contrastingly, different sets of proteins showed temperature-dependent abundance patterns with proteins related to immune response showing lower abundance and catabolic pro-growth processes showing higher abundance in animals reared at 29 °C relative to 23 °C. Conclusion: Although time was a stronger driver than temperature of metamorphic proteome changes, temperature-induced proteome differences led to pro-growth physiology corresponding to larger oyster size at 29 °C, and to altered specific metamorphic processes and possible pathogen presence at 23 °C. These findings offer high resolution insight into why oysters may experience high mortality rates during this life transition in both field and culture settings. The proteome resource generated by this study provides data-driven guidance for future work on developmental changes in molluscs. Furthermore, the analytical approach taken here provides a foundation for effective shotgun proteomic analyses across a variety of taxa. [ABSTRACT FROM AUTHOR]

Background Nuclear receptors are a superfamily of transcription factors important in key biological, developmental and reproductive processes. Several of these receptors are ligand- activated and through their ability to bind endogenous and exogenous ligands, are potentially vulnerable to xenobiotics. Molluscs are key ecological species in defining aquatic and terrestrial habitats and are sensitive to xenobiotic compounds in the environment. However, the understanding of nuclear receptor presence, function and xenobiotic disruption in the phylum Mollusca is limited. Results Here, forty-three nuclear receptor sequences were mined from the genome of the Pacific oyster, Crassostrea gigas. They include members of NR0-NR5 subfamilies, notably lacking any NR6 members. Phylogenetic analyses of the oyster nuclear receptors have been conducted showing the presence of a large novel subfamily group not previously reported, which is named NR1P. Homologues to all previous identified nuclear receptors in other mollusc species have also been determined including the putative heterodimer partner retinoid X receptor, estrogen receptor and estrogen related receptor. Conclusion C. gigas contains a highly diverse set of nuclear receptors including a novel NR1 group, which provides important information on presence and evolution of this transcription factor superfamily in invertebrates. The Pacific oyster possesses two members of NR3, the sex steroid hormone receptor analogues, of which there are 9 in humans. This provides increasing evidence that steroid ligand specific expansion of this family is deuterostome specific. This new knowledge on divergence and emergence of nuclear receptors in C. gigas provides essential information for studying regulation of molluscan gene expression and the potential effects of xenobiotics. [ABSTRACT FROM AUTHOR]

Background: With a diversity of pigmented shell morphotypes governed by Mendelian patterns of inheritance, the common grove snail, Cepaea nemoralis, has served as a model for evolutionary biologists and population geneticists for decades. Surprisingly, the molecular mechanisms by which C. nemoralis generates this pigmented shelled diversity, and the degree of evolutionary conservation present between molluscan shell-forming proteomes, remain unknown. Results: Here, using next generation sequencing and high throughput proteomics, we identify and characterize the major proteinaceous components of the C. nemoralis shell, the first shell-proteome for a pulmonate mollusc. The recent availability of several marine molluscan shell-proteomes, and the dataset we report here, allow us to identify 59 evolutionarily conserved and novel shell-forming proteins. While the C. nemoralis dataset is dominated by proteins that share little to no similarity with proteins in public databases, almost half of it shares similarity with proteins present in other molluscan shells. In addition, we could not find any indication that a protein (or class of proteins) is directly associated with shell pigmentation in C. nemoralis. This is in contrast to the only other partially characterized molluscan-shell pigmentation mechanism employed by the tropical abalone Haliotis asinina. Conclusions: The unique pulmonate shell-forming proteome that we report here reveals an abundance of both mollusc-specific and pulmonate-specific proteins, suggesting that novel coding sequences, and/or the extensive divergence of these sequences from ancestral sequences, supported the innovation of new shell types within the Conchifera. In addition, we report here the first evidence that molluscs use independently evolved mechanisms to pigment their shells. This proteome provides a solid foundation from which further studies aimed at the functional characterization of these shell-forming proteins can be conducted. [ABSTRACT FROM AUTHOR]