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Biomineralization processes in bivalves, particularly the initial production of molecular components (such as matrix deposition and calcification) in the early stages of shell development are highly complex and well-organized. This study investigated the temporal dynamics of organic matrix and calcium carbonate (CaCO 3 ) deposition in Pacific oysters (Crassostrea gigas) across various development stages. The shell-field initiated matrix secretion during the gastrula stage. Subsequent larval development triggered central shell-field calcification, accompanied by expansion of the calcium ring from its interior to the periphery. Notably, the expression patterns of CgTyrp-2 and CgTyr closely correlated with matrix deposition and calcification during early developmental stages, with peak expression occurring in oyster's gastrula and D-veliger stages. Subsequently, the CRISPR/Cas9 system was utilized to knock out CgTyrp-2 and CgTyr with more distinct phenotypic alterations observed when both genes were concurrently knocked out. The relative gene expression was analyzed post-knockout, indicating that the knockout of CgTyr or CgTyrp-2 led to reduced expression of CgChs1, along with increased expression of CgChit4. Furthermore, when dual-sgRNAs were employed to knockout CgTyrp-2, a large deletion (2 kb) within the CgTyrp-2 gene was identified. In summary, early shell formation in C. gigas is the result of a complex interplay of multiple molecular components with CgTyrp-2 and CgTyr playing key roles in regulating CaCO 3 deposition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although the mechanisms of molluscan shell growth have been studied using mathematical models, little is known about the molecular basis underpinning shell morphogenesis. Here, we performed Wnt activation experiments to elucidate the potential roles of Wnt signaling in the shell growth of Lymnaea stagnalis. In general, we observed following three types of shell malformations in both dose- and developmental stage-dependent manners: (i) cap-shaped shell, (ii) cap-shaped shell with hydropic soft tissues, and (iii) compressed shell with a smaller number of coiling. We analyzed the morphologies of these malformed shells using the growing tube model, revealing that the compressed malformations show significantly larger values for T (torsion), with no significant changes in the values for the remaining parameters E (expansion) and C (curvature). We also found that cap-shaped malformations have significantly larger values for E, suggesting that the effects of BIO on shell formation may change during growth. Since the changes in T and/or E parameter values can greatly alter the shell morphologies from a planispiral or a cap-shaped one to various three-dimensional helices, changes in shell developmental processes possibly controlled by Wnt signaling may account for at least a part of the evolution of diverse shell forms in molluscs., (© 2024. The Author(s).)

Introduction: Bivalve molluscs are abundant in marine and freshwater systems and contribute essential ecosystem services. They are characterized by an exuberant diversity of biomineralized shells and typically have two symmetric valves (a.k.a shells), but oysters (Ostreidae), some clams (Anomiidae and Chamidae) and scallops (Pectinida) have two asymmetrical valves. Predicting and modelling the likely consequences of ocean acidification on bivalve survival, biodiversity and aquaculture makes understanding shell biomineralization and its regulation a priority., Objectives: This study aimed to a) exploit the atypical asymmetric shell growth of some bivalves and through comparative analysis of the genome and transcriptome pinpoint candidate biomineralization-related genes and regulatory long non-coding RNAs (LncRNAs) and b) demonstrate their roles in regulating shell biomineralization/growth., Methods: Meta-analysis of genomes, de novo generated mantle transcriptomes or transcriptomes and proteomes from public databases for six asymmetric to symmetric bivalve species was used to identify biomineralization-related genes. Bioinformatics filtering uncovered genes and regulatory modules characteristic of bivalves with asymmetric shells and identified candidate biomineralization-related genes and lncRNAs with a biased expression in asymmetric valves. A shell regrowth model in oyster and gene silencing experiments, were used to characterize candidate gene function., Results: Shell matrix genes with asymmetric expression in the mantle of the two valves were identified and unique cis-regulatory lncRNA modules characterized in Ostreidae. LncRNAs that regulate the expression of the tissue inhibitor of metalloproteinases gene family (TIMPDR) and of the shell matrix protein domain family (SMPDR) were identified. In vitro and in vivo silencing experiments revealed the candidate genes and lncRNA were associated with divergent shell growth rates and modified the microstructure of calcium carbonate (CaCO 3 ) crystals., Conclusion: LncRNAs are putative regulatory factors of the bivalve biomineralization toolbox. In the Ostreidae family of bivalves biomineralization-related genes are cis-regulated by lncRNA and modify the planar growth rate and spatial orientation of crystals in the shell., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Mollusca is a morphologically diverse phylum, exhibiting an immense variety of calcium carbonate structures. Proteomic studies of adult shells often report high levels of rapidly-evolving, 'novel' shell matrix proteins (SMPs), which are hypothesized to drive shell diversification. However, relatively little is known about the phylogenetic distribution of SMPs, or about the function of individual SMPs in shell construction. To understand how SMPs contribute to shell diversification a thorough characterization of SMPs is required. Here, we build tools and a foundational understanding of SMPs in the marine gastropod species Crepidula fornicata and Crepidula atrasolea because they are genetically-enabled mollusc model organisms. First, we established a staging system of shell development in C. atrasolea for the first time. Next, we leveraged previous findings in C. fornicata combined with phylogenomic analyses of 95 metazoan species to determine the evolutionary lineage of its adult SMP repertoire. We found that 55% of C. fornicata's SMPs belong to molluscan orthogroups, with 27% restricted to Gastropoda, and only 5% restricted at the species level. The low percentage of species-restricted SMPs underscores the importance of broad-taxon sampling and orthology inference approaches when determining homology of SMPs. From our transcriptome analysis, we found that the majority of C. fornicata SMPs that were found conserved in C. atrasolea were expressed in both larval and adult stages. We then selected a subset of SMPs of varying evolutionary ages for spatial-temporal analysis using in situ hybridization chain reaction (HCR) during larval shell development in C. atrasolea. Out of the 18 SMPs analyzed, 12 were detected in the larval shell field. These results suggest overlapping larval vs. adult SMP repertoires. Using multiplexed HCR, we observed five SMP expression patterns and three distinct cell populations within the shell field. These patterns support the idea that modular expression of SMPs could facilitate divergence of shell morphological characteristics. Collectively, these data establish an evolutionary and developmental framework in Crepidula that enables future comparisons of molluscan biomineralization to reveal mechanisms of shell diversification., (© 2024. The Author(s).)

Understanding past coastal variability is valuable for contextualizing modern changes in coastal settings, yet existing Holocene paleoceanographic records for the North American Pacific Coast commonly originate from offshore marine sediments and may not represent the dynamic coastal environment. A potential archive of eastern Pacific Coast environmental variability is the intertidal mussel species Mytilus californianus. Archaeologists have collected copious stable isotopic (δ18O and δ13C) data from M. californianus shells to study human history at California's Channel Islands. When analyzed together, these isotopic data provide windows into 9000 years of Holocene isotopic variability and M. californianus life history. Here we synthesize over 6000 δ18O and δ13C data points from 13 published studies to investigate M. californianus shell isotopic variability across ontogenetic, geographic, seasonal, and millennial scales. Our analyses show that M. californianus may grow and record environmental information more irregularly than expected due to the competing influences of calcification, ontogeny, metabolism, and habitat. Stable isotope profiles with five or more subsamples per shell recorded environmental information ranging from seasonal to millennial scales, depending on the number of shells analyzed and the resolution of isotopic subsampling. Individual shell profiles contained seasonal cycles and an accurate inferred annual temperature range of ~ 5°C, although ontogenetic growth reduction obscured seasonal signals as organisms aged. Collectively, the mussel shell record reflected millennial-scale climate variability and an overall 0.52‰ depletion in δ18Oshell from 8800 BP to the present. The archive also revealed local-scale oceanographic variability in the form of a warmer coastal mainland δ18Oshell signal (-0.32‰) compared to a cooler offshore islands δ18Oshell signal (0.33‰). While M. californianus is a promising coastal archive, we emphasize the need for high-resolution subsampling from multiple individuals to disentangle impacts of calcification, metabolism, ontogeny, and habitat and more accurately infer environmental and biological patterns recorded by an intertidal species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Vriesman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recent work on microbe-host interactions has revealed an important nexus between the environment, microbiome, and host fitness. Marine invertebrates that build carbonate skeletons are of particular interest in this regard because of predicted effects of ocean acidification on calcified organisms, and the potential of microbes to buffer these impacts. Here we investigate the role of sulfate-reducing bacteria, a group well known to affect carbonate chemistry, in Pacific oyster (Magallana gigas) shell formation. We reared oyster larvae to 51 days post fertilization and exposed organisms to control and sodium molybdate conditions, the latter of which is thought to inhibit bacterial sulfate reduction. Contrary to expectations, we found that sodium molybdate did not uniformly inhibit sulfate-reducing bacteria in oysters, and oysters exposed to molybdate grew larger shells over the experimental period. Additionally, we show that microbiome composition, host gene expression, and shell size were distinct between treatments earlier in ontogeny, but became more similar by the end of the experiment. Although additional testing is required to fully elucidate the mechanisms, our work provides preliminary evidence that M. gigas is capable of regulating microbiome dysbiosis caused by environmental perturbations, which is reflected in shell development., Competing Interests: The authors have declared that no competing interests exist.

Snails are model organisms for studying the genetic, molecular, and developmental bases of left-right asymmetry in Bilateria. However, the development of their typical helicospiral shell, present for the last 540 million years in environments as different as the abyss or our gardens, remains poorly understood. Conversely, ammonites typically have a bilaterally symmetric, planispiraly coiled shell, with only 1% of 3,000 genera displaying either a helicospiral or a meandering asymmetric shell. A comparative analysis suggests that the development of chiral shells in these mollusks is different and that, unlike snails, ammonites with asymmetric shells probably had a bilaterally symmetric body diagnostic of cephalopods. We propose a mathematical model for the growth of shells, taking into account the physical interaction during development between the soft mollusk body and its hard shell. Our model shows that a growth mismatch between the secreted shell tube and a bilaterally symmetric body in ammonites can generate mechanical forces that are balanced by a twist of the body, breaking shell symmetry. In gastropods, where a twist is intrinsic to the body, the same model predicts that helicospiral shells are the most likely shell forms. Our model explains a large diversity of forms and shows that, although molluscan shells are incrementally secreted at their opening, the path followed by the shell edge and the resulting form are partly governed by the mechanics of the body inside the shell, a perspective that explains many aspects of their development and evolution., Competing Interests: The authors declare no competing interest.

The turtle carapace is composed of severely deformed fused dorsal vertebrae, ribs, and bone plates. In particular, the lateral growth in the superficial layer of turtle ribs in the dorsal trunk causes an encapsulation of the scapula and pelvis. The recent study suggested that the carapacial ridge (CR) is a new model of epithelial-mesenchymal transition which is essential for the arrangement of the ribs. Therefore, it is necessary to explore the regulatory mechanism of carapacial ridge development to analyze the formation of the turtle shell. However, the current understanding of the regulatory network underlying turtle carapacial ridge development is poor due to the lack of both systematic gene screening at different carapacial ridge development stages and gene function verification studies. In this study, we obtained genome-wide gene transcription and gene translation profiles using RNA sequencing and ribosome nascent-chain complex mRNA sequencing from carapacial ridge tissues of Chinese soft-shell turtle at different development stages. A correlation analysis of the transcriptome and translatome revealed that there were 129, 670, and 135 codifferentially expressed genes, including homodirection and opposite-direction differentially expressed genes, among three comparison groups, respectively. The pathway enrichment analysis of codifferentially expressed genes from the Kyoto Encyclopedia of Genes and Genomes showed dynamic changes in signaling pathways involved in carapacial ridge development. Especially, the results revealed that the Wnt signaling pathway and MAPK signaling pathway may play important roles in turtle carapacial ridge development. In addition, Wnt and Fgf were expressed during the carapacial ridge development. Furthermore, we discovered that Wnt5 a regulated carapacial ridge development through the Wnt5a/JNK pathway. Therefore, our studies uncover that the morphogenesis of the turtle carapace might function through the co-operation between conserved WNT and FGF signaling pathways. Consequently, our findings revealed the dynamic signaling pathways acting on the carapacial ridge development of Chinese soft-shell turtle and provided new insights into uncover the molecular mechanism underlying turtle shell morphogenesis.

The New Zealand green-lipped mussel aquaculture industry is largely dependent on the supply of young mussels that wash up on Ninety Mile Beach (so-called Kaitaia spat), which are collected and trucked to aquaculture farms. The locations of source populations of Kaitaia spat are unknown and this lack of knowledge represents a major problem because spat supply may be irregular. We combined genotypic (microsatellite) and phenotypic (shell geochemistry) data in a geospatial framework to determine if this new approach can help identify source populations of mussels collected from two spat-collecting and four non-spat-collecting sites further south. Genetic analyses resolved differentiated clusters (mostly three clusters), but no obvious source populations. Shell geochemistry analyses resolved six differentiated clusters, as did the combined genotypic and phenotypic data. Analyses revealed high levels of spatial and temporal variability in the geochemistry signal. Whilst we have not been able to identify the source site(s) of Kaitaia spat our analyses indicate that geospatial testing using combined genotypic and phenotypic data is a powerful approach. Next steps should employ analyses of single nucleotide polymorphism markers with shell geochemistry and in conjunction with high resolution physical oceanographic modelling to resolve the longstanding question of the origin of Kaitaia spat.

Background: A major goal of evolutionary developmental biology is to discover general models and mechanisms that create the phenotypes of organisms. However, universal models of such fundamental growth and form are rare, presumably due to the limited number of physical laws and biological processes that influence growth. One such model is the logarithmic spiral, which has been purported to explain the growth of biological structures such as teeth, claws, horns, and beaks. However, the logarithmic spiral only describes the path of the structure through space, and cannot generate these shapes., Results: Here we show a new universal model based on a power law between the radius of the structure and its length, which generates a shape called a 'power cone'. We describe the underlying 'power cascade' model that explains the extreme diversity of tooth shapes in vertebrates, including humans, mammoths, sabre-toothed cats, tyrannosaurs and giant megalodon sharks. This model can be used to predict the age of mammals with ever-growing teeth, including elephants and rodents. We view this as the third general model of tooth development, along with the patterning cascade model for cusp number and spacing, and the inhibitory cascade model that predicts relative tooth size. Beyond the dentition, this new model also describes the growth of claws, horns, antlers and beaks of vertebrates, as well as the fangs and shells of invertebrates, and thorns and prickles of plants., Conclusions: The power cone is generated when the radial power growth rate is unequal to the length power growth rate. The power cascade model operates independently of the logarithmic spiral and is present throughout diverse biological systems. The power cascade provides a mechanistic basis for the generation of these pointed structures across the tree of life.

Bivalve shells are increasingly used as archives for high-resolution paleoclimate analyses. However, there is still an urgent need for quantitative temperature proxies that work without knowledge of the water chemistry-as is required for δ18O-based paleothermometry-and can better withstand diagenetic overprint. Recently, microstructural properties have been identified as a potential candidate fulfilling these requirements. So far, only few different microstructure categories (nacreous, prismatic and crossed-lamellar) of some short-lived species have been studied in detail, and in all such studies, the size and/or shape of individual biomineral units was found to increase with water temperature. Here, we explore whether the same applies to properties of the crossed-acicular microstructure in the hinge plate of Arctica islandica, the microstructurally most uniform shell portion in this species. In order to focus solely on the effect of temperature on microstructural properties, this study uses bivalves that grew their shells under controlled temperature conditions (1, 3, 6, 9, 12 and 15°C) in the laboratory. With increasing temperature, the size of the largest individual biomineral units and the relative proportion of shell occupied by the crystalline phase increased. The size of the largest pores, a specific microstructural feature of A. islandica, whose potential role in biomineralization is discussed here, increased exponentially with culturing temperature. This study employs scanning electron microscopy in combination with automated image processing software, including an innovative machine learning-based image segmentation method. The new method greatly facilitates the recognition of microstructural entities and enables a faster and more reliable microstructural analysis than previously used techniques. Results of this study establish the new microstructural temperature proxy in the crossed-acicular microstructures of A. islandica and point to an overarching control mechanism of temperature on the micrometer-scale architecture of bivalve shells across species boundaries., Competing Interests: The authors have declared that no competing interests exist.

Across the evolutionary history of insects, the shift from nitrogen-rich carnivore/omnivore diets to nitrogen-poor herbivorous diets was made possible through symbiosis with microbes. The herbivorous turtle ants Cephalotes possess a conserved gut microbiome which enriches the nutrient composition by recycling nitrogen-rich metabolic waste to increase the production of amino acids. This enrichment is assumed to benefit the host, but we do not know to what extent. To gain insights into nitrogen assimilation in the ant cuticle we use gut bacterial manipulation, 15 N isotopic enrichment, isotope-ratio mass spectrometry, and 15 N nuclear magnetic resonance spectroscopy to demonstrate that gut bacteria contribute to the formation of proteins, catecholamine cross-linkers, and chitin in the cuticle. This study identifies the cuticular components which are nitrogen-enriched by gut bacteria, highlighting the role of symbionts in insect evolution, and provides a framework for understanding the nitrogen flow from nutrients through bacteria into the insect cuticle.

The beetle horn primordium is a complex and compactly folded epithelial sheet located beneath the larval cuticle. Only by unfolding the primordium can the complete 3D shape of the horn appear, suggesting that the morphology of beetle horns is encoded in the primordial folding pattern. To decipher the folding pattern, we developed a method to manipulate the primordial local folding on a computer and clarified the contribution of the folding of each primordium region to transformation. We found that the three major morphological changes (branching of distal tips, proximodistal elongation, and angular change) were caused by the folding of different regions, and that the folding mechanism also differs according to the region. The computational methods we used are applicable to the morphological study of other exoskeletal animals.

The C1q protein, which contains the globular C1q (gC1q) domain, is involved in the innate immune response, and is found abundantly in the shell, and it participates in the shell formation. In this study, a novel gC1q domain-containing gene was identified from Pinctada fucata martensii (P. f. martensii) and designated as PmC1qDC-1. The full-length sequence of PmC1qDC-1 was 902 bp with a 534 bp open reading frame (ORF), encoding a polypeptide of 177 amino acids. Quantitative real-time PCR (qRT-PCR) result showed that PmC1qDC-1 was widely expressed in all tested tissues, including shell formation-associated tissue and immune-related tissue. PmC1qDC-1 expression was significantly high in the blastula and gastrula and especially among the juvenile stage, which is the most important stage of dissoconch shell formation. PmC1qDC-1 expression was located in the outer epithelial cells of mantle pallial and mantle edge and irregular crystal tablets were observed in the nacre upon knockdown of PmC1qDC-1 expression at mantle pallial. Moreover, the recombined protein PmC1qDC-1 increased the rate of calcium carbonate precipitation. Besides, PmC1qDC-1 expression was significantly up-regulated in the mantle pallial at 6 h and was significantly up-regulated in the mantle edge at 12 h and 24 h after shell notching. The expression level of PmC1qDC-1 in mantle edge was significantly up-regulated at 48 h after LPS stimulation and was significantly up-regulated at 12 h, 24 h and 48 h after poly I:C stimulation. Moreover, PmC1qDC-1 expression was significantly up-regulated in hemocytes at 6 h after lipopolysaccharide (LPS) and poly I:C challenge. These findings suggest that PmC1qDC-1 plays a crucial role both in the shell formation and the innate immune response in pearl oysters, providing new clues for understanding the shell formation and defense mechanism in mollusk.

Precisely targeted measurements of trace elements using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) reveal inter-chamber heterogeneities in specimens of the planktic foraminifer Trilobatus (Globigerinoides) sacculifer. We find that Mg/Ca ratios in the final growth chamber are generally lower compared to previous growth chambers, but final chamber Mg/Ca is elevated in one of thirteen sample intervals. Differences in distributions of Mg/Ca values from separate growth chambers are observed, occurring most often at lower Mg/Ca values, suggesting that single-chamber measurements may not be reflective of the specimen's integrated Mg/Ca. We compared LA-ICPMS Mg/Ca values to paired, same-individual Mg/Ca measured via inductively coupled plasma optical emission spectrometry (ICP-OES) to assess their correspondence. Paired LA-ICPMS and ICP-OES Mg/Ca show a maximum correlation coefficient of R = 0.92 (p < 0.05) achieved by applying a weighted average of the last and penultimate growth chambers. Population distributions of paired Mg/Ca values are identical under this weighting. These findings demonstrate that multi-chamber LA-ICPMS measurements can approximate entire specimen Mg/Ca, and is thus representative of the integrated conditions experienced during the specimen's lifespan. This correspondence between LA-ICPMS and ICP-OES data links these methods and demonstrates that both generate Mg/Ca values suitable for individual foraminifera palaeoceanographic reconstructions.

Background: Albino mutations are commonly observed in the animal kingdom, including in bivalves. In the black-lipped pearl oyster Pinctada margaritifera, albino specimens are characterized by total or partial absence of colouration resulting in typical white shell phenotype expression. The relationship of shell colour with resulting cultured pearl colour is of great economic interest in P. margaritifera, on which a pearl industry is based. Hence, the albino phenotype provides a useful way to examine the molecular mechanisms underlying pigmentation., Results: Whole transcriptome RNA-sequencing analysis comparing albino and black wild-type phenotypes at three stages over the culture cycle of P. margaritifera revealed a total of 1606, 798 and 187 differentially expressed genes in whole juvenile, adult mantle and pearl sac tissue, respectively. These genes were found to be involved in five main molecular pathways, tightly linked to known pigmentation pathways: melanogenesis, calcium signalling pathway, Notch signalling pathway, pigment transport and biomineralization. Additionally, significant phenotype-associated SNPs were selected (N = 159), including two located in the Pif biomineralization gene, which codes for nacre formation. Interestingly, significantly different transcript splicing was detected between juvenile (N = 1366) and adult mantle tissue (N = 313) in, e.g., the tyrosinase Tyr-1 gene, which showed more complex regulation in mantle, and the Notch1 encoding gene, which was upregulated in albino juveniles., Conclusion: This multiple RNA-seq approach provided new knowledge about genes associated with the P. margaritifera albino phenotype, highlighting: 1) new molecular pathways, such as the Notch signalling pathway in pigmentation, 2) associated SNP markers with biomineraliszation gene of interest like Pif for marker-assisted selection and prevention of inbreeding, and 3) alternative gene splicing for melanin biosynthesis implicating tyrosinase.

Nipponites is a heteromorph ammonoid with a complex and unique morphology that obscures its mode of life and ethology. The seemingly aberrant shell of this Late Cretaceous nostoceratid seems deleterious. However, hydrostatic simulations suggest that this morphology confers several advantages for exploiting a quasi-planktic mode of life. Virtual, 3D models of Nipponites mirabilis were used to compute various hydrostatic properties through 14 ontogenetic stages. At each stage, Nipponites had the capacity for neutral buoyancy and was not restricted to the seafloor. Throughout ontogeny, horizontally facing to upwardly facing soft body orientations were preferred at rest. These orientations were aided by the obliquity of the shell's ribs, which denote former positions of the aperture that were tilted from the growth direction of the shell. Static orientations were somewhat fixed, inferred by stability values that are slightly higher than extant Nautilus. The initial open-whorled, planispiral phase is well suited to horizontal backwards movement with little rocking. Nipponites then deviated from this bilaterally symmetric coiling pattern with a series of alternating U-shaped bends in the shell. This modification allows for proficient rotation about the vertical axis, while possibly maintaining the option for horizontal backwards movement by redirecting its hyponome. These particular hydrostatic properties likely result in a tradeoff between hydrodynamic streamlining, suggesting that Nipponites assumed a low energy lifestyle of slowly pirouetting in search for planktic prey. Each computed hydrostatic property influences the others in some way, suggesting that Nipponites maintained a delicate hydrostatic balancing act throughout its ontogeny in order to facilitate this mode of life., Competing Interests: The authors have declared that no competing interests exist.

Securing economically and ecologically significant molluscs, as our oceans warm due to climate change, is a global priority. South eastern Australia receives warm water in a strengthening East Australia Current and so resident species are vulnerable to elevated temperature and marine heat waves. This study tested whether prior exposure to elevated temperature can enhance resilience of oysters to ocean warming. Two Australian species, the flat oyster, Ostrea angasi, and the Sydney rock oyster, Saccostrea glomerata, were obtained as adults and "heat shocked" by exposure to a dose of warm water in the laboratory. Oysters were then transferred to elevated seawater temperature conditions where the thermal outfall from power generation was used as a proxy to investigate the impacts of ocean warming. Shell growth, condition index, lipid content and survival of flat oysters and condition of Sydney rock oysters were all significantly reduced by elevated seawater temperature in the field. Flat oysters grew faster than Sydney rock oysters at ambient temperature, but their growth and survival was more sensitive to elevated temperature. "Stress inoculation" by heat shock did little to ameliorate the negative effects of increased temperature, although the survival of heat-shocked flat oysters was greater than non-heat shocked oysters. Further investigations are required to determine if early exposure to heat stress can enhance resilience of oysters to ocean warming., Competing Interests: The authors have declared that no competing interests exist.

Marine bivalves secrete calcified shells to protect their soft bodies from predation and damages, which is of great importance for their survival, and for the safety of the coastal ecosystem. In recent years, larval shell formation of marine bivalves has been severely affected by ocean acidification (OA), and previous study indicated that OA might affect such process by disrupting endogenous energy metabolism. Developmental stages from trochophore to D-shape larvae are extremely important for initial shell formation in oyster since a calcified shell was formed to cover the chitin one. In the present study, metabolomic and transcriptomic approaches were employed to investigate the energy metabolism of oyster larvae during initial shell (prodissoconch I, PDI shell) formation and under experimental OA treatment. Totally 230 chemical compounds were identified from the present dataset, most of which were highly expressed in the "middle" stage (early D-shape larvae) which was critical for PDI shell formation since a calcified shell was formed to cover the chitin one. Several compounds such as glucose, glutarylcarnitine (C5), β-hydroxyisovaleroylcarnitine, 5-methylthioadenosine (MTA), myristoleate (14:1n5) and palmitoleate (16:1n7) were identified, which were involved in energy metabolic processes including amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. In addition, mRNA expressions of genes related to protein metabolism, glycolysis, lipid degradation, calcium transport and organic matrix formation activities were significantly down-regulated upon experimental OA. These results collectively suggested that formation of the initial shell in oyster larvae required endogenous energy coming from amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. These metabolic activities could be severely inhibited by experimental OA, which might alter the allocation of endogenous energy. Insufficient endogenous energy supply then suppressed the mobilization of calcium and resulted in a failure or delay in PDI shell formation.

This work provides methods for chitosan production with larval exoskeletons from Hermetia illucens and characterizes the produced material. The study aims to provide optimal reaction conditions for the purification of chitin from insect material and evaluates the key characteristics of the insect-based chitosan compared to crab shell chitosan. H. illucens is a sustainable source of high-quality protein, lipids and compost currently applied as aquaculture feed and soil improver. The ecdysis of the H. illucens larvae results in larval exoskeletons (LE) rich in chitin and suitable for chitosan production. Using procedures based on crab shell processing, two steps were performed to purify chitin from the larval exoskeletons. The results showed that formic acid is a suitable demineralization agent, requiring 5 mol per kg larval exoskeletons and thorough washing afterwards for 89 % demineralization effectivity (DME). The effect of the deproteinization variables were explored via design of experiments and analyzed using linear regression resulting in chitin contents of the deproteinized material of 83 % in small-scale and 87 % in 10 L-scale, respectively. Heterogeneous and homogeneous deacetylation was performed to convert chitin in different chitosan fractions. Heterogeneous deacetylation at 120 °C resulted in a deacetylation degree (DD) of 72 % and a yield of 43 % at maximum with regard to the total chitin applied. Homogeneous deacetylation at 4 °C resulted in a low chitosan yield of 13 % and a DD of 34 %. However, chitosan solubilized in acetic acid revealed superior film forming properties and a high viscosity. The results indicated that the chitosan from insects has comparable properties than those produced from crab shells, although the properties are also strongly dependent on the manufacturing conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Brachiopods and mollusks are 2 shell-bearing phyla that diverged from a common shell-less ancestor more than 540 million years ago. Brachiopods and bivalve mollusks have also convergently evolved a bivalved shell that displays an apparently mundane, yet striking feature from a developmental point of view: When the shell is closed, the 2 valve edges meet each other in a commissure that forms a continuum with no gaps or overlaps despite the fact that each valve, secreted by 2 mantle lobes, may present antisymmetric ornamental patterns of varying regularity and size. Interlocking is maintained throughout the entirety of development, even when the shell edge exhibits significant irregularity due to injury or other environmental influences, which suggests a dynamic physical process of pattern formation that cannot be genetically specified. Here, we derive a mathematical framework, based on the physics of shell growth, to explain how this interlocking pattern is created and regulated by mechanical instabilities. By close consideration of the geometry and mechanics of 2 lobes of the mantle, constrained both by the rigid shell that they secrete and by each other, we uncover the mechanistic basis for the interlocking pattern. Our modeling framework recovers and explains a large diversity of shell forms and highlights how parametric variations in the growth process result in morphological variation. Beyond the basic interlocking mechanism, we also consider the intricate and striking multiscale-patterned edge in certain brachiopods. We show that this pattern can be explained as a secondary instability that matches morphological trends and data., Competing Interests: The authors declare no competing interest.

Bivalve biomineralization is a highly complex and organized process, involving several molecular components identified in adults and larval stages. However, information is still scarce on the ontogeny of the organic matrix before calcification occurs. In this work, first shell formation was investigated in the mussel Mytilus galloprovincialis . The time course of organic matrix and CaCO 3 deposition were followed at close times post fertilization (24, 26, 29, 32, 48 h) by calcofluor and calcein staining, respectively. Both components showed an exponential trend in growth, with a delay between organic matrix and CaCO 3 deposition. mRNA levels of genes involved in matrix deposition (chitin synthase; tyrosinase- TYR) and calcification (carbonic anhydrase; extrapallial protein) were quantified by qPCR at 24 and 48 hours post fertilization (hpf) with respect to eggs. All transcripts were upregulated across early development, with TYR showing highest mRNA levels from 24 hpf. TYR transcripts were closely associated with matrix deposition as shown by in situ hybridization. The involvement of tyrosinase activity was supported by data obtained with the enzyme inhibitor N-phenylthiourea. Our results underline the pivotal role of shell matrix in driving first CaCO 3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis .

Animal morphogenesis can be summarized as a reconfiguration of a mass of cells. Although extracellular matrices that include rigid skeletal elements, such as cartilage/bones and exoskeletons, have important roles in morphogenesis, they are also secreted in situ by accumulated cells or epithelial cells. In contrast, recent studies of the skeleton construction of sponges (Porifera) illuminate a conceptually different mechanism of morphogenesis in which cells manipulate rather fine rigid materials (spicules) to form larger structures. Here, two different types of sponge skeleton formation using calcareous spicules or siliceous spicules are compared with regard to the concept of the production of rigid materials and their use in skeletons. The comparison highlights the advantages of their different strategies of forming sponge skeletons., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Environmental factors are encoded in shells of marine bivalves in the form of geochemical properties, shell microstructure and shell growth rate. Few studies have investigated how shell growth is affected by habitat conditions in natural populations of the commercial clam Chamelea gallina. Here, skeletal parameters (micro-density and apparent porosity) and growth parameters (bulk density, linear extension and net calcification rates) were investigated in relation to shell sizes and environmental parameters along a latitudinal gradient in the Adriatic Sea (400 km). Net calcification rates increased with increasing solar radiation, sea surface temperature and salinity and decreasing Chlorophyll concentration in immature and mature shells. In immature shells, which are generally more porous than mature shells, enhanced calcification was due to an increase in bulk density, while in mature shells was due to an increase in linear extension rates. The presence of the Po river in the Northern Adriatic Sea was likely the main driver of the fluctuations observed in environmental parameters, especially salinity and Chlorophyll concentration, and seemed to negatively affect the growth of C. gallina.

Mollusk shells are an ideal model system for understanding the morpho-elastic basis of morphological evolution of invertebrates' exoskeletons. During the formation of the shell, the mantle tissue secretes proteins and minerals that calcify to form a new incremental layer of the exoskeleton. Most of the existing literature on the morphology of mollusks is descriptive. The mathematical understanding of the underlying coupling between pre-existing shell morphology, de novo surface deposition and morpho-elastic volume growth is at a nascent stage, primarily limited to reduced geometric representations. Here, we propose a general, three-dimensional computational framework coupling pre-existing morphology, incremental surface growth by accretion, and morpho-elastic volume growth. We exercise this framework by applying it to explain the stepwise morphogenesis of seashells during growth: new material surfaces are laid down by accretive growth on the mantle whose form is determined by its morpho-elastic growth. Calcification of the newest surfaces extends the shell as well as creates a new scaffold that constrains the next growth step. We study the effects of surface and volumetric growth rates, and of previously deposited shell geometries on the resulting modes of mantle deformation, and therefore of the developing shell's morphology. Connections are made to a range of complex shells ornamentations., Competing Interests: The authors have declared that no competing interests exist.

The present study evaluated the association among traditional biochemical biomarkers with biometric, morphometric, and elemental composition of Lottia subrugosa (patelliform gastropod) shells from three multi-impacted coastal areas in Brazil. The study was carried out in Todos os Santos Bay (TSB), Santos/São Vicente Estuarine System (SESS) and Paranaguá Estuarine Complex (CEP), using three sampling sites to seek contamination gradients in each area. Results showed that all biomarkers evaluated responded to environmental contamination, regardless the presence (SESS and CEP) or absence (TSB) of a gradient of contamination. The responses found using biometric and morphometric parameters were consistent with the traditional biomarkers of exposure and effects (lipid peroxidation and DNA damage). Indeed, changes in elemental composition of L. subrugosa shells suggest that exposure to contaminated environments is probably responsible for the alterations detected. Despite the simplicity and lower cost of biometric and morphometric analyzes, these parameters are influenced by natural environmental conditions from which biases may arise. Therefore, these tools should be evaluated through experimental studies before it can be used in future assessments. However, the findings from the present study were observed in three aquatic systems distributed over a wide range of latitudes, which indicates that gastropod shells reflect effects resulting from environmental contamination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The multiplicity of cell types comprising multicellular organisms begs the question as to how cell type identities evolve over time. Cell type phylogenetics informs this question by comparing gene expression of homologous cell types in distantly related taxa. We employ this approach to inform the identity of larval skeletogenic cells of echinoderms, a clade for which there are phylogenetically diverse datasets of spatial gene expression patterns. We determined ancestral spatial expression patterns of alx1, ets1, tbr, erg , and vegfr , key components of the skeletogenic gene regulatory network driving identity of the larval skeletogenic cell. Here we show ancestral state reconstructions of spatial gene expression of extant eleutherozoan echinoderms support homology and common ancestry of echinoderm larval skeletogenic cells. We propose larval skeletogenic cells arose in the stem lineage of eleutherozoans during a cell type duplication event that heterochronically activated adult skeletogenic cells in a topographically distinct tissue in early development., Competing Interests: The authors declare no competing interests.

Gastropod shell morphologies are famously diverse but generally share a common geometry, the logarithmic coil. Variations on this morphology have been modeled mathematically and computationally but the developmental biology of shell morphogenesis remains poorly understood. Here we characterize the organization and growth patterns of the shell-secreting epithelium of the larval shell of the basket whelk Tritia (also known as Ilyanassa ). Despite the larval shell's relative simplicity, we find a surprisingly complex organization of the shell margin in terms of rows and zones of cells. We examined cell division patterns with EdU incorporation assays and found two growth zones within the shell margin. In the more anterior aperture growth zone, we find that inferred division angles are biased to lie parallel to the shell edge, and these divisions occur more on the margin's left side. In the more posterior mantle epithelium growth zone, inferred divisions are significantly biased to the right, relative to the anterior-posterior axis. These growth zones, and the left-right asymmetries in cleavage patterns they display, can explain the major modes of shell morphogenesis at the level of cellular behavior. In a gastropod with a different coiling geometry, Planorbella sp., we find similar shell margin organization and growth zones as Tritia , but different left-right asymmetries than we observed in the helically coiled shell of Tritia These results indicate that differential growth patterns in the mantle edge epithelium contribute to shell shape in gastropod shells and identify cellular mechanisms that may vary to generate shell diversity in evolution., Competing Interests: The authors declare no conflict of interest.

Background: The most critical step in the pearl formation during aquaculture is issued to the proliferation and differentiation of outer epithelial cells of mantle graft into pearl sac. This pearl sac secretes various matrix proteins to produce pearls by a complex physiological process which has not been well-understood yet. Here, we aimed to unravel the genes involved in the development of pearl sac and pearl, and the sequential expression patterns of different shell matrix proteins secreted from the pearl sac during pearl formation by pearl oyster Pinctada fucata using high-throughput transcriptome profiling., Results: Principal component analysis (PCA) showed clearly different gene expression profiles between earlier (before 1 week) and later stages (1 week to 3 months) of grafting. Immune-related genes were highly expressed between 0 h - 24 h (donor dependent) and 48 h - 1 w (host dependent), and in the course of wound healing process pearl sac was developed by two weeks of graft transplantation. Moreover, for the first time, we identified some stem cell marker genes including ABCG2, SOX2, MEF2A, HES1, MET, NRP1, ESR1, STAT6, PAX2, FZD1 and PROM1 that were expressed differentially during the formation of pearl sac. The expression profiling of 192 biomineralization-related genes demonstrated that most of the shell matrix proteins (SMPs) involved in prismatic layer formation were first up-regulated and then gradually down-regulated indicating their involvement in the development of pearl sac and the onset of pearl mineralization. Most of the nacreous layer forming SMPs were up-regulated at 2 weeks after the maturation of pearl sac. Nacrein, MSI7 and shematrin involved in both layer formation were highly expressed during 0 h - 24 h, down-regulated up to 1 week and then up-regulated again after accomplishment of pearl sac formation., Conclusions: Using an RNA-seq approach we unraveled the expression pattern of the key genes involved in the development of pearl sac and pearl as a result of host immune response after grafting. These findings provide valuable information in understanding the molecular mechanism of pearl formation and immune response in P. fucata.

Background: Marine bivalves undergo complex development processes, such as shell morphology conversion and changes of anatomy and life habits. In this study, the transcriptomes of pearl oyster Pinctada fucata martensii and Pacific oyster Crassostrea gigas at different development stages were analyzed to determine the key molecular events related to shell formation, settlement and metamorphosis., Result: According to the shell matrix proteome, biomineralization-related genes exhibited a consensus expression model with the critical stages of shell formation. Differential expression analysis of P. f. martensii, revealed the negative regulation and feedback of extracellular matrixs as well as growth factor pathways involved in shell formation of larvae, similar to that in C. gigas. Furthermore, neuroendocrine pathways in hormone receptors, neurotransmitters and neuropeptide receptors were involved in shell formation, settlement and metamorphosis., Conclusion: Our research demonstrated the main clusters of regulation elements related to shell formation, settlement and metamorphosis. The regulation of shell formation and metamorphosis could be coupled forming the neuroendocrine-biomineralization crosstalk in metamorphosis. These findings could provide new insights into the regulation in bivalve development.

The explosion of the Deepwater Horizon (DWH) oil drilling rig resulted in the release of crude oil into the Gulf of Mexico. This event coincided with the spawning season of the Eastern oyster, Crassostrea virginica. Although oil bound to sediments constitutes an important source of polycyclic aromatic hydrocarbon (PAH) exposure to benthic organisms, toxicity of sediment-associated DWH oil has not been investigated in any bivalve species. Here, we evaluated the sublethal effects of acute exposure of gametes, embryos and veliger larvae of the Eastern oyster to different concentrations of unfiltered elutriates of sediment contaminated with DWH oil. Our results suggest that gametes, embryos and veliger larvae are harmed by exposure to unfiltered elutriates of contaminated sediment. Effective concentrations for fertilization inhibition were 40.6 μg tPAH50 L -1 and 173.2 μg tPAH50 L -1 for EC20 1h and EC50 1h values, respectively. Embryo exposure resulted in dose-dependent abnormalities (EC20 and EC50 values were 77.7 μg tPAH50 L -1 and 151 μg tPAH50 L -1 , respectively) and reduction in shell growth (EC20 24h value of 1180 μg tPAH50 L -1 ). Development and growth of veliger larvae were less sensitive to sediment-associated PAHs compared to embryos. Fertilization success and abnormality of larvae exposed as embryos were the most sensitive endpoints for assessing the toxicity of oil-contaminated sediment. Bulk of measured polycyclic aromatic hydrocarbons were sediment-bound and caused toxic effects at lower tPAH50 concentrations than high energy water accommodated fractions (HEWAF) preparations from the same DWH oil. This study suggests risk assessments would benefit from further study of suspended contaminated sediment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The molluscan shell is a fascinating biomineral consisting of a highly organized calcium carbonate composite. Biomineralization is elaborately controlled and involves several macromolecules, especially matrix proteins, but little is known about the regulatory mechanisms. The matrix protein Shematrin-2, expression of which peaks in the mantle tissues and in the shell components of the pearl oyster Pinctada fucata , has been suggested to be a key participant in biomineralization. Here, we expressed and purified Shematrin-2 from P. fucata and explored its function and transcriptional regulation. An in vitro functional assay revealed that Shematrin-2 binds the calcite, aragonite, and chitin components of the shell, decreases the rate of calcium carbonate deposition, and changes the morphology of the deposited crystal in the calcite crystallization system. Furthermore, we cloned the Shematrin-2 gene promoter, and analysis of its sequence revealed putative binding sites for the transcription factors CCAAT enhancer-binding proteins (Pf-C/EBPs) and nuclear factor-Y (NF-Y). Using transient co-transfection and reporter gene assays, we found that cloned and recombinantly expressed Pf-C/EBP-A and Pf-C/EBP-B greatly and dose-dependently up-regulate the promoter activity of the Shematrin-2 gene. Importantly, Pf-C/EBP-A and Pf-C/EBP-B knockdowns decreased Shematrin-2 gene expression and induced changes in the inner-surface structures in prismatic layers that were similar to those of antibody-based Shematrin-2 inhibition. Altogether, our data reveal that the transcription factors Pf-C/EBP-A and Pf-C/EBP-B up-regulate the expression of the matrix protein Shematrin-2 during shell formation in P. fucata , improving our understanding of the transcriptional regulation of molluscan shell development at the molecular level., (© 2018 Chen et al.)

Understanding developmental processes is foundational to clarifying the mechanisms by which convergent evolution occurs. Here, we show how a key convergently evolving trait is slowly 'acquired' in growing turtles. Many functionally relevant traits emerge late in turtle ontogeny, owing to design constraints imposed by the shell. We investigated this trend by examining derived patterns of shell formation associated with the multiple (at least 8) origins of shell kinesis in small-bodied turtles. Using box turtles as a model, we demonstrate that the flexible hinge joint required for shell kinesis differentiates gradually and via extensive repatterning of shell tissue. Disproportionate changes in shell shape and size substantiate that this transformation is a delayed ontogenetic response (3-5 years post-hatching) to structural alterations that arise in embryogenesis. These findings exemplify that the translation of genotype to phenotype may reach far beyond embryonic life stages. Thus, the temporal scope for developmental origins of adaptive morphological change might be broader than generally understood. We propose that delayed trait differentiation via tissue repatterning might facilitate phenotypic diversification and innovation that otherwise would not arise due to developmental constraints., (© 2018 The Author(s).)

Shell matrix proteins (SMPs) have important functions in biomineralization. In the past decades, the roles of SMPs were gradually revealed. In 2015, our group identified 72 unique SMPs in Pinctada fucata, among which Alveoline-like (Alv) protein was reported to have homologous genes in Pinctada maxima and Pinctada margaritifera. In this study, the full-length cDNA sequence of Alv and the functional analysis of Alv protein during shell formation were explored. The deduced protein (Alv), which has a molecular mass of 24.9 kDa and an isoelectric point of 11.34, was characterized, and the functional analyses was explored in vivo and in vitro. The Alv gene has high expression in mantle and could response to notching damage. The functional inhibition of Alv protein in vivo by injecting recombinant Alv (rAlv) antibodies destroyed prism structure but accelerated nacre growth. Western blot and immunofluorescence staining showed that native Alv exists in the EDTA-insoluble matrix of both prismatic and nacreous layers and has different distribution patterns in the inner or outer prismatic layer. Taken together, the characterization and functional analyses of matrix protein Alv could expand our understanding of basic matrix proteins and their functions during shell formation.

Marine bivalves inhabiting naturally pCO 2 -enriched habitats can likely tolerate high levels of acidification. Consequently, elucidating the mechanisms behind such resilience can help to predict the fate of this economically and ecologically important group under near-future scenarios of CO 2 -driven ocean acidification. Here, we assess the effects of four environmentally realistic pCO 2 levels (900, 1500, 2900 and 6600 μatm) on the shell production rate of Mya arenaria juveniles originating from a periodically pCO 2 -enriched habitat (Kiel Fjord, Western Baltic Sea). We find a significant decline in the rate of shell growth as pCO 2 increases, but also observe unchanged shell formation rates at moderate pCO 2 levels of 1500 and 2900 μatm, the latter illustrating the capacity of the juveniles to partially mitigate the impact of high pCO 2 . Using recently developed geochemical tracers we show that M. arenaria exposed to a natural pCO 2 gradient from 900 to 2900 μatm can likely concentrate HCO 3 - in the calcifying fluid through the exchange of HCO 3 - /Cl - and simultaneously maintain the pH homeostasis through active removal of protons, thereby being able to sustain the rate of shell formation to a certain extent. However, with increasing pCO 2 beyond natural maximum the bivalves may have limited capacity to compensate for changes in the calcifying fluid chemistry, showing significant shell growth reduction. Findings of the present study may pave the way for elucidating the underlying mechanisms by which marine bivalves acclimate and adapt to high seawater pCO 2 ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Atlantic surfclam (Spisula solidissima) is a commercially important species in North American waters, undergoing biological and ecological shifts. These are attributed, in part, to environmental modifications in its habitat and driven by climate change. Investigation of shell growth patterns, trace elements, and isotopic compositions require an examination of growth lines and increments preserved in biogenic carbonates. However, growth pattern analysis of S. solidissima is challenging due to multiple disturbance lines caused by environmental stress, erosion in umbonal shell regions, and constraints related to sample size and preparation techniques. The present study proposes an alternative method for describing chronology. First, we analyzed growth patterns using growth lines within the shell and hinge. To validate the assumption of annual periodicity of growth line formation, we analyzed the oxygen isotope composition of the outer shell layer of two specimens (46°54'20"N; 56°18'58"W). Maximum δ18Oshell values occurred at the exact same location as internal growth lines in both specimens, confirming that they are formed annually and that growth ceases during winter. Next, we used growth increment width data to build a standardized growth index (SGI) time-series (25-year chronology) for each of the three parts of the shell. Highly significant correlations were found between the three SGI chronologies (p < 0.001; 0.55 < τ < 0.68) of all specimens. Thus, ligament growth lines provide a new method of determining ontogenetic age and growth rate in S. solidissima. In a biogeographic approach, the shell growth performance of S. solidissima in Saint-Pierre and Miquelon was compared to those in other populations along its distribution range in order to place this population in a temporal and regional context., Competing Interests: One author is affiliated with TBM environnement/Somme. This does not alter our adherence to all PLOS ONE policies on sharing data and materials.

Animal size is a highly variable trait regulated by complex interactions between biological and environmental processes. Despite the importance of understanding the mechanistic bases of growth, predicting size variation in early stages of development remains challenging. Pedigreed lines of the Pacific oyster ( Crassostrea gigas ) were crossed to produce contrasting growth phenotypes to analyze the metabolic bases of growth variation in larval stages. Under controlled environmental conditions, substantial growth variation of up to 430% in shell length occurred among 12 larval families. Protein was the major biochemical constituent in larvae, with an average protein-to-lipid content ratio of 2.8. On average, 86% of protein synthesized was turned over (i.e. only 14% retained as protein accreted), with a regulatory shift in depositional efficiency resulting in increased protein accretion during later larval growth. Variation in protein depositional efficiency among families did not explain the range in larval growth rates. Instead, changes in protein synthesis rates predicted 72% of growth variation. High rates of protein synthesis to support faster growth, in turn, necessitated greater allocation of the total ATP pool to protein synthesis. An ATP allocation model is presented for larvae of C. gigas that includes the major components (82%) of energy demand: protein synthesis (45%), ion pump activity (20%), shell formation (14%) and protein degradation (3%). The metabolic trade-offs between faster growth and the need for higher ATP allocation to protein synthesis could be a major determinant of fitness for larvae of different genotypes responding to the stress of environmental change., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

In bivalves, the mantle tissue secretes organic matrix and inorganic ions into the extrapallial space (EPS) to form the shells. In addition, more and more evidences indicate the participation of hemocytes in shell mineralization, but no direct evidence has been reported that verifies the presence of hemocytes in the EPS, and their exact roles in biomineralization remain uncertain. Here, we identified hemocytes from the EPS of Pinctada fucata. Numerous components involved in cellular and humoral immunity were identified by proteome analysis, together with several proteins involved in calcium metabolism. The hemocytes exerted active phagocytosis and significantly upregulated the expression of immune genes after immune stimulation. A group of granulocytes were found to contain numerous calcium-rich vesicles and crystals, which serve as a calcium pool. During shell regeneration, some genes involved in calcium metabolism are upregulated. Strikingly, most of the shell matrix proteins were absent in the hemocytes, suggesting that they might not be solely responsible for directing the growth of the shell. Taken together, our results provided comprehensive information about the function of hemocytes in immunity and shell formation.

The three-spined stickleback Gasterosteus aculeatus is an important model for studying microevolution and parallel adaptation to freshwater environments. Marine and freshwater forms differ markedly in their phenotype, especially in the number of lateral plates, which are serially repeated elements of the exoskeleton. In fishes, thyroid hormones are involved in adaptation to salinity, as well as the developmental regulation of serially repeated elements. To study how thyroid hormones influence lateral plate development, we manipulated levels of triiodothyronine and thiourea during early ontogeny in a marine and freshwater population with complete and low plate phenotypes, respectively. The development of lateral plates along the body and keel was heterochronic among experimental groups. Fish with a low dosage of exogenous triiodothyronine and those treated with thiourea exhibited retarded development of bony plates compared to both control fish and those treated with higher a triiodothyronine dosage. Several triiodothyronine-treated individuals of the marine form expressed the partial lateral plate phenotype. Some individuals with delayed development of lateral plates manifested 1-2 extra bony plates located above the main row of lateral plates.

Characterization of morphological variation in the shells of extant Eastern Box Turtles, Terrapene carolina, provides a baseline for comparison to fossil populations. It also provides an example of the difficulties inherent to recognizing intraspecific diversity in the fossil record. The degree to which variation in fossils of T. carolina can be accommodated by extant variation in the species has been disagreed upon for over eighty years. Using morphometric analyses of the carapace, I address the relationship between modern and fossil T. carolina in terms of sexual dimorphism, geographic and subspecific variation, and allometric variation. Modern T. carolina display weak male-biased sexual size dimorphism. Sexual shape dimorphism cannot be reliably detected in the fossil record. Rather than a four-part subspecific division, patterns of geographic variation are more consistent with clinal variation between various regions in the species distribution. Allometric patterns are qualitatively similar to those documented in other emydid turtles and explain a significant amount of shape variation. When allometric patterns are accounted for, Holocene specimens are not significantly different from modern specimens. In contrast, several geologically older specimens have significantly different carapace shape with no modern analogue. Those large, fossilized specimens represent extinct variation occupying novel portions of morphospace. This study highlights the need for additional documentation of modern osteological variation that can be used to test hypotheses of intraspecific evolution in the fossil record.

Kinase-family with sequence similarity 20, member C (Fam20C) is a protein kinase, which can phosphorylate biomineralization related proteins in vertebrate animals. However, the function of Fam20C in invertebrate animals especially the role in biomineralization is still unknown. Herein, we cloned the cDNA of fam20C from the pearl oyster, Pinctada fucata. It is showed that the expression of fam20C in the mantle edge was much higher than other tissues. In situ hybridization showed that fam20C was expressed mostly in the outer epithelial cells of the middle fold, indicating it may play important roles in the shell formation. Besides, fam20C expression increased greatly in the D-shape stage of pearl oyster development, when the shell was first formed. During the shell repair process, the expression level of fam20C increased 1.5 times at 6 h after shell notching. Knockdown of fam20C in vivo by RNA interference resulted in abnormally stacking of calcium carbonate crystals at the edges of nacre tablets, showing direct evidence that fam20C participates in the shell formation. This study provides an insight into the role of kinase protein in the shell formation in mollusk and broaden our understanding of biomineralization mechanism.

Background: Despite recent work to characterize gene expression changes associated with larval development in oysters, the mechanism by which the larval shell is first formed is still largely unknown. In Crassostrea gigas, this shell forms within the first 24 h post fertilization, and it has been demonstrated that changes in water chemistry can cause delays in shell formation, shell deformations and higher mortality rates. In this study, we use the delay in shell formation associated with exposure to CO 2 -acidified seawater to identify genes correlated with initial shell deposition., Results: By fitting linear models to gene expression data in ambient and low aragonite saturation treatments, we are able to isolate 37 annotated genes correlated with initial larval shell formation, which can be categorized into 1) ion transporters, 2) shell matrix proteins and 3) protease inhibitors. Clustering of the gene expression data into co-expression networks further supports the result of the linear models, and also implies an important role of dynein motor proteins as transporters of cellular components during the initial shell formation process., Conclusions: Using an RNA-Seq approach with high temporal resolution allows us to identify a conceptual model for how oyster larval calcification is initiated. This work provides a foundation for further studies on how genetic variation in these identified genes could affect fitness of oyster populations subjected to future environmental changes, such as ocean acidification.

Shape variability represents an important direct response of organisms to selective environments. Here, we use a combination of geometric morphometrics and generalised additive mixed models (GAMMs) to identify spatial patterns of natural shell shape variation in the North Atlantic and Arctic blue mussels, Mytilus edulis and M. trossulus, with environmental gradients of temperature, salinity and food availability across 3980 km of coastlines. New statistical methods and multiple study systems at various geographical scales allowed the uncoupling of the developmental and genetic contributions to shell shape and made it possible to identify general relationships between blue mussel shape variation and environment that are independent of age and species influences. We find salinity had the strongest effect on the latitudinal patterns of Mytilus shape, producing shells that were more elongated, narrower and with more parallel dorsoventral margins at lower salinities. Temperature and food supply, however, were the main drivers of mussel shape heterogeneity. Our findings revealed similar shell shape responses in Mytilus to less favourable environmental conditions across the different geographical scales analysed. Our results show how shell shape plasticity represents a powerful indicator to understand the alterations of blue mussel communities in rapidly changing environments.

The turtle shell is often described as an evolutionary novelty that facilitated the radiation of the clade Testudines. The scutes, or keratinous plates, of the turtle shell are hypothesized to be patterned by reaction-diffusion dynamics, and this property of their development provides explanatory power to mechanisms of anomalous variation. A mathematical model of scute development predicts that anomalous variation in the phylogenetically stable pattern of scutes is achieved by environmental influence on the developmental program. We test this prediction with data on patterns of scute variation from natural nests and controlled incubation of sea turtle eggs in Florida and Western Australia. We find that high temperatures are sufficient to produce anomalous patterns in turtle scutes, and that this correlation is even stronger when conditions are dry. Furthermore, we find that the patterns of variation are not random; greater anomalous variation is found in the midline vertebral scutes and during a critical period of turtle development., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Understanding mollusk calcification sensitivity to ocean acidification (OA) requires a better knowledge of calcification mechanisms. Especially in rapidly calcifying larval stages, mechanisms of shell formation are largely unexplored-yet these are the most vulnerable life stages. Here we find rapid generation of crystalline shell material in mussel larvae. We find no evidence for intracellular CaCO 3 formation, indicating that mineral formation could be constrained to the calcifying space beneath the shell. Using microelectrodes we show that larvae can increase pH and [CO 3 2- ] beneath the growing shell, leading to a ~1.5-fold elevation in calcium carbonate saturation state (Ω arag ). Larvae exposed to OA exhibit a drop in pH, [CO 3 2- ] and Ω arag at the site of calcification, which correlates with decreased shell growth, and, eventually, shell dissolution. Our findings help explain why bivalve larvae can form shells under moderate acidification scenarios and provide a direct link between ocean carbonate chemistry and larval calcification rate.

The potential release of nanoparticles (NPs) into aquatic environments represents a growing concern for their possible impact on aquatic organisms. In this light, exposure studies during early life stages, which can be highly sensitive to environmental perturbations, would greatly help identifying potential adverse effects of NPs. Although in the marine bivalve Mytilus spp. the effects of different types of NPs have been widely investigated, little is known on the effects of NPs on the developing embryo. In M. galloprovincialis, emerging contaminants were shown to affect gene expression profiles during early embryo development (from trocophorae-24 hpf to D-veligers-48 hpf). In this work, the effects of amino-modified polystyrene NPs (PS-NH 2 ) on mussel embryos were investigated. PS-NH 2 affected the development of normal D-shaped larvae at 48 hpf (EC 50  = 0.142 mg/L). Higher concentrations (5-20 mg/L) resulted in high embryotoxicity/developmental arrest. At concentrations ≅ EC 50 , PS-NH 2 affected shell formation, as shown by optical and polarized light microscopy. In these conditions, transcription of 12 genes involved in different biological processes were evaluated. PS-NH 2 induced dysregulation of transcription of genes involved in early shell formation (Chitin synthase, Carbonic anhydrase, Extrapallial Protein) at both 24 and 48 hpf. Decreased mRNA levels for ABC transporter p-glycoprotein-ABCB and Lysozyme were also observed at 48 hpf. SEM observations confirmed developmental toxicity at higher concentrations (5 mg/L). These data underline the sensitivity of Mytilus early embryos to PS-NH 2 and support the hypothesis that calcifying larvae of marine species are particularly vulnerable to abiotic stressors, including exposure to selected types of NPs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Biologists have been fascinated with the extreme products of sexual selection for decades. However, relatively few studies have characterized patterns of selection acting on ornaments and weapons in the wild. Here, we measure selection on a wild population of weapon-bearing beetles (frog-legged leaf beetles: Sagra femorata) for two consecutive breeding seasons. We consider variation in both weapon size (hind leg length) and in relative weapon size (deviations from the population average scaling relationship between hind leg length and body size), and provide evidence for directional selection on weapon size per se and stabilizing selection on a particular scaling relationship in this population. We suggest that whenever growth in body size is sensitive to external circumstance such as nutrition, then considering deviations from population-level scaling relationships will better reflect patterns of selection relevant to evolution of the ornament or weapon than will variation in trait size per se. This is because trait-size versus body-size scaling relationships approximate underlying developmental reaction norms relating trait growth with body condition in these species. Heightened condition-sensitive expression is a hallmark of the exaggerated ornaments and weapons favored by sexual selection, yet this plasticity is rarely reflected in the way we think about-and measure-selection acting on these structures in the wild., (© 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.)

Interest in the origin and evolution of the turtle shell has resulted in a most unlikely clade becoming an important research group for investigating morphological diversity in developmental biology. Many turtles generate a two-component shell that nearly surrounds the body in a bony exoskeleton. The ectoderm covering the shell produces epidermal scutes that form a phylogenetically stable pattern. In some lineages, the bones of the shell and their ectodermal covering become reduced or lost, and this is generally associated with different ecological habits. The similarity and diversity of turtles allows research into how changes in development create evolutionary novelty, interacting modules, and adaptive physiology and anatomy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Biomineralization, including shell formation, is dedicatedly regulated by matrix proteins. PfY2, a matrix protein detected in the ethylene diamine tetraacetic acid (EDTA)-soluble fraction from both prismatic layer and nacreous layer, was discovered by our group using microarray. It may play dual roles during biomineralization. However, the molecular mechanism is still unclear. In this research, we studied the function of PfY2 on crystallization in vivo and in vitro, revealing that it might be a negative regulator during shell formation. Notching experiment indicated that PfY2 was involved in shell repairing and regenerating process. Repression of PfY2 gene affected the structure of prismatic and nacreous layer simultaneously, confirming its dual roles in shell formation. Recombinant protein rPfY2 significantly suppressed CaCO 3 precipitation rate, participated in the crystal nucleation process, changed the morphology of crystals and inhibited the transformation of amorphous calcium carbonate (ACC) to stable calcite or aragonite in vitro. Our results may provide new evidence on the biomineralization inhibition process.

The 'brain regionalization genes' Six3/6, Otx, Pax2/5/8, Gbx, and Hox1 are expressed in a similar fashion in the deuterostome, ecdysozoan, and the cephalopod brain, questioning whether this holds also true for the remaining Mollusca. We investigated developmental Gbx-expression in representatives of both molluscan sister groups, the Aculifera and Conchifera. Gbx is expressed in the posterior central nervous system of an aculiferan polyplacophoran and solenogaster but not in a conchiferan bivalve suggesting that Gbx, together with Six3/6, Otx, Pax2/5/8, and Hox1, is involved in central nervous system regionalization as reported for other bilaterians. Gbx is, however, also expressed in the anterior central nervous system, i.e. the anlagen of the cerebral ganglia, in the solenogaster, a condition not reported for any other bilaterian so far. Strikingly, all Gbx-orthologs and the other 'posterior brain regionalization genes' such as Pax2/5/8 and Hox1 are expressed in the mantle that secretes shell(s) and spicules of mollusks (except cephalopods). In bivalves, the ancestral condition has even been lost, with Gbx and Pax2/5/8 not being expressed in the developing central nervous system anymore. This suggests an additional role in the formation of the molluscan shell field(s) and spicule-bearing cells, key features of mollusks.