Shell Repair Research Articles

Neuropeptides regulate shell growth in the Mediterranean mussel (Mytilus galloprovincialis)

In bivalves, which are molluscs enclosed in a biomineralized shell, a diversity of neuropeptide precursors has been described but their involvement in shell growth has been largely neglected. Here, using a symmetric marine bivalve, the Mediterranean mussel (Mytilus galloprovincialis), we uncover a role for the neuroendocrine system and neuropeptides in shell production. We demonstrate that the mantle is rich in neuropeptide precursors and that a complex network of neuropeptide-secreting fibres innervates the mantle edge a region highly involved in shell growth. We show that shell damage and shell repair significantly modify neuropeptide gene expression in the mantle edge and the nervous ganglia (cerebropleural ganglia, CPG). When the CPG nerve commissure was severed, shell production was impaired after shell damage, and modified neuropeptide gene expression, the spatial organization of nerve fibres in the ganglia and mantle and biomineralization enzyme activity in the mantle edge. Injection of CALCIa and CALCIIa peptides rescued the impaired shell repair phenotype providing further support for their role in biomineralization. We propose that the regulatory mechanisms identified are likely to be conserved across bivalves and other shelled molluscs since they all share a similar nervous system, a common mantle biomineralization toolbox, and shell structure.

Effects of ocean acidification on the interaction between calcifying oysters (Ostrea chilensis) and bioeroding sponges (Cliona sp.)

Ocean acidification can negatively affect a broad range of physiological processes in marine shelled molluscs. Marine bioeroding organisms could, in contrast, benefit from ocean acidification due to reduced energetic costs of bioerosion. Ocean acidification could thus exacerbate negative effects (e.g. reduced growth) of ocean acidification and shell borers on oysters. The aim of this study was to assess the impact of ocean acidification on the oyster Ostrea chilensis, the boring sponge Cliona sp., and their host-parasite relationship. We exposed three sets of organisms 1) O. chilensis, 2) Cliona sp., and 3) O. chilensis infested with Cliona sp. to pHT 8.03, 7.83, and 7.63. Reduced pH had no significant effect on calcification, respiration and clearance rate of uninfested O. chilensis. Low pH significantly reduced calcification leading to net dissolution of oyster shells at pHT 7.63 in sponge infested oysters. Net dissolution was likely caused by increased bioerosion by Cliona sp. at pHT 7.63. Additionally, declining pH and sponge infestation had a significant negative antagonistic effect (less negative than predicted additively) on clearance rate. This interaction suggests that sponge infested oysters increase clearance rates to cope with higher energy demand of increased shell repair resulting from higher boring activity of Cliona sp. at low seawater pH. O. chilensis body condition was unaffected by sponge infestation, pH, and the interaction of the two. The reduction in calcification rate suggests sponge infestation and ocean acidification together would exacerbate direct (reduced growth) and indirect (e.g., increased predation) negative effects on oyster health and survival. Our results indicate that ocean acidification by the end of the century could have severe consequences for marine molluscs with boring organisms.

Among-individual differences in risk-taking behavior are associated with shell repair rate but not growth in a marine gastropod

Among-individual differences in risk-taking behavior are associated with shell repair rate but not growth in a marine gastropod

Management of shell repair in Indian pond terrapin: Case study

Management of shell repair in Indian pond terrapin: Case study

The molecular response of Mytilus coruscus mantle to shell damage under acute acidified sea water revealed by iTRAQ based quantitative proteomic analysis

Mytilus coruscus is an economically important marine bivalve that lives in estuarine sea areas with seasonal coastal acidification and frequently suffers shell injury in the natural environment. However, the molecular responses and biochemical properties of Mytilus under these conditions are not fully understood. In the present study, we employed tandem mass spectrometry combined with isobaric tagging to identify differentially expressed proteins in the mantle tissue of M. coruscus under different short-term treatments, including shell-complete mussels raised in normal seawater (pH 8.1), shell-damaged mussels raised in normal seawater (pH 8.1), and acidified seawater (pH 7.4). A total of 2694 proteins were identified in the mantle, and analysis of their relative abundance from the three different treatments revealed alterations in the proteins involved in immune regulation, oxidation-reduction processes, protein folding and processing, energy provision, and cytoskeleton. The results obtained by quantitative proteomic analysis of the mantle allowed us to delineate the molecular strategies adopted by M. coruscus in the shell repair process in acidified environments, including an increase in proteins involved in oxidation-reduction processes, protein processing, and cell growth at the expense of proteins involved in immune capacity and energy metabolism. SignificanceThe impact of global ocean acidification on calcifying organisms has become a major ecological and environmental problem in the world. Mytilus coruscus is an economically important marine bivalve living in estuary sea area with seasonal coastal acidification, and frequently suffering shell injury in natural environment. Molecular responses of M coruscus under the shell damage and acute acidification is still largely unknown. For this reason, iTRAQ based quantitative proteomic and histological analysis of the mantle from M. coruscus under shell damage and acute acidification were performed, for revealing the proteomic response and possible adaptation mechanism of Mytilus under combined shell damage and acidified sea water, and understanding how the mussel mantle implement a shell-repair process under acidified sea water. Our study provides important data for understanding the shell repair process and proteomic response of Mytilus under ocean acidification, and providing insights into potential adaptation of mussels to future global change.

Metabolic profiling of Mytilus coruscus mantle in response of shell repairing under acute acidification.

Mytilus coruscus is an economically important marine bivalve mollusk found in the Yangtze River estuary, which experiences dramatic pH fluctuations due to seasonal freshwater input and suffer from shell fracture or injury in the natural environment. In this study, we used intact-shell and damaged-shell M. coruscus and performed metabolomic analysis, free amino acids analysis, calcium-positive staining, and intracellular calcium level tests in the mantle to investigate whether the mantle-specific metabolites can be induced by acute sea-water acidification and understand how the mantle responds to acute acidification during the shell repair process. We observed that both shell damage and acute acidification induced alterations in phospholipids, amino acids, nucleotides, organic acids, benzenoids, and their analogs and derivatives. Glycylproline, spicamycin, and 2-aminoheptanoic acid (2-AHA) are explicitly induced by shell damage. Betaine, aspartate, and oxidized glutathione are specifically induced by acute acidification. Our results show different metabolic patterns in the mussel mantle in response to different stressors, which can help elucidate the shell repair process under ocean acidification. furthermore, metabolic processes related to energy supply, cell function, signal transduction, and amino acid synthesis are disturbed by shell damage and/or acute acidification, indicating that both shell damage and acute acidification increased energy consumption, and disturb phospholipid synthesis, osmotic regulation, and redox balance. Free amino acid analysis and enzymatic activity assays partially confirmed our findings, highlighting the adaptation of M. coruscus to dramatic pH fluctuations in the Yangtze River estuary.

Transcriptomic response of Mytilus coruscus mantle to acute sea water acidification and shell damage

Mytilus coruscus is an economically important marine calcifier living in the Yangtze River estuary sea area, where seasonal fluctuations in natural pH occur owing to freshwater input, resulting in a rapid reduction in seawater pH. In addition, Mytilus constantly suffers from shell fracture or injury in the natural environment, and the shell repair mechanisms in mussels have evolved to counteract shell injury. Therefore, we utilized shell-complete and shell-damaged Mytilus coruscus in this study and performed transcriptomic analysis of the mantle to investigate whether the expression of mantle-specific genes can be induced by acute seawater acidification and how the mantle responds to acute acidification during the shell repair process. We found that acute acidification induced more differentially expressed genes than shell damage in the mantle, and the biomineralization-related Gene Ontology terms and KEGG pathways were significantly enriched by these DEGs. Most DEGs were upregulated in enriched pathways, indicating the activation of biomineralization-related processes in the mussel mantle under acute acidification. The expression levels of some shell matrix proteins and antimicrobial peptides increased under acute acidification and/or shell damage, suggesting the molecular modulation of the mantle for the preparation and activation of the shell repairing and anti-infection under adverse environmental conditions. In addition, morphological and microstructural analyses were performed for the mantle edge and shell cross-section, and changes in the mantle secretory capacity and shell inner film system induced by the two stressors were observed. Our findings highlight the adaptation of M. coruscus in estuarine areas with dramatic fluctuations in pH and may prove instrumental in its ability to survive ocean acidification.

Ca2+ addition facilitates the shell repair with eggs production of Pomacea canaliculata through biomineralization and food intaking regulation

Pomacea canaliculata was by far one of the most harmful invasive organisms in the world, causing serious harm to aquatic crops and ecosystem. Calcium carbonate is a common component of aquatic environment, which is important for the growth of Pomacea canaliculata. Therefore, the objective of this study was to investigate the response characteristics of P. canaliculata suffered shell breakage to the addition of calcium carbonate in water environment. In this experiment, we explored the effects of calcium carbonate addition on the P. canaliculata shell repair rate, food intake, egg production, shell strength, and calcium content through breaking the snails shell and the addition of calcium carbonate treatment. The results showed that snail broken-shell repaired mostly within 21 days. The snails experienced a significant increase in shell repair rates during earlier days of the treatment, especially for female snails. Food intake of snails exhibited different patterns when their shells were broken and calcium carbonate was added. Shell breakage treatment combined with calcium carbonate addition significantly increased the diameter of snail eggs compared with the control and the calcium carbonate addition treatment without shell-broken snail group. There was no significant difference in shell strength or calcium content of male snails between the treatments. The study suggests that P. canaliculata exhibits a sex-dependent response pattern when subjected to shell damage and calcium carbonate addition. The findings can provide some references to better understand the invasion mechanism and survival strategy of the P. canaliculata.

Agricultural Use of Insecticides Alters Homeostatic Behaviors and Cognitive Ability in Lymnaea stagnalis.

Lymnaea stagnalis is an ecologically important, stress-sensitive, freshwater mollusk that is at risk for exposure to insecticides via agricultural practices. We provide insight into the impact insecticides have on L. stagnalis by comparing specific behaviors including feeding, locomotion, shell regeneration, and cognition between snails collected at two different sites: one contaminated by insecticides and one not. We hypothesized that each of the behaviors would be altered in the insecticide-exposed snails and that similar alterations would be induced when control snails were exposed to the contaminated environment. We found no significant differences in locomotion, feeding, and shell regeneration of insecticide-exposed L. stagnalis compared with nonexposed individuals. Significant changes in feeding and shell repair were observed in nonexposed snails inhabiting insecticide-contaminated pond water. Most importantly, snails maintained and trained in insecticide-contaminated pond water did not form configural learning, but this cognitive deficit was reversed when these snails were maintained in insecticide-free pond water. Our findings conclude that insecticides have a primarily negative impact on this higher form of cognition in L. stagnalis. Environ Toxicol Chem 2023;00:1-12. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Shell injuries, repair and malformation in the early Cambrian mollusc Helcionella antiqua from Scania, Sweden

ABSTRACT Three cases of repaired injuries and malformation in specimens of the helcionelloid mollusc Helcionella antiqua (Kiær, 1917) from the lower Cambrian (Cambrian Series 2, Stage 4) Gislöv Formation of southern Sweden document some of the oldest known durophagous attacks on Palaeozoic molluscs. Two of the injuries are developed as clefts, of which one had a severe effect on the continued growth of the shell. The third example is a large embayment removing large portions of the supra-apical part of the shell. A similar repaired injury is known in the slightly older mollusc Marocella mira Geyer, 1986. from Antarctica and Australia. The morphology of the injuries and the hydrodynamically quiet depositional setting suggests that the shell damage was caused by failed predatory attacks. The location of the repaired injuries suggests that the attacks may have targeted the head region of the molluscs, thus supporting an endogastrically coiled orientation of the shell in Helcionella. Only three repaired injuries in 252 Helcionella specimens were found, giving a shell repair frequency of 1.2%. All three examples occur in the larger size classes. The size-frequency distribution (N = 182) is strongly right skewed, which could suggest high input of juvenile specimens into the assemblage. The assemblage is interpreted as a time averaged and mixed death assemblage, albeit with good correspondence with the living shelly assemblage, due to a relatively thin, homogenous unit that may suggest within-habitat time averaging.

Middle and Upper Ordovician linguliformean and craniiformean brachiopods from the Brabant Massif, Belgium: Infaunal giants, encrusting forms and durophagy

Linguliformean and craniiformean brachiopods from the Middle and Upper Ordovician of the Brabant Massif (Belgium) are described for the first time and their palaeoecological and palaeobiogeographical implications are discussed. The restricted and generally poorly preserved material was collected from the Abbaye de Villers (Dapingian–Darriwilian) Formation and from the Katian Huet and Fauquez formations. The dark graptolitic mudstones of the latter unit yielded the most diverse assemblage including seven pseudolingulid, obolid, discinid, and craniopsid species while the Abbaye de Villers and Huet formations only yielded one species each. Due to the scarcity of internal morphological details available, comparison of the pseudolingulid and obolid specimens under investigation here was undertaken by running a Principal Component Analysis using a Log-Shape Ratio transformation of linear measurements. The study of valve shape changes at various growth stages helped identify these Belgian specimens at the family and generic levels. Finally, two unusually long (up to 20 mm) shell repair scars are documented in Pseudolingula and reflect predatory attacks at the anterior margin during early growth stages.

Evidence of biotic interactions through shell repair on Early Cretaceous gastropods from west-central Argentina

Evidence of durophagous predatory behavior on benthic invertebrates in the Upper Jurassic–Lower Cretaceous of the Neuquén Basin is scarce, despite the abundant record of potential predators. Herein, we document shell repair in one gastropod from lower Barremian marginal-marine deposits in northern Neuquén Basin, Argentina. This is the first report of shell repair on Early Cretaceous gastropods from Argentina and of shell repair frequencies from the Lower Cretaceous. Paleoanculosa macrochilinoides shells from three sections of the La Tosca Member (Huitrín Formation) in Mendoza province were studied. We described and interpreted the repaired breakage, calculated repair frequencies which were compared among localities, tested for geographic differences in size distribution of total samples and of repaired vs. undamaged shells, and assessed whether this species reached a size refuge. Studied shell repair consists of fractures cutting through growth lines roughly diagonally, from suture to suture, and near the aperture, thus representing apertural damage. Given its stereotypical nature, the damage represents the record of a biotic interaction, likely sublethal predation, instead of diagenetic compaction or damage by physical disturbance. Repair frequencies are low, indicating that shell architecture made P. macrochilinoides susceptible to lethal predatory attacks, or else that there were few predator-prey encounters. There are no major geographic differences regarding repair frequency, size distribution, and preservation. Likely, P. macrochilinoides did not reach a size refuge. This study provides evidence from both a time slice and geographic area with scarce data on crushing predation and from an in-between phase within the Mesozoic Marine Revolution.

Low-complexity domain-containing protein (LCDP), induced accumulation of calcium carbonate individual crystals to irregular polycrystals in the triangle sail mussel Hyriopsis cumingii

The nacre layer is composed of sheet-like aragonite crystals, with often loosely arranged polycrystal aragonite sheets which may induce poor mechanical properties in shells. In this study, a full-length low-complexity domain-containing protein (LCDP) cDNA from the triangle sail mussel Hyriopsis cumingii was generated and its role in shell formation investigated. The full-length cDNA was 1058 bp; it had an open reading frame (ORF) of 714 bp encoding 237 amino acids and contained a 20-amino acid signal peptide at the N-terminus and two low-complexity domains. H. cumingii LCDP was not homologous with other species. Tissue expression analyses showed that LCDP was specifically expressed in the mantle. In shell repair assays, significantly higher LCDP expression was observed in the shell repair group from days 12–21 (p < 0.01). After LCDP silencing, aragonite flake shapes in pearl layers became irregular with disordered deposition, while calcium carbonate (CaCO3) crystal surfaces in prismatic layers became rougher and organic matrices between crystals appeared skeletonized, indicating the importance of biomineralization. Our in vitro CaCO3 crystallization assays showed that LCDP induced single crystals to polycrystals, probably via loose arrangement between aragonite flakes. These results provide new insights on freshwater mollusk biomineralization and a theoretical basis for improved pearl quality.

Limited impact of a bioeroding sponge, Cliona sp., on Ostrea chilensis from Foveaux Strait, New Zealand.

Bioeroding sponges can cause extensive damage to aquaculture and wild shellfish fisheries. It has been suggested that heavy sponge infestations that reach the inner cavity of oysters may trigger shell repair and lead to adductor detachment. Consequently, energy provision into shell repair could reduce the energy available for other physiological processes and reduce the meat quality of commercially fished oysters. Nevertheless, the impacts of boring sponges on oysters and other shellfish hosts are inconclusive. We studied the interaction between boring sponges and their hosts and examined potential detrimental effects on an economically important oyster species Ostrea chilensis from Foveaux Strait (FS), New Zealand. We investigated the effect of different infestation levels with the bioeroding sponge Cliona sp. on commercial meat quality, condition, reproduction, and disease susceptibility. Meat quality was assessed with an index based on visual assessments used in the FS O. chilensis fishery. Meat condition was assessed with a common oyster condition index, while histological methods were used to assess sex, gonad stage, reproductive capacity, and pathogen presence. Commercial meat quality and condition of O. chilensis were unaffected by sponge infestation. There was no relationship between sex ratio, gonad developmental stage, or gonad index and sponge infestation. Lastly, we found no evidence that sponge infestation affects disease susceptibility in O. chilensis. Our results suggest that O. chilensis in FS is largely unaffected by infestation with Cliona sp. and therefore reinforces the growing body of evidence that the effects of sponge infestation can be highly variable among different host species, environments, and habitats.

Hc-transgelin is a novel matrix protein gene involved in the shell biomineralization of triangle sail mussel (Hyriopsis cumingii)

In triangle sail mussel (Hyriopsis cumingii), shell biomineralization is a complicated process that involves multiple gene products. Shell matrix proteins are involved in the formation of the organic framework and play an important role in the regulation of calcium carbonate deposition. In this study, A new shell matrix protein gene Hc-transgelin was identified in H. cumingii. The full-length cDNA of Hc-transgelin was 1200 bp, including a 501 bp open reading frame, which encoded 166 amino acids. Hc-transgelin is rich in lysine, it accounts for 11.40% of the protein. The predicted transgelin protein contained a conserved calmodulin homologous domain. A tissue-specific expression assay indicated that Hc-transgelin exhibited significantly highest expression in the mantle. Furthermore, Hc-transgelin in situ hybridization detected positive signals at the edge of the mantle outer fold, where nacre and prismatic layer biomineralization occur. An RNA interference assay showed that the shape of aragonite flakes in nacre changed and their growth was inhibited, and cracks appeared in the prismatic layer organic sheath when the expression of Hc-transgelin was suppressed. In a shell repair assay, a higher expression of Hc-transgelin appeared from day 12 to day 25 when the nacre accumulated quickly. These findings indicate that Hc-transgelin may be involved in the formation of aragonite flakes in the nacre and play a role in the formation of the organic sheath in the prismatic layer of the shell. This study provides new insights into the role of the Hc-transgelin gene and also contributes to the molecular understanding of mollusk shell formation.

The effect of environmental factors on shell growth and repair in Buccinum undatum

The processes and factors which affect shell growth and repair in molluscs are poorly understood. In this study, the capabilities of shell growth and repair in the marine gastropod Buccinum undatum were investigated experimentally by implementing laboratory-controlled mechanical damage to the shell margin/lip. Three key factors, life stage (juvenile or adult), seawater temperature (5–15 °C) and food availability (unfed, weekly, or daily feeding), were investigated in a series of controlled laboratory experiments to establish their roles in the processes of shell growth and repair. Significant differences in rates of shell growth and repair between food and temperature regimes were observed, with the greatest difference occurring with different life stages. Rates of shell growth in non-damaged whelks were slightly faster but not significantly different from damaged individuals in any of the experiments. Tank-reared juveniles maintained in the highest seawater temperature regime (15 °C) displayed significantly faster rates of shell repair (F = 6.47, p < 0.05) than conspecifics held at lower seawater temperatures. Through characterising both biological and environmental factors affecting shell growth and repair, it is demonstrated that there are multiple aspects influencing shell growth and shell repair. It is important to be able to understand and establish differences in rates of growth to better manage this commercial species.

Mussels Repair Shell Damage despite Limitations Imposed by Ocean Acidification

Bivalves frequently withstand shell damage that must be quickly repaired to ensure survival. While the processes that underlie larval shell development have been extensively studied within the context of ocean acidification (OA), it remains unclear whether shell repair is impacted by elevated pCO2. To better understand the stereotypical shell repair process, we monitored mussels (Mytilus edulis) with sublethal shell damage that breached the mantle cavity within both field and laboratory conditions to characterize the deposition rate, composition, and integrity of repaired shell. Results were then compared with a laboratory experiment wherein mussels (Mytilus trossulus) repaired shell damage in one of seven pCO2 treatments (400–2500 µatm). Shell repair proceeded through distinct stages; an organic membrane first covered the damaged area (days 1–15), followed by the deposition of calcite crystals (days 22–43) and aragonite tablets (days 51–69). OA did not impact the ability of mussels to close drill holes, nor the microstructure, composition, or integrity of end-point repaired shell after 10 weeks, as measured by µCT and SEM imaging, energy-dispersive X-ray (EDX) analysis, and mechanical testing. However, significant interactions between pCO2, the length of exposure to treatment conditions, the strength and inorganic content of shell, and the physiological condition of mussels within OA treatments were observed. These results suggest that while OA does not prevent adult mussels from repairing or mineralizing shell, both OA and shell damage may elicit stress responses that impose energetic constraints on mussel physiology.

Announcing the 2021 Journal of Experimental Biology Outstanding Paper Prize shortlist and winner.

Announcing the 2021 Journal of Experimental Biology Outstanding Paper Prize shortlist and winner.

Ecdysone signal pathway participates in shell formation in pearl oysters Pinctada fucata martensii

Ecdysone exists in arthropods, Mollusca and other invertebrates and plays vital roles in exoskeleton formation of Ecdysozoa. However, little is known about its functions in bivalve species. Herein, we identified ecdysone from the serum of pearl oyster Pinctada fucata martensii and obtained the coding sequence of ecdysone receptor (PmEcR) and homologue of its heterodimer protein retinoid X receptor (PmRXR). The deduced amino acid sequences of PmEcR and PmRXR contained a DNA-binding and ligand-binding domain and were very similar to the orthologs of other species. Moreover, PmEcR and PmRXR were located in the nuclei and cytoplasm of HEK-293T cells. PmEcR and PmRXR were highly expressed in early embryos and biomineralized mantle tissue. Moreover, the serum concentration of ecdysone significantly increased at 2, 4, 6, and 8 h post-shell notching. The expression of PmEcR in the mantle tissue was significantly induced at the corresponding time points, while that of PmRXR was significantly induced at 6 h. Ecdysone stimulation remarkably induced the expression of growth factors (BMP2 and BMP7), transcription factors (PmRunt and AP-1), and shell matrix protein genes (chitinase, lysine-rich matrix protein (KRMP), TYR2, and PmCOLVI), which indicated that ecdysone signaling plays important roles in shell repair. However, yeast two-hybrid assay and bimolecular fluorescence complementation showed that PmEcR and PmRXR did not form dimers, suggesting the different molecular interactions of EcR in bivalves. These findings provide insights into the function of ecdysone and its regulation pathway in bivalve species.

Foil tape—a simple, versatile tool for turtle shell repair

The repair of shell fractures in turtles is often delayed due to the time, labor and resources involved in many current shell repair methods, resulting in increased stress, handling and recovery time for the patient. This article introduces a new repair technique using aluminium foil tape combined with cyanoacrylate glue, which allows quick, simple and long-lasting closure of fresh shell injuries. Strips of malleable but inelastic foil tape are cut to size and positioned across the fracture at critical junctures, then glued in place on either side of the wound and burnished down to conform to surface irregularities and insure consistent adhesion. This technique is non-invasive, requires no curing time and can be customized for a wide range of turtle sizes and injuries, either as a stand-alone method or a preliminary stabilization tool. In the author’s experience, it has proven to be consistently effective at reducing fractures and staying in place until removal, when it is easily peeled off with no residual damage. The speed, ease and endurance of the foil tape method may encourage more widespread repair of fresh shell fractures and, in so doing, optimize recovery time and results for chelonian patients.