Ocean acidification disrupts the biomineralization process in the oyster Crassostrea virginica via intracellular calcium signaling dysregulation

Ocean acidification disrupts the biomineralization process in the oyster Crassostrea virginica via intracellular calcium signaling dysregulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ocean acidification disrupts the biomineralization process in the oyster Crassostrea virginica via intracellular calcium signaling dysregulation Wei Xu, Chi Huang, Joseph Matt, Christopher Hollenbeck, Leisha Martin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7180529/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Anthropogenically increased atmospheric carbon dioxide ( p CO 2 ) leads to ocean acidification, disrupting calcification in marine calcifiers by reducing the saturation state of calcium carbonate. Calcium is not only a crucial component in the shell and skeleton structure but also serves as an essential second messenger for regulating biomineralization across many species. Ocean acidification is well-studied as causing shell dissolution in a diversity of bivalve species by disordering calcium deposition. However, it remains unclear whether the calcium-mediated signaling pathway regulating biomineralization is also affected. This study assessed eastern oyster ( Crassostrea virginica ) to determine how calcium signaling responds to elevated p CO₂ and influences shell formation. Under elevated p CO 2 , increased intracellular calcium concentration was found in primary epithelial cell cultures from oyster mantle. Meanwhile, we observed upregulation of calmodulin, a primary sensor of intracellular calcium, while its downstream effector, calcineurin, was downregulated. In addition, four conserved shell matrix proteins (SMPs), representing shell construction conditions, were significantly upregulated in the CO 2 -exposed mantle cells. In vivo , larval C. virginica exhibited developmental stage-dependent alterations in calcium signaling and SMPs disarrangement stimulated by p CO 2 . We hypothesize that dysregulation of calcium signaling disrupts the expressions of SMPs and causes oyster shell deformation. Pharmaceutical blockage of the calcium-calmodulin binding induced abnormal expression of related genes and shell matrix changes consistent with those caused by elevated p CO 2 , both in vivo and in vitro . Importantly, calcineurin restored SMPs expression in CO 2 -treated mantle cells. These findings suggest that shell deformities under ocean acidification are related to disruption of the calcium-calmodulin signaling pathway, inhibiting calcineurin activity and affecting SMPs production. Biological sciences/Developmental biology/Experimental organisms/Non-model organisms Biological sciences/Evolution/Evolutionary developmental biology Biomineralization Calcium Signaling Shell Matrix Proteins Ocean Acidification Primary Mantel Cell Culture Oyster Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Biomineralization in marine calcifiers, such as mollusks, corals, and echinoderms, form shells or skeletons through their ability to precipitate calcium carbonate (CaCO 3 ) 1 , 2 . As one of the major calcifier groups, marine bivalves have important ecological functions and commercial value worldwide 3 , 4 . Biomineralization is fundamental to marine bivalve shell formation, which serves as a physical barrier against predators, environmental stressors, and desiccation. The shells of bivalves are composed of 95% CaCO 3 and 1–5% organic matrix 5 , 6 . Previous studies have shown that the shell formation process is initiated by mantle tissues, where the CaCO 3 is secreted to facilitate shell growth 7 , 8 . The mantle epithelial cells produce components of the shell organic matrix, which regulate the nucleation, regulation, and structural organization of CaCO 3 9,10 . Over the past decades, it has been speculated that a cascade of genetic regulation controls the shell formation process in marine bivalves 11 – 14 , 10 . More than 250 shell formation-related genes are predicted from the genome of Magallana gigas (previously Crassostrea gigas ) 15 , 16 . In particular, shell matrix protein (SMP) production and ion transportation pathways are suggested to be key genetic processes in regulating biomineralization of bivalves 11 , 17 – 21 . Several signaling pathways, including Wnt 22 , TGF-β/BMP 23 , and a subunit of guanine nucleotide-binding protein (G-protein) 24 are also implicated in organizing shell formation process in various marine bivalve species. In addition, some transcription factors, including homeobox genes, NF-κB, zinc finger proteins, and Smad family proteins, are identified as regulators of SMPs in marine bivalve 25 – 29 . However, the precise genetic pathways controlling shell formation remain unclear. Calcium is not only an essential component of biomineralizing organisms but also serves as a crucial intracellular second messenger regulating numerous signaling pathways. Many studies have reported that calcium signaling-related genes share a high degree of conservation throughout evolution between mollusks and mammals (Li et al., 2009; Li et al., 2016) . Calmodulin (CaM), a primary calcium sensor in calcium signaling pathways, has been identified in various bivalve species 32 – 35 , and mantle tissue related to the shell formation process shows the highest expression of CaM (Li et al., 2005). CaM and CaM-like proteins are reported to be present in the bivalve shell layer, and have been implicated in aragonite nucleation in conjunction with other shell organic matrix 18 , 36 . As a calcium/CaM-dependent phosphatase, calcineurin (CaN) is directly activated upon binding of the calcium-CaM complex 37 , 38 . CaN is also detected mainly localized in the inner epithelial cells of the mantle 30 , and is reported to play a role in regulating the shell formation process in P. fucata 39 . This process is homologous to the role of CaN in bone formation and absorption processes in vertebrate species 40 – 42 . Anthropogenic activities significantly increase atmospheric CO 2 levels, with projections indicating a rise from 400 ppm to 1000 ppm by the year 2100 43 . The ocean absorbs approximately 30% of the rapidly rising atmospheric CO 2 , leading to ocean acidification (OA) 44 – 46 . The dissolution of CO 2 in seawater results in altering pH levels and carbonate system in the ocean 47 . There is strong evidence that marine organisms are suffering from increased acidification 48 , 49 . Compared with vertebrate species, OA negatively affects more biological responses in invertebrate species, especially in calcification and biomechanics. The shifts of pH and carbonate system in the ocean ecosystem disrupt biomineralization processes in marine calcifiers by the dissolution of calcareous structures 51 and reduction of carbonate ions for CaCO 3 precipitation 44 , 52 , 53 . In addition to promoting dissolution of CaCO 3 shell and decreasing availability of carbonate ions, there is strong evidence suggesting that OA poses a threat to the shell formation process in marine bivalves by affecting calcium absorption and deposition in mantle tissue 54 , 1 , 55 – 57 . The fluctuations of intracellular calcium and transcriptional changes in calcium signaling-related genes in the gill, mantle, or hemocytes of various marine bivalve species are suggested as a compensatory mechanism to maintain calcium deposition for shell formation under the stress of OA 58 , 35 , 59 – 61 . Several studies have suggested that simulated OA conditions may disrupt cellular signaling pathways associated with immune responses in marine organisms by altering intracellular calcium concentration in hemocytes 62 – 64 . Therefore, there is a plausible hypothesis that OA conditions may disturb the intracellular calcium homeostasis and dysregulate the genetic mechanism of the calcium signaling pathway, ultimately impairing the bivalve shell formation. Due to the important characteristics of calcium for numerous intracellular processes and as the major component of bivalve shells, the role of intracellular calcium in biomineralization under OA conditions was explored in this study. We examined how OA affected bivalve biomineralization through the calcium signaling pathway by establishing an in vitro epithelial cell-dominated model using primary mantle cell culture from the eastern oyster, Crassostrea virginica . An in vivo analysis of biomineralization-related response to OA conditions across early developmental stages of C. virginica was also conducted to elucidate the role of calcium signaling in early shell formation. Both in vitro and in vivo results indicate that OA stress dysregulates the calcium signaling pathway by disrupting intracellular calcium levels in the mantle epithelial cells. The abnormal production and activity of CaM and CaN in the calcium signaling pathway subsequently affects the expression of SMPs, which disrupts the organic matrix function in the shell structure. Our findings suggest that OA-induced shell deformation in marine bivalves is associated with the dysregulation of intracellular calcium signaling pathway. 2. Results 2.1 Intracellular calcium influx in C. virginica mantle cells under elevated CO 2 To investigate the impact of OA on the cellular functions in biomineralization-related organs, the mantle tissues of C. virginica were dissected for primary cell culture (Fig. 1 A). Various cell types, including epithelial-like cells, hemocyte-like cells, and fibroblast-like cells, migrated from the explant within 48 hours (Fig. 1 B). The mantle cells from non-adhered explants or the cell dissociation were detached within 24 hours, whereas the cells that migrated from adhered explants remained viable for over two weeks. Hemocyte-like cells were progressively diminished from the adherent cell populations following culture medium replacement after 3–4 days. Less than 10% of fibroblast-like cells were detected in the migrated cell populations around each collagenase-treated explant (Fig. 2 B, Supplementary Data, Figure S3C). After 14 days, the epithelial-like cells migrated around the explants, forming approximately 70% confluence in the surrounding areas and becoming the dominant cell type (Fig. 1 C&D, Supplementary Data, Figure S3C). While most epithelial-like cells degenerated within two weeks, some remained viable for up to a month. To determine whether the elevated p CO 2 condition (simulated OA) can dysregulate calcium signaling pathways in the oyster mantle cells, the intracellular calcium level was monitored in CO 2 -treated mantle cells using calcium indicator Fluo4-AM. Following a short-term CO 2 treatment (1 hour), the average fluorescent intensity of the calcium signal in mantle cells significantly increased by 2.2% compared to their pre-exposure levels (Fig. 1 E&F). Additionally, flow cytometry analysis showed that the average fluorescence intensity of calcium in the mantle cells under long-term CO 2 exposure (24 hours) was 82.17%, significantly higher than the mantle cells in the control group (Fig. 1 G&H). 2.2 Dysregulation of calcium signaling pathway and SMPs in C. virginica mantle cells under elevated p CO 2 Two calcium signaling-related proteins, CaM ( Cv-CaM ) and CaN ( Cv-CaN ), were assessed to evaluate the cellular responses of calcium signaling pathway to increased p CO 2 level in oyster mantle cells. Expression patterns of the Cv-CaM were significantly upregulated at the mRNA level by 2.47 \(\:\pm\:\) 0.31-fold ( p = 0.0004) under 1.5% CO 2 condition compared with mantle cells with an ambient CO 2 environment (Fig. 2 A). However, Cv-CaN , the downstream gene of Cv-CaM , showed a contrasting pattern of mRNA expression. The expression of Cv-CaN was downregulated significantly to 0.75 \(\:\pm\:\) 0.08-fold ( p = 0.0493) in 1.5% CO 2 -treated mantle cells compared to the control group (Fig. 2 B). Similarly, the protein expression of CaM and CaN quantified by the immunofluorescence (IF) analysis showed a pattern consistent with the transcriptional analysis. The CaM expression at the protein level increased by 112.53% in the C. virginica mantle cells cultured in 1.5% atmospheric CO 2 exposure compared to the cells in the control group (Fig. 2 C, p = 0.0042). The C. virginica mantle cells dramatically decreased production of CaN protein by 86.46% under the 1.5% CO 2 condition (Fig. 2 D, p = 0.023). In the mantle epithelial cells, the transcription of SMPs, which are well-studied for their role in mollusks shell formation, were utilized to assess the responses of shell formation to elevated CO 2 concentration. Despite species-specific variations in SMPs, SMP repertories are reported to share four functional domains, including von Willebrand factor type A domains (VWA), chitin-binding-2 domains (CB-2), carbonic anhydrase domains (CA), and tyrosinase domains in diverse molluscan species 65 , 66 . The presence of these shared domains in different SMPs suggests a conserved biomineralization toolkit across bivalve species. Therefore, we selected four conserved SMPs-encoding genes to represent the shell construction conditions at the molecular level, including Pif97 ( Cv-Pif97 , VWA and CB-2 domains) 13 , Tyrosinase ( Cv-Tyr , Tyrosinase domain) 67 , Nacrein ( Cv-Nacrein , CA domain) 68 , 69 , and Chitin synthase ( Cv-Chit ) 70 , 71 . The mRNA expression of the four SMPs encoding genes were all significantly upregulated under the 1.5% CO 2 exposure compared to the control groups. The increased mRNA expression levels were 8.70 \(\:\pm\:\) 1.51-fold ( p = 0.0001), 9.85 \(\:\pm\:\) 2.19-fold ( p = 0.0007), 10.31 \(\:\pm\:\) 3.49-fold ( p = 0.02), and 11.98 \(\:\pm\:\) 3.27-fold ( p = 0.013) for Cv - Nacrein , Cv - Pif97, Cv - Chits , and Cv-Tyr , respectively (Fig. 2 E-H). 2.3 Elevated p CO 2 disturbed early shell formation and shell organic matrix biosynthesis in vivo To investigate the effects of elevated p CO₂ on early shell formation in vivo , developmental morphology and shell composition of C. virginica larvae were examined across critical early stages. With increased p CO 2 exposure, the early shell development during the period from trochophore (12 hours old) to D-shaped (36 hours old) C. virginica larvae (Supplementary Data, Figure S1 C) exhibited significant morphological changes. The trochophore larvae failed to develop into a D-shape morphology and showed asymmetric shell shape (Fig. 3 A&B). Larval shell deformity patterns, including “concave” hinge and protruding mantle on the fringe 72 , 73 were also detected under elevated p CO 2 concentration (Supplementary Data, Figure S4). In addition, the combination of Calcofluor/Calcein staining was utilized to assess the production of organic matrix and CaCO 3 , respectively. Compared to the distribution of the calcofluor signal (blue, indicator of organic matrix) on the outer shell surface of the larvae in control group, the location of calcofluor staining in OA treated D-shaped larvae dispersedly distributed across the entire shell surface (Fig. 3 A&B). Notably, shell calcification also irregularly displayed a brighter calcein (green, indicator of calcium) signal around hinge areas under simulated OA conditions. 2.4 Abnormal gene expression profiles of early shell formation under elevated atmospheric p CO 2 during C. virginica larval development At the four primary developmental stages of C. virginica larvae (Supplementary Data, Figure S1 C), including trochophore (T, 12 hours), D-shape (D, 36 hours), umbonal (U, 10 days), and pediveliger (P, 20 days) stages, the transcriptional pattern of Cv-CaM , Cv-CaN , and four SMPs biomarkers encoded genes showed differential responses to the elevated CO 2 environment (Fig. 3 C&D). Although the upregulation of Cv-CaM by elevated pCO 2 condition was observed at the trochophore (3.01 \(\:\pm\:\) 0.691 fold of control, p = 0.028) and the pediveliger stages (2.40 \(\:\pm\:\) 0.669 fold of control, p = 0.048), there was downregulation of the CaM expression pattern at the D-shape stages (0.84 \(\:\pm\:\) 0.09 fold of control, p = 0.033) and no significant changes in the umbonal stages (Fig. 5 A). On the other hand, significant downregulation of Cv- CaN was observed in all the larval development stages under the OA simulation (fold changes ranging from 0.47 \(\:\pm\:\) 0.11 to 0.86 \(\:\pm\:\) 0.13, p < 0.05), except D-shape larvae, which showed similar expression pattern between control and OA treatment groups (Fig. 3 D). While the mRNA levels of Cv-Nacrein and Cv-Pif97 showed no significant changes at the D-shape and the pediveliger stages, they were both significantly upregulated at the trochophore stage (Nacrein: 3.76 \(\:\pm\:\) 0.87 fold of control, p = 0.016; Pif97: 3.56 \(\:\pm\:\) 0.79 fold of control, p = 0.041) and downregulated at the umbonal stage (Nacrein: 0.36 \(\:\pm\:\) 0.08 fold of control, p = 0.008; Pif97: 0.33 \(\:\pm\:\) 0.07 fold of control, p = 0.017) under the OA simulation (Figs. 3 E&F). Compared with the control group, expression of Cv-Tyr in the CO 2 -treated larvae only displayed significant downregulation at the umbonal stage (0.43 \(\:\pm\:\) 0.09-fold of control, p = 0.004, Fig. 3 G). Chitin synthase, another SMP regulating the synthesis of extracellular matrix components, showed significant downregulation at the trochophore (0.73 \(\:\pm\:\) 0.10-fold of control, p = 0.049) and umbonal stages (0.47 \(\:\pm\:\) 0.10-fold of control, p = 0.0007) for the OA treatment but no significant changes at the D-shape and pediveliger stages (Fig. 3 H). 2.5 Elevated p CO 2 treatment and pharmacological calcium-CaM signaling disruption led to comparable dysregulation of calcium signaling-related genes and SMPs-encoding genes in vivo The results of elevated p CO 2 treatment above showed that the simulated OA condition not only dysregulated the calcium-signaling pathway but also affected shell development by changing the expression of SMPs. Thus, we assumed the modified expression of SMPs resulted from the dysregulated calcium signaling pathway under increased p CO 2 concentration and ultimately led to shell deformation. To test this hypothesis, we assayed the relationship between the calcium signaling pathway and SMPs expression in the mantle epithelial cells by inhibiting the calcium-CaM binding site using W-7. Similar to the mantle cells under elevated p CO 2 exposure, the cells treated by W-7 also demonstrated significant upregulation of CaM and downregulation of CaN at both mRNA and protein levels (Figs. 4 A-B & G-H). With 25 µM W-7 treatment, the mRNAs of Cv-CaM were upregulated by 4.10 \(\:\pm\:\) 1.40-fold ( p = 0.022, Fig. 4 A). Compared to the control group, there was 0.86 \(\:\pm\:\) 0.32-fold ( p = 0.045) downregulation of Cv-CaN under the W-7 treatment at the mRNA level (Fig. 4 B). Additionally, the significant upregulations of Cv - Nacrein (13.75 \(\:\pm\:\) 5.89-fold of control, p = 0.037), Cv - Pif97 (12.71 \(\:\pm\:\) 5.04 fold of control, p = 0.017), Cv-Tyr (43.99 \(\:\pm\:\) 28.53-fold of control, p = 0.043) and Cv - Chits (5.70 \(\:\pm\:\) 2.31 fold of control, p = 0.046) were also detected in the mantle cells under the W-7 treatment (Figs. 4 C-F), which is consistent with their mRNA expression pattern under the OA simulation treatment. Similarly, there was 110.51% higher protein production of CaM in the C. virginica mantle cells under the W-7 treatment than in the control group (Fig. 4 G). In contrast to the CaM protein level, the protein level of CaN under the W-7 treatment decreased by 92.98% compared with the cells under the control condition (Fig. 4 H). Notably, with the blockage of CaM binding site on calcium by W-7, the phosphatase enzymatic activity of CaN was significantly decreased (0.511 \(\:\pm\:\) 0.035-fold of control, p = 0.00208, Fig. 4 I). Likewise, the elevated environmental CO 2 level also reduced the phosphatase enzymatic activity of CaN to 0.534 \(\:\pm\:\) 0.0097-fold of control in the mantle cells ( p = 0.00151, Fig. 4 I). 2.6 Inhibition of calcium-CaM signaling disrupted the early shell formation of D-shape C. virginica larvae With the W-7 blockage of calcium-CaM binding, C. virginica larvae also demonstrated disorganization of shell organic matrix production, similar to those larvae in the elevated p CO 2 experiment. After 24 hours of W-7 treatment, the organization of the organic shell matrix, indicated by the calcofluor signal (blue), exhibited a distinct pattern in the D-shape larvae (Fig. 5 A-D). In the control group, the calcofluor signal was visibly concentrated in the center of the shell field and gradually faded toward the front end of the shell edge (Fig. 5 A). Under 1 \(\:\mu\:\) M W-7 treatment, the calcofluor signal was more uniformly distributed across the whole shell surface and showed higher intensity in the center (Fig. 5 B). In contrast, larvae exposed to 2 \(\:\mu\:\) M W-7 displayed a reduced calcofluor signal compared to the above treatments. The organic matrix appeared to have irregular patterns, with asymmetric growth of the organic matrix and lower intensity of the calcofluor signal compared to the control group. However, there was no obvious malformation of the calcification process in the oyster shell (Fig. 5 C). A dramatic change was detected under 5 µM W-7 treatment, where the shell exhibited substantial calcein signal around the center. Nevertheless, the organic matrix was confined to a thin layer along the margins of the valve, with a faint calcofluor signal observed at the center (Fig. 5 D). The ratio between matrix-occupied and calcified areas in a single valve from each larva was measured during early larval development. Despite there being no significant difference in the average ratio of matrix to calcified areas between the larvae in the control group and 1 \(\:\mu\:\) M W-7 treatment groups, the ratio significantly reduced by 57.22% under 2 \(\:\mu\:\) M and 45.31% under 5 \(\:\mu\:\) M W-7 treatment compared to the control group (Fig. 5 E). Notably, compared with the calcofluor signal intensity on the shell surface in the control group, the average fluorescent intensity was significantly enhanced by 59.79% under 1 µM W-7 treatment. In contrast, the average intensity decreased substantially by 56.34% and 48.80% in the larvae exposed to 2 µM and 5 µM W-7 treatments, respectively (Fig. 5 F). 2.7 Rescue of calcium signaling pathway and SMP production in mantle cells under CO 2 stress by CaN In the C.virginica mantle epithelial cells, both elevated p CO 2 and W-7 experiments displayed decreased protein production and phosphatase activity of CaN due to the dysregulated calcium signaling pathway. Based on this result, we further determined that the inhibition of CaN under the elevated p CO 2 concentration affected the SMPs expression using a CaN addition rescue experiment. Expression of the Cv-CaM and four SMPs in CO 2 -exposed C. virginica mantle cells in the presence of CaN were consistent with the mantle cells under ambient air conditions. The mRNA expression of Cv-CaM in the CO 2 -treated mantle cells with 20U ( p = 0.0001) and 100U ( p = 0.0025) CaN addition was significantly downregulated to control levels compared to the cells without CaN treatment, respectively (Fig. 6 A). However, Cv-CaN mRNA expression showed no significant difference between cells treated with elevated CO 2 alone and those exposed to both CO 2 and CaN (Fig. 6 B). Compared to mantle cells exposed to increased CO 2 without CaN treatment, Cv-Nacrein mRNA expression in CO 2 -exposed mantle cells was significantly downregulated with 20U ( p = 0.00153) and 100U ( p = 0.00164) CaN addition (Fig. 6 C). Similarly, Cv-Pif97 mRNA expression in CO 2 -exposed mantle cells treated with 20U and 100U CaN returned to control levels, significantly lower than in the mantle cells treated with elevated CO 2 alone (20U CaN: p = 0.00579; 100U CaN: p = 0.01577) (Fig. 6 D). Under elevated CO 2 conditions, Cv-Tyr mRNA expression in mantle cells with 20U ( p = 0.0495) and 100U ( p = 0.0414) CaN addition was significantly lower than in mantle cells without CaN treatment (Fig. 6 E). Furthermore, the Cv-Chits also displayed a significantly reduced pattern at the mRNA level in CO 2 -stimulated mantle cells treated with CaN compared to those exposed to CO 2 alone (20U CaN: p = 0.0441, 100U CaN: p = 0.0485) (Fig. 6 F). There was also no significant difference between the CO 2 -treated cells in the presence of CaN and the mantle cells in the control group. 3. Discussion Despite calcium being the most abundant mineral element in the marine bivalve shell structure, its role extends beyond providing substrate resources for biomineralization, as it also regulates various physiological processes as a secondary messenger intracellularly 74 – 76 . Under the stress of OA, the dysregulation patterns of intracellular calcium concentration are observed in oyster hemocytes 58 , 59 , suggesting that marine bivalves experience fluctuations in both intracellular and extracellular calcium levels in response to changes in carbonate chemistry in the ocean induced by the OA conditions. Recent studies highlight the crucial role of the calcium-CaM signaling pathway in vertebrate bone formation by regulating osteoblast differentiation and proliferation 40 , 42 , 77 , 78 . Similarly, several important calcium signaling-related genes, including CaM and CaN, are reported to be involved in shell formation in marine bivalves 36 , 17 , 18 , 39 . In this study, we identified an intracellular calcium influx pattern in oyster mantle epithelial cells, triggering dysregulation of the calcium-CaM signaling pathway under elevated p CO 2 conditions. As a result of the calcium-CaM signaling pathway disruption, dramatic morphological changes in shell shape and the shell organic matrix were observed, which are consistent with previous reports of OA effects on shell formation 72 , 73 . To link calcium signaling with the genetic regulation of C. virginica shell formation in response to OA, we established a more efficient in vitro model using oyster mantle cells. Compared to previous methods for isolating mantle cells from oysters using cell dissociation and earlier mantle tissue explant techniques 35 , 79 , our method significantly reduced the populations of other cell types, such as fibroblast cells and hemocytes (Supplementary Data, Figure S3). The high-quality, long-term in vitro maintenance of oyster mantle epithelial cells in this study provided a more robust system for monitoring prolonged, low-level environmental changes. Under elevated p CO 2 conditions, the pH of the oyster cell culture medium dropped from 7.8 to 7.4 within 48 hours of treatment (Supplementary data, Figure S5). A corresponding increase in intracellular calcium concentration in the mantle epithelial cells (Figs. 1 E-H) is consistent with an increased trend of extracellular calcium in the hemolymph of adult oysters exposed to elevated p CO 2 for 60 days 59 . These results suggest that the elevated CO 2 level may induce calcium influx in the oyster mantle epithelial cells as a compensatory response to maintain calcium homeostasis in fluctuating ambient conditions 61 . Along with CaM, several calcium-binding proteins have been shown to be upregulated in the oyster mantle under elevated CO 2 conditions 35 , 59 , 80 . Several studies indicate that the OA conditions disrupt the carbonate system and calcium level in marine bivalves 51 , 81 . Consequently, this study suggested that the dynamic of calcium concentrations in the intracellular and extracellular environments of oyster mantle epithelial cells triggered the differential expression of calcium-binding proteins and alteration of the calcium signaling pathway. In the calcium signaling pathways, CaM plays an essential role in various cellular processes in marine bivalves, such as the processes of environmental stress acclimation 82 , immune response 83 , and neuroendocrine regulation 84 . However, most studies on the biomineralization-related function of CaM focus on its role as an SMP in organizing the structure of shell layers and the transformation of CaCO 3 crystals during marine bivalve shell development. Our study demonstrated the upregulation of CaM at both mRNA and protein levels in isolated mantle epithelial cells under increased p CO 2 exposure (Figs. 2 A-D). These results are consistent with previous findings that reported upregulation of several CaM isoforms in oyster mantle tissue following CO 2 exposure in adult oysters 80 and at the cellular level 35 . However, in our in vivo oyster larvae models, only the trochophore and pediveliger stages exhibited upregulation of CaM in response to OA conditions (Fig. 3 C). Notably, these two stages are involved in initial shell formation and the preparation of metamorphosis for settlement 85 . Further, several CaM-like proteins have shown downregulation patterns in mantle tissue due to a potential calcium compensation strategy among the gill and mantle induced by CO 2 exposure 61 . Given the diverse cellular functions of CaM at the organism level, the in vitro mantle epithelial cell model demonstrated a higher efficiency and specificity in revealing the responses of the mantle epithelium cellular processes to the stress of simulated acidification conditions. As an essential downstream effector of the calcium-CaM signaling pathway, CaN is revealed to play an important role in marine bivalve shell formation by regulating the BMP signaling pathway 39 . In a previous study, two CaN subunits were isolated from the pearl oyster P. fucata : subunit A, which functions as the phosphatase catalysis unit, and subunit B, which serves as the calcium-binding site 30 . To explore the role of phosphatase activity in CaN during shell formation, we used the CaN subunit A homolog from C. virginica to assess the responses of phosphatase-related genes to OA treatment (Supplementary Data, Table S3). CaN expression was downregulated at the mRNA and protein levels in oyster mantle cells with increased CO 2 exposure (Fig. 2 B&D). Similarly, CaN downregulation was observed across oyster larval development stages in acidified conditions (Fig. 3 D), which is consistent with a previous study documenting the decreased transcription level of CaN in M. gigas D-shaped larvae exposed to decreased pH stress 64 . These shared patterns among the in vitro and in vivo models suggest that the dysregulation of the calcium-CaM signaling pathway is induced by elevated p CO 2 . In addition to the dysregulation of calcium-CaM, p CO 2 exposure also affected the expression of four conserved SMPs associated with bivalve shell formation in both in vitro and in vivo oyster models (Figs. 2 & 3 ). The upregulation of Cv-Nacrein in oyster mantle epithelial cells suggests a potential reduction of oyster calcification rate 11 , 68 . While in vitro analysis also revealed an upregulation pattern of Cv-Nacrein during initial shell formation at the trochophore stage (Fig. 3 E), these findings contrasted with previous studies that significant nacrein upregulation was only detected after the pediveliger stage 69 , 86 . The unexpected early upregulation was potentially related to a disruption of crystal formation during shell construction 68 , 87 . Since Pif97 regulates shell formation by inhibiting calcite crystal growth and amorphous CaCO 3 stabilization 13 , 88 , the increased expression level of this gene in the mantel epithelial cells suggested an OA-induced disruption of shell framework construction. The consistent upregulation of Cv-Pif97 during trochophore development under p CO 2 exposure indicated a negative impact of OA on the transformation and crystallization of CaCO 3 for early shell formation 71 , 89 , 90 . Compared to the upregulation of chitin synthase in the C. virginica mantle epithelial cells under OA conditions (Fig. 2 G), the Cv-Chits at both trochophore and umbonal stages exposed to elevated p CO 2 exhibited the opposite expression pattern (Fig. 3 H). Previous studies find chitin decomposition during early shell formation in acidified-treated trochophore oysters 71 , 91 . Given the evolutionary diversity of chitin synthase isoforms among mollusks, the contrasting Cv-Chits expression between the in vitro and in vivo models under OA suggests alternative biological functions of chitin synthetase beyond the biomineralization process in adult C. virginica 92 . Similarly, the tyrosinase ( Cv-Tyr ) also showed contrasting expression patterns between in vivo and in vitro models. While there was no significant impact of elevated CO 2 stress on the expression of Cv-Tyr at the trochophore stage (Fig. 3 G), the upregulation of Cv-Tyr in the mantle cells under the OA stress (Fig. 2 H) is in accordance with the increased mRNA expression of tyrosinase at the trochophore stage in Crassostrea angulata responding to OA 93 . The upregulation of Cv-Tyr revealed a potential disruption of the shell matrix production of oysters 67 . The reversed expression pattern of Cv-Tyr in larval C. virginica at the umbonal stage indicated a potential deformation of periostracum, the initial shell layer during the oyster’s early development 94 , 95 , which suggests potentially different shell formation mechanisms compared to adult oysters. Collectively, these findings indicate that elevated p CO 2 disrupts multiple aspects of shell biomineralization. The variable transcriptional level of the SMPs during C. virginica larval development also implied potentially different acclimation strategies for the shell formation process across larval stages due to the different forms of mineralization related to the developmental phases of oyster larvae 69 , 96 . This study also illustrated a potential connection between the dysregulated calcium-CaM signaling pathway and the abnormal expression of biomineralization-related SMPs in oysters under OA conditions. By blocking the calcium-binding sites on CaM, the CaM antagonist W-7 induced mantel epithelial cells to produce CaM while suppressing CaN protein production. The W-7 exposure functioned similarly to OA stress (Figs. 2 & 4 ). In addition, decreased phosphatase activity of CaN in the C. virginica mantle epithelial cells was also detected under both the elevated p CO 2 and the W-7 treatments (Fig. 5 ). These results suggested that dysregulation of the calcium-CaM signaling pathway inhibited CaN activity. Although no direct evidence linked CaM to the expression of CaN, a possible explanation involves the upregulation of calcium/CaM dependent protein kinase II (CaMKII), which was detected in both CO 2 -exposed and W-7 treated C. virginica mantle cells (Supplementary Data, Figure S6). CaMKII activation is found to inhibit the production of the Nuclear Factor of Activated T-cells (NFAT), which is identified as a transcription factor of CaN expression 97 . Interestingly, the activity of NFAT is also triggered by the dephosphorylation through CaN 98 . As the hypothesized downstream products of the calcium signaling pathway, the four SMPs in mantle epithelial cells were also regulated consistently between the treatments of the W-7 and increased p CO 2 . The anomalous changes in the organic matrix were attributed to the abnormal production of SMPs, which organize the further calcification step by transforming and organizing CaCO 3 deposition 99 . The presence of the chitin-binding domains in the Pif97 13 and tyrosinase 100 and a calcium-binding domain in nacrein 68 further suggests the role for these SMPs in oyster shell construction. Collectively, these results highlight the negative impact of the dysregulated calcium-CaM pathway on the production of SMPs and the organization of the organic matrix framework on the oyster shell surface. The CaN rescue experiments demonstrated that elevated CaN levels in the oyster mantle cells resulted in upregulated SMP expression in the CO 2 -treated cells returning to the control levels (Fig. 6 C-F). The reduced CaN activity under OA conditions was linked in this study to the transcription of SMP encoding genes, including Cv-Nacrein , Cv-Pif97 , Cv-Tyr , and Cv-Chits in C. virginica . As mentioned above, the decreased CaN production was possibly caused by the suppression of NFAT activity 101 , 102 , which interferes with several biomineralization-related signaling pathways, such as the BMP/Smads 103 , 39 , Wnt 104 , and NF-κB signaling pathways 105 . For example, previous studies show that Rel/ NF-κB mediates the transcription of nacrein in P. fucata 106 , 28 . The transcription of Pif97 is demonstrated to be related to the BMP/Smads signaling pathway 26 , 107 . The inhibition of CaN is found to decrease the biosynthesis of dopamine 108 and subsequently affect the synthesis of tyrosinase and chitin 91 . Surprisingly, the transcription levels of Cv-CaN in the CO 2 -treated mantle cells, following the addition of CaN, displayed a similar expression pattern to mantle cells without CaN treatment (Fig. 6 B). One possible explanation for the reduced CaM expression in the CO 2 -exposed mantle cells treated with CaN (Fig. 6 A) is that the external introduction of CaN was activated by binding to calcium-bound CaM. This binding likely decreased the availability of elevated free calcium, which in turn led to lower CaM gene expression. Based on the results of this study, we propose a model of the calcium-CaM signaling pathway regulating SMP production to explain the impact of future OA conditions on the biomineralization process in C. virginica (Fig. 6 G). Under OA stress, the dissolution of shell in marine bivalves leads to increased extracellular calcium levels and carbonate ions in the extrapallial fluid to support calcification 52 , 59 , 81 . To maintain the calcium homeostasis between the intra- and extracellular environment, the mantle epithelial cells of marine bivalves release large amounts of calcium intracellularly and enhance the calcium transportation from the ambient environment through the cell membrane (Wang et al., 2017; Chandra Rajan et al., 2021; Li et al., 2023). To counteract shell dissolution under long-term OA stress, oysters are shown to increase extracellular calcium (serum level) and upregulate CaM production, which plays a critical role in calcium deposition and transportation 59 , 61 . However, at the same time, this persistent upregulation of CaM may dysregulate the calcium-CaM signaling pathway by inhibiting the activities of CaN-NFAT and triggering the CaMKII-CREB pathway due to elevated concentrations of the calcium-bound CaM complex. Similar effects are suggested in human cardiac myocytes 97 . Finally, the decreased CaN activity results in the abnormal expression pattern of several SMPs by interrupting relevant signaling pathways, including the BMP signaling pathway (Pif97, Zhao et al., 2014; Shi et al., 2020), NF-κB signaling pathway (nacrein, Sun et al., 2015), and TGF- \(\:\beta\:\) signaling pathway (chitin synthase and tyrosinase, Liu et al., 2020). 4. Conclusion In summary, our results identified increased intracellular calcium and the dysregulation of calcium-CaM in the C. virginica mantle epithelial cells under a simulated OA environment as a strategy to maintain calcium homeostasis. The persistent calcium regulation, driven by the upregulation of CaM, reduced CaN protein production and phosphatase activity in the oyster mantle epithelial cells. The reduced CaN activity induced by elevated CO 2 exposure disrupted the production of various SMPs involved in the shell formation process and, consequently, disorganized the shell organic matrix framework and the shell construction process. This study clarifies the role of the calcium signaling pathway in marine bivalve shell formation. These findings, especially the essential role of CaN in regulating oyster SMP production and shell organic matrix organization, serve as valuable data for understanding the cellular response to OA conditions in marine bivalves and for developing potential strategies for mitigation. 5. Materials and Methods 5.1 Oyster mantle cell culture Adult C. virginica oysters of south Texas origin were obtained from the Texas A&M AgriLife Research Mariculture Facility (Corpus Christi, TX) and acclimated for two weeks in a recirculating system with 1 µm-filtered seawater from Corpus Christi Bay, Texas (~ 30ppt salinity) at 22 ~ 24 ◦ C. Before primary cell culture, oysters were incubated overnight in sterilized calcium and magnesium free artificial seawater solution (CMFSS) (Supplementary Data, Table S1 ) 35 , supplemented with 100units/ml of penicillin and 100µg/ml of streptomycin (CMFSS-Pen/Strep, pH = 7.7). Pre-treated oysters were dissected, and connective tissues along the mantle pallial were collected (Fig. 1 A). Mantle tissues were rinsed with CMFSS-Pen/Strep, then filtered with a 100 µm cell strainer. The rinsed mantle pieces were digested with 0.2% collagenase Type I (Thermo Fisher Scientific) in oyster cell culture basal medium (Supplementary Data, Table S1 ) to separate fibroblast cells from mantle explants. After 3 hours of digestion with collagenase I at 37 \(\:℃\) , the tissues were collected and minced into 1–2 mm 3 explants using a sterilized scalpel. Four to five explants were placed in a 25 mm 3 tissue culture flask (Thermo Fisher Scientific) for 45 minutes to adhere. The explants were then covered with 1 mL oyster cell culture medium supplemented with 10% Fetal Bovine Serum (FBS, Avantor, US), 100 units/ml of penicillin, and 100 µg/mL of streptomycin (oyster cell culture working medium). Primary cultures were incubated at 28°C in a humidified incubator without CO 2 for 48 hours to allow cell migration. 5.2 Stimulation of oyster mantle cells with increased environmental CO 2 C. virginica mantle cells were observed under a microscope on the 2nd day post-explantation (dpe) to confirm attachment. On the 3rd dpe, the medium was replaced with 1.5 ml fresh oyster cell culture medium + + before the elevated p CO 2 treatment. Cells from explants were exposed to ambient air (Control) or 1.5% CO 2 on the 5th dpe. After 48-hour exposure, mantle cells were harvested for RNA isolation using TRIzol Reagent (Molecular Research Center, Cincinnati) or fixed in 4% paraformaldehyde (PFA) (Thermo Fisher Scientific) for immunofluorescent staining. The explants were removed by filtering through 40 µm cell strainers before RNA isolation in TRIzol. Mantle cells from eight C. virginica oysters were used for the control and the CO 2 exposure groups. 5.3 Intracellular calcium level measurement in oyster mantle cells under elevated pCO 2 treatment Short-term (1 hour) and long-term (24 hours) CO 2 exposure experiments were conducted to evaluate intracellular calcium changes under elevated p CO 2 in C. virginica mantle cells. Intracellular calcium levels were measured using Fluo-4 AM (Thermo Fisher Scientific) following the manufacturer’s protocol. Briefly, a 2:1 ratio of 1 \(\:\mu\:\) M Fluo-4 AM and 1 \(\:\mu\:\) M Polyethylenimine (PEI) mixture was added to the oyster cell culture working medium and incubated for 30 minutes before staining. For short-term exposure, the calcium dye was incubated with isolated mantle cells for 1 hour at 28°C on the 5th dpe. Then, the Fluo-4 AM stained mantle cells were treated with 1.5% CO 2 for 1 hour. The fluorescent intensity in the stained cells was quantified before and after the CO 2 exposure under a fluorescent microscope (Olympus CKX53). At least five 10x magnified images derived from one oyster were captured for calcium measurement. The fluorescent signal of intracellular calcium was measured by calculating the mean grey value of the cells in each image via ImageJ 109 . For long-term CO 2 exposure, untreated and 24-hour 1.5% CO 2 -exposed mantle cells were incubated with Fluo-4 AM in oyster cell culture medium for 1 hour following the staining protocol above. The oyster mantle cells were harvested with 0.05% trypsin (Corning Inc.), and explants were removed using 40 \(\:\mu\:\) m cell strainers. Trypsinized mantle cells were rinsed and resuspended with Dulbecco's phosphate-buffered saline (DPBS, Corning Inc.). The calcium fluorescence in each cell was subsequently quantified by flow cytometry (BD Accuri™ C6 Plus) with excitation wavelength at 495 nm and emission wavelength at 518 nm, analyzing 10000 events. Mantle cells isolated from four C. virginica oysters were used in short- and long-term CO 2 exposure experiments. 5.4 Assay of oyster shell formation process under W-7 treatment in mantle cells N-(6-aminohexyl)-5-chloro-naphthalene sulfonilamide hydrochloride (W-7), a specific CaM antagonist inhibiting calcium-CaM binding site on its target proteins CaN 110 , was used to investigate the role calcium-CaM signaling pathway in C. virginica mantle cells. The mantle cells were treated with 25 µM W-7 (EMD Millipore Corp, IC₅₀ = 28 µM for calcium-CaM-dependent phosphodiesterase inhibition) on the 5th dpe for 24 hours. A 100 mM stock solution of W-7 was prepared by dissolving the W-7 powder in dimethyl sulfoxide (DMSO) and stored at − 20°C. The working concentration of 25 µM W-7 was prepared by diluting the stock solution in oyster cell culture working medium. Treated mantle cells were harvested for RNA isolation or immunofluorescent staining, as described in section 2.2 . The mantle cells from eight C. virginica oysters were used in the W-7 treatment group. 5.5 Immunofluorescence analysis of CaM and CaN in oyster mantle cells Immunofluorescence (IF) was used to visualize and quantify the expression of CaM and CaN at the protein level of C. virginica mantle cells in the control and treatment groups. After the CO 2 exposure or W-7 treatment, the mantle cells were rinsed with phosphate-buffered saline (PBS) and fixed in 4% PFA for 1 hour. The fixed samples were then blocked with 10% lamb serum in 1% Triton-PBS (PBST) for 2 hours at room temperature. Primary antibodies of CaM and CaN (purchased from the Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA) diluted in PBST (1:100) were applied to cover the samples in each culture flask, respectively and incubated at 4°C overnight. The primary antibodies used in this study contained mouse-anti-fungus CaM and mouse-anti-human CaN. After removing the primary antibodies and rinsing with PBS, the secondary antibody (goat anti-mouse) conjugated with Alexa flour TM 594 (red) dye (Thermo Fisher Scientific) was diluted in PBST (1: 400) and incubated with the samples for 2 hours in a dark area at room temperature. A blue nuclear dye, 4′,6-diamidino-2-phenylindole (DAPI, Biotium), was used to fluorescently stain the nuclear DNA to localize the individual cells with 1:5000 dilution in PBST. The samples were monitored under the Olympus BX53 fluorescent microscope after IF staining. Three pictures were captured for each culture flask under 20 \(\:\times\:\) magnification. The exposure time of the image was adjusted to 50 ms under the UV channel and 250 ms under the Texas Red channel. The same exposure time was used on all images of each antibody. Pictures were also taken from secondary antibodies-stained samples without the primary antibodies as a negative control to avoid the disturbance of autofluorescence. The total fluorescent signal of CaM and CaN in each cell was measured using ImageJ 109 . The protein expression level was calculated as the corrected total cell fluorescence (CTCF), which was calculated as Integrated Density – (Area of selected cell \(\:\times\:\) Mean fluorescence of background readings). 5.6 CaN phosphatase activity assay in C. virginica mantle cells Post-treatment C. virginica mantle cells from sections 2.2 and 2.4 were harvested in 1 mL of 0.05% trypsin (Corning Inc.), centrifuged to form a pellet, and lysed in 50 µL of calcineurin assay buffer (Enzo) for protein isolation. Calcineurin phosphatase activity was measured using the calcineurin phosphatase assay system (BML-AK904, Enzo) with 3 µg of protein from mantle cells, following the manufacturer's instructions. The activity was quantified spectrophotometrically by measuring the release of free phosphate from the calcineurin-specific RII phosphopeptide substrate using the BIOTEK Cytation 5 image reader (Agilent). 5.7 Larval oysters production and simulated acidification treatments South Texas C. virginica oyster larvae were collected from the Texas A&M AgriLife Mariculture Center, following the center’s spawning and culture protocols. Four early life stages of oysters, including trochophore, D-shaped, umbonal, and pediveliger stages, were collected respectively in 12 hours, 48 hours, 10 days, and 20 days post-fertilization. A light microscope was used to ensure that > 70% of the larvae had reached the particular development stages (Supplementary Data, Figure S1 ). Oyster larvae were exposed to two different p CO 2 conditions, including current p CO 2 (425 ppm) and predicted p CO 2 (1000 ppm) levels based on IPCC 43 . Each experimental trial involved larvae placed in 1-liter plastic beakers with 700mL of filtered seawater. A simulated OA environment was set up in a glovebox filled with CO 2 gas to mimic the elevated CO 2 in the atmosphere (Supplementary Data, Figure S1 ). A programmable CO 2 controller (ICC-500T, INKBIRD) was utilized to maintain a consistent atmospheric CO 2 concentration of 1000 \(\:\pm\:\) 50 ppm. Four replicates of larvae were treated with ambient air and elevated CO 2 conditions, respectively. The larval samples were allocated to different groups for 24 hours of treatment during the trochophore stage or 48 hours treatment at later larval stages, to ensure adequate CO 2 dissolution. The number of larvae in each replicate was optimized based on the guidelines from the Mariculture Center (Supplementary Data, Table S2). After a certain period of CO 2 exposure, the larvae from each treatment replicate were harvested in 7 ml TRIzol Reagent for RNA isolation. 5.8 Evaluation of shell organic matrix synthesis by larval oysters under CO 2 exposure Shell formation in 12-hour-old trochophores under the CO 2 exposure was observed after 24-hour treatment. Early D-shape larvae were evaluated for the synthesis of shell organic matrix materials and calcification of the shell using fluorescent dyes calcein and calcofluor, respectively 67 , 73 . Calcein (MP Biomedicals), a calcium-dependent fluorescent signal (UV channel, Exc: 408 nm/Em: 450–490 nm), was utilized for CaCO 3 staining of the calcified shell (final concentration 10 mM in 0.01% DMSO). Calcofluor White Stain (Sigma-Aldrich), a chitin-staining fluorescent signal (FITC channel, Exc: 488 nm/Em: 520–560 nm), was used to visualize the shell organic matrix (final concentration 0.02 mM in 0.01% DMSO). Before staining, overnight fixed larvae were washed with DPBS to remove the 4% PFA. Calcein was applied to the rinsed larvae for 2 hours. After the DPBS rinsing to remove the excess Calcein dye, the larval samples were stained with the Calcofluor White Stain. After 30 minutes of Calcofluor staining, the larvae were rinsed again with DPBS to remove Calcofluor and immediately imaged with a fluorescent microscope. 5.9 CaM-inhibition assay for larval oyster shell development W-7 was used to assess the function of the calcium-CaM signaling pathway in the biomineralization process during the early development of C. virginica larvae. Approximately 100 early D-shape larvae (24 hours old) were separately treated with 2 mL of different concentrations of W-7 (1 µM, 2 µM, and 5 µM) diluted in filtered seawater for 24 hours in the dark at room temperature. After treatment, 10 larvae per group were fixed in 4% PFA at 4°C overnight for shell biogenesis analysis. The early shell development was assessed using Calcein and Calcofluor White Stain, as described in section 2.8. The areas occupied by calcein and calcofluor on a single valve of each larva were manually outlined in ImageJ to determine the ratio of calcein to calcofluor as an indicator of shell biogenesis 67 , 73 . In addition, the fluorescence intensity of calcofluor in larval samples was quantified to estimate the production of shell matrix materials. 5.10 Recovery experiment of CaN on regulating SMPs expression in C. virginica mantle cells under elevated CO 2 C. virginica mantle cells treated with CaN during elevated CO 2 exposure were used to investigate the involvement of CaN in regulating the expression of SMPs under simulated OA conditions. A 5000U human CaN stock solution (BML-SE163, Enzo), where 1U of calcineurin catalyzes the dephosphorylation of 1 mM substrate per minute, was mixed with 10 µM PEI (1:1 ratio) for 30 minutes incubation to facilitate cell delivery. The mixture was then diluted with oyster cell culture working medium to prepare 20U and 100U CaN-supplemented cell culture medium. On the 5th dpe, the mantle cells were exposed to 1.5% CO 2 exposure in CaN- supplemented media for 48 hours. Treated cells were harvested for RNA isolation, as described in section 2.2 . The mantle cells from four C. virginica oysters were used in both the 20U and 100U CaN treatment groups. 5.11 Quantification of gene expression in C. virginica mantle cells and C. virginica larvae Total RNA was extracted from C. virginica mantle cells across different treatment groups using a standard Trizol-chloroform protocol 111 , followed by DNA removal with the Ambion TURBO DNA-free Kit (Thermo Fisher Scientific). SuperScript IV Reverse Transcriptase (Invitrogen) was used to synthesize cDNA. The cDNA samples were used for SYBR Green qPCR analysis on a QuantStudio 3 Real-Time PCR System (Applied Biosystems). The qPCR program was run at 50°C for 2 min, 94°C for 2 min, followed by 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The 2 − ΔΔCt method was used to analyze the gene expression data. The mRNA expression levels of CaM, CaN, and four SMP-encoding genes, were measured under different treatment groups. Candidate genes previously characterized from molluscan species, including P. fucata , M. gigas , and Atrina rigida were used to perform BLASTp analysis against the whole genome derived from C. virginica to identify the homologous mRNA sequences of CaM ( Cv - CaM ), CaN ( Cv - CaN ), Pif97 ( Cv - Pif97 ), Nacrein ( Cv - Nacrein ), Tyrosinase ( Cv-Tyr ), and Chitin synthase ( Cv - Chits ) (Supplementary Data, Table S3). The targeted genes for qPCR analysis were identified based on their amino acid similarity from the BLASTp results. The specificities of the primers designed for the qPCR analysis by Integrated DNA Technologies (IDT) were confirmed by running the amplicons on a 7% acrylamide TBE gel (Supplementary Data, Figure S2). All the Ct values of the biomarkers were normalized by those of the housekeeping gene Elongation factor 1α gene ( Cv-ef1α ) 35 . The Trizol-stored larval samples from sections 2.7 and 2.9 were homogenized using a microtube homogenizer (Bertin technologies). The homogenized samples were utilized for qPCR analysis to evaluate the relative expression levels of the biomineralization-related biomarkers (Table S3) following the procedures detailed above. 5.12 Statistical analysis Comparisons of intracellular calcium fluorescent signal, gene expressions of calcium signaling pathway related genes and SMPs at mRNA level, IF quantification of Cv -CaM and Cv -CaN, organic matrix/shell area ratio, Calcofluor fluorescent intensity, and CaN phosphatase activity were all conducted between the control and treatment groups using one-way ANOVA in R version 3.6.2 112,113 . All quantitative data were expressed as mean \(\:\pm\:\) standard error (SE). In all the statistical evaluations, P < 0.05 was considered statistically significant. Declarations Acknowledgement We would like to thank Dr. Michael Wetz’s group at the TAMU-Corpus Christi, Harte Research Institute for assisting us with the flow cytometry work. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7180529","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":498671243,"identity":"affcebca-4f7f-4c06-9ea2-25ebd8456ff9","order_by":0,"name":"Wei Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAnklEQVRIiWNgGAWjYJCCAx+gDAlidTAenJFAohbmwzwkaTG4kWNw2PaHXbTBAeaDt3mI0nLmjMHhnITk3A0H2JKtidJidrwHpIUZqIXHTJo4LYd5DA5bJNQDtfB/I1ILyBaGhMMgW9iI02J/5ljBwZ6047kzD7MZW84hRovkjOTNH37YVOf2HW9+eOMNMVoQgJk05aNgFIyCUTAK8AEAK340b+w+hkMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-0023-4045","institution":"Texas A\u0026M University","correspondingAuthor":true,"prefix":"","firstName":"Wei","middleName":"","lastName":"Xu","suffix":""},{"id":498671244,"identity":"2cd36fd7-6406-4e41-b362-06a061ec76b8","order_by":1,"name":"Chi Huang","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Chi","middleName":"","lastName":"Huang","suffix":""},{"id":498671245,"identity":"db9c421b-ac66-4a35-aeb2-1ff7275de7af","order_by":2,"name":"Joseph Matt","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Matt","suffix":""},{"id":498671246,"identity":"e8a73f32-bbb4-401b-bc75-82bde5625ffa","order_by":3,"name":"Christopher Hollenbeck","email":"","orcid":"","institution":"Texas A\u0026M University Corpus Christi","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Hollenbeck","suffix":""},{"id":498671247,"identity":"794e1e84-208d-4c45-8e4b-3c0bf370e627","order_by":4,"name":"Leisha Martin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Leisha","middleName":"","lastName":"Martin","suffix":""}],"badges":[],"createdAt":"2025-07-21 19:50:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7180529/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7180529/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-026-09861-y","type":"published","date":"2026-03-17T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89393297,"identity":"ea45f41f-cc03-48d5-ad47-aab58522b016","added_by":"auto","created_at":"2025-08-19 13:23:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":586945,"visible":true,"origin":"","legend":"\u003cp\u003eCalcium flux in C. virginica mantle cells under elevated CO\u003csub\u003e2\u003c/sub\u003e stress. (A) Soft body of the eastern oyster C. virginica. The muscle, heart, gill, and mantle were blue arrowed, respectively. Dissection location of mantle tissue for cell isolation was indicated by a red dashed line. (B) C. virginica mantle explant and isolated mantle cells after 48 hours of seeding. White arrows = epithelial-like cells; blue arrows = hemocyte-like cells; black arrows = fibroblast-like cells. (C) C. virginica mantle cells cultured on day 15. (D) A magnified view of C showed a majority of epithelial-like cells. (E) Loading of Fluo-4/AM in the isolated mantel epithelial cells before and after 1.5% CO\u003csub\u003e2\u003c/sub\u003e short-term exposure. (F) Comparison of labeled calcium signal intensities in the C. virginica mantel cells before and after 1.5% CO\u003csub\u003e2\u003c/sub\u003e 1-hour short-term exposure (n = 5 images per oyster). (G) Fluorescence intensity of intracellular calcium in C. virginica mantle cells recorded by flow cytometer between the control condition and the 24-hour long-term CO\u003csub\u003e2\u003c/sub\u003e exposure. (H) Mean fluorescence intensity of intracellular calcium level (n = 4 oysters). *P \u0026lt; 0.05. White scale bar: 100 m.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7180529/v1/d2b99075fad6c3cfd654125a.jpg"},{"id":89393304,"identity":"63f1a6f3-c1aa-4380-8f51-5bf61d0897af","added_by":"auto","created_at":"2025-08-19 13:23:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":393254,"visible":true,"origin":"","legend":"\u003cp\u003eResponses of calcium\u003csup\u003e \u003c/sup\u003esignaling-related genes and SMPs in C. virginica mantle cells under simulated CO\u003csub\u003e2\u003c/sub\u003e condition (n = 8 oysters). (A-B) The relative mRNA expression of Cv-CaM (A) and Cv-CaN (B) in C. virginica mantle cells under simulated CO\u003csub\u003e2\u003c/sub\u003e condition. (C-D) Protein levels of Cv-CaM (C) and Cv-CaN (C) were respectively compared among autofluorescence (Auto), control, and 1.5% CO\u003csub\u003e2\u003c/sub\u003e exposure with IF quantification via ImageJ (n = 3 images per oyster). (E-F) The relative mRNA expression level of Cv-Nacrein(E), Cv-Pif (F), Cv-Chits (G), and Cv-Tyr (H) in C. virginica mantle cells between ambient air and elevated pCO\u003csub\u003e2\u003c/sub\u003e conditions. All CO\u003csub\u003e2\u003c/sub\u003e treatments were applied to cells for 48 hours. * P \u0026lt; 0.05. White scale bar: 100 m. Error bar: standard error of the mean.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7180529/v1/4830129247d114c3559543cf.jpg"},{"id":89393317,"identity":"2bc68bcf-9f22-45ae-a8da-1a87f4c4ea63","added_by":"auto","created_at":"2025-08-19 13:23:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":364694,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological and molecular changes of early shell formation during C. virginica larvae development to the simulated OA condition at the morphological and molecular level. (A-B) Morphological characteristics of early D-shape larvae (36-hour old) under the control (A) and OA (B) conditions corresponding to the organic matrix production (calcofluor staining) and the CaCO\u003csub\u003e3\u003c/sub\u003e deposition (calcein staining). (C-H) Expression pattern of two calcium signaling-related genes Cv-CaM (C) and Cv-CaN (D) and four SMP-encoding genes Cv-Nacrein (E), Cv-Pif97 (F), Cv-Tyr (G), and Cv-Chits (H) at mRNA level across developmental stages. T, trochophore stage; D, D-shaped stage; U, umbonal stage; P, pediveliger stage. The trochophore stage larvae were exposed to elevated CO\u003csub\u003e2\u003c/sub\u003e for 24 hours, and the rest of the larval stages were exposed for 48 hours. *P \u0026lt; 0.05. White scale bar: 100 m. Error bar: standard error of the mean.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7180529/v1/8a6aeced21bf7abf8e9743b4.jpg"},{"id":89393321,"identity":"40b726ae-e0ba-4574-846b-ed28697c6455","added_by":"auto","created_at":"2025-08-19 13:23:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":387175,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of CaM inhibitor (W-7) and OA condition on calcium signaling and biomineralization-related markers gene expression and CaN activity in C. virginica mantle epithelial cells. (A-F) Expression of Cv-CaM (A), Cv-CaN (B), Cv-Nacrein (C), Cv-Pif (D), Cv-Tyr (E), and Cv-Chits (F) in C. virginica mantle cells between ambient air and W-7 treatment. (G-H) Protein levels of Cv-CaM (G) and Cv-CaN (H) were respectively compared among autofluorescence (Auto), control, and 1.5% CO\u003csub\u003e2\u003c/sub\u003e exposure with IF quantification via ImageJ (n = 3 images per oyster). (E-F) (I) CaN phosphatase activity under the simulated OA condition (1.5% CO\u003csub\u003e2\u003c/sub\u003e) and the W-7 treatment (25 M). Significant difference between the two groups (P \u0026lt; 0.05) was demonstrated by different letters over the column in I. All W-7 and CO\u003csub\u003e2\u003c/sub\u003e treatments were applied to cells for 48 hours. *P \u0026lt; 0.05. White scale bar: 100 m. Error bar: standard error of the mean.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7180529/v1/7ad0861d0942443a719e7ee3.jpg"},{"id":89393326,"identity":"20d0c310-1c20-44b7-b51c-d7a33d5312b6","added_by":"auto","created_at":"2025-08-19 13:23:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":328469,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of CaM inhibitor (W-7) treatments on early shell development from trochophore to D-shape stages in C. virginica. (A-D) Brightfield, calcofluor fluorescent staining (Shell organic matrix), calcein fluorescent staining (CaCO3 deposition), and merged images of early D-shaped oyster larvae under control (A), 1 mM W-7 (B), 2mM W-7 (C), and 5 mM W-7 (D) conditions. (E) Ratio of areas occupied by the shell matrix and calcified shell in the C. virginica D-shaped larvae after different W-7 treatments. (F) Fluorescent intensity of calcofluor corresponding to shell organic matrix production in C. virginica D-shaped larvae after different W-7 treatments. All the larvae were treated with W-7 for 24 hours. \u0026nbsp;*P \u0026lt; 0.05. White scale bar: 100 mm.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7180529/v1/cd4d028a2ab9383807d3a721.jpg"},{"id":89393280,"identity":"55338bcb-bbbf-429a-9dbb-d082e122cf43","added_by":"auto","created_at":"2025-08-19 13:23:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":606635,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of different CaN concentrations (20U and 100U) in regulating the transcription of SMPs in \u003cem\u003eC. virginica\u003c/em\u003e mantel epithelium cells under the elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e condition. (A-F) Comparison of relative mRNA expression of \u003cem\u003eCv-CaM\u003c/em\u003e (A), \u003cem\u003eCv-CaN\u003c/em\u003e (B), \u003cem\u003eCv-Nacrein\u003c/em\u003e (C), \u003cem\u003eCv-Pif\u003c/em\u003e (D), \u003cem\u003eCv-Tyr\u003c/em\u003e (E), and \u003cem\u003eCv-Chits\u003c/em\u003e (F) in the elevated CO\u003csub\u003e2 \u003c/sub\u003eexposed \u003cem\u003eC. virginica\u003c/em\u003e mantle cells with and without the presence of additional CaN. Significant difference between two groups (P \u0026lt; 0.05) was demonstrated by different letters over the column. All CO\u003csub\u003e2\u003c/sub\u003e treatments with or without the addition of CaN were applied to cells for 48 hours. *P \u0026lt; 0.05. Error bar: standard error of the mean. (G) Schematic model illustrating the potential impact of OA on the dysregulation of the calcium-CaM signaling pathway and disrupting the organization of SMPs in \u003cem\u003eC. virginica\u003c/em\u003e mantle epithelium cells (Created with \u003ca href=\"https://BioRender.com\"\u003ehttps://BioRender.com\u003c/a\u003e).\u003c/p\u003e","description":"","filename":"16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7180529/v1/2fda7e0c0bb85233baa7d1c8.jpg"},{"id":108576177,"identity":"82646e98-1917-4c97-afd6-94db3df1da2f","added_by":"auto","created_at":"2026-05-06 07:11:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3330689,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7180529/v1/7ef067ea-3520-44e6-8133-576497f239b0.pdf"},{"id":89394171,"identity":"e014e485-6bdc-460a-ad4a-bf7a0b4d8a23","added_by":"auto","created_at":"2025-08-19 13:31:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24787947,"visible":true,"origin":"","legend":"Supplementary Data","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7180529/v1/5859df5ff295951ea5694d23.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Ocean acidification disrupts the biomineralization process in the oyster \u003ci\u003eCrassostrea virginica\u003c/i\u003e via intracellular calcium signaling dysregulation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBiomineralization in marine calcifiers, such as mollusks, corals, and echinoderms, form shells or skeletons through their ability to precipitate calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. As one of the major calcifier groups, marine bivalves have important ecological functions and commercial value worldwide \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Biomineralization is fundamental to marine bivalve shell formation, which serves as a physical barrier against predators, environmental stressors, and desiccation. The shells of bivalves are composed of 95% CaCO\u003csub\u003e3\u003c/sub\u003e and 1\u0026ndash;5% organic matrix \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that the shell formation process is initiated by mantle tissues, where the CaCO\u003csub\u003e3\u003c/sub\u003e is secreted to facilitate shell growth \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The mantle epithelial cells produce components of the shell organic matrix, which regulate the nucleation, regulation, and structural organization of CaCO\u003csub\u003e3\u003c/sub\u003e \u003csup\u003e9,10\u003c/sup\u003e .\u003c/p\u003e\u003cp\u003eOver the past decades, it has been speculated that a cascade of genetic regulation controls the shell formation process in marine bivalves \u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. More than 250 shell formation-related genes are predicted from the genome of \u003cem\u003eMagallana gigas\u003c/em\u003e (previously \u003cem\u003eCrassostrea gigas\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In particular, shell matrix protein (SMP) production and ion transportation pathways are suggested to be key genetic processes in regulating biomineralization of bivalves \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Several signaling pathways, including Wnt \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, TGF-β/BMP \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and a subunit of guanine nucleotide-binding protein (G-protein) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e are also implicated in organizing shell formation process in various marine bivalve species. In addition, some transcription factors, including homeobox genes, NF-κB, zinc finger proteins, and Smad family proteins, are identified as regulators of SMPs in marine bivalve \u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, the precise genetic pathways controlling shell formation remain unclear.\u003c/p\u003e\u003cp\u003eCalcium is not only an essential component of biomineralizing organisms but also serves as a crucial intracellular second messenger regulating numerous signaling pathways. Many studies have reported that calcium signaling-related genes share a high degree of conservation throughout evolution between mollusks and mammals \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(Li et al., 2009; Li et al., 2016)\u003c/span\u003e. Calmodulin (CaM), a primary calcium sensor in calcium signaling pathways, has been identified in various bivalve species \u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and mantle tissue related to the shell formation process shows the highest expression of CaM (Li et al., 2005). CaM and CaM-like proteins are reported to be present in the bivalve shell layer, and have been implicated in aragonite nucleation in conjunction with other shell organic matrix \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. As a calcium/CaM-dependent phosphatase, calcineurin (CaN) is directly activated upon binding of the calcium-CaM complex \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. CaN is also detected mainly localized in the inner epithelial cells of the mantle \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and is reported to play a role in regulating the shell formation process in \u003cem\u003eP. fucata\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This process is homologous to the role of CaN in bone formation and absorption processes in vertebrate species \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAnthropogenic activities significantly increase atmospheric CO\u003csub\u003e2\u003c/sub\u003e levels, with projections indicating a rise from 400 ppm to 1000 ppm by the year 2100 \u003csup\u003e43\u003c/sup\u003e. The ocean absorbs approximately 30% of the rapidly rising atmospheric CO\u003csub\u003e2\u003c/sub\u003e, leading to ocean acidification (OA) \u003csup\u003e\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The dissolution of CO\u003csub\u003e2\u003c/sub\u003e in seawater results in altering pH levels and carbonate system in the ocean \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. There is strong evidence that marine organisms are suffering from increased acidification \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Compared with vertebrate species, OA negatively affects more biological responses in invertebrate species, especially in calcification and biomechanics. The shifts of pH and carbonate system in the ocean ecosystem disrupt biomineralization processes in marine calcifiers by the dissolution of calcareous structures \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and reduction of carbonate ions for CaCO\u003csub\u003e3\u003c/sub\u003e precipitation \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn addition to promoting dissolution of CaCO\u003csub\u003e3\u003c/sub\u003e shell and decreasing availability of carbonate ions, there is strong evidence suggesting that OA poses a threat to the shell formation process in marine bivalves by affecting calcium absorption and deposition in mantle tissue \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The fluctuations of intracellular calcium and transcriptional changes in calcium signaling-related genes in the gill, mantle, or hemocytes of various marine bivalve species are suggested as a compensatory mechanism to maintain calcium deposition for shell formation under the stress of OA \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Several studies have suggested that simulated OA conditions may disrupt cellular signaling pathways associated with immune responses in marine organisms by altering intracellular calcium concentration in hemocytes \u003csup\u003e\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Therefore, there is a plausible hypothesis that OA conditions may disturb the intracellular calcium homeostasis and dysregulate the genetic mechanism of the calcium signaling pathway, ultimately impairing the bivalve shell formation.\u003c/p\u003e\u003cp\u003eDue to the important characteristics of calcium for numerous intracellular processes and as the major component of bivalve shells, the role of intracellular calcium in biomineralization under OA conditions was explored in this study. We examined how OA affected bivalve biomineralization through the calcium signaling pathway by establishing an \u003cem\u003ein vitro\u003c/em\u003e epithelial cell-dominated model using primary mantle cell culture from the eastern oyster, \u003cem\u003eCrassostrea virginica\u003c/em\u003e. An \u003cem\u003ein vivo\u003c/em\u003e analysis of biomineralization-related response to OA conditions across early developmental stages of \u003cem\u003eC. virginica\u003c/em\u003e was also conducted to elucidate the role of calcium signaling in early shell formation. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e results indicate that OA stress dysregulates the calcium signaling pathway by disrupting intracellular calcium levels in the mantle epithelial cells. The abnormal production and activity of CaM and CaN in the calcium signaling pathway subsequently affects the expression of SMPs, which disrupts the organic matrix function in the shell structure. Our findings suggest that OA-induced shell deformation in marine bivalves is associated with the dysregulation of intracellular calcium signaling pathway.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Intracellular calcium influx in \u003cem\u003eC. virginica\u003c/em\u003e mantle cells under elevated CO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eTo investigate the impact of OA on the cellular functions in biomineralization-related organs, the mantle tissues of \u003cem\u003eC. virginica\u003c/em\u003e were dissected for primary cell culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Various cell types, including epithelial-like cells, hemocyte-like cells, and fibroblast-like cells, migrated from the explant within 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The mantle cells from non-adhered explants or the cell dissociation were detached within 24 hours, whereas the cells that migrated from adhered explants remained viable for over two weeks. Hemocyte-like cells were progressively diminished from the adherent cell populations following culture medium replacement after 3\u0026ndash;4 days. Less than 10% of fibroblast-like cells were detected in the migrated cell populations around each collagenase-treated explant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Supplementary Data, Figure S3C). After 14 days, the epithelial-like cells migrated around the explants, forming approximately 70% confluence in the surrounding areas and becoming the dominant cell type (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026amp;D, Supplementary Data, Figure S3C). While most epithelial-like cells degenerated within two weeks, some remained viable for up to a month.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether the elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e condition (simulated OA) can dysregulate calcium signaling pathways in the oyster mantle cells, the intracellular calcium level was monitored in CO\u003csub\u003e2\u003c/sub\u003e-treated mantle cells using calcium indicator Fluo4-AM. Following a short-term CO\u003csub\u003e2\u003c/sub\u003e treatment (1 hour), the average fluorescent intensity of the calcium signal in mantle cells significantly increased by 2.2% compared to their pre-exposure levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026amp;F). Additionally, flow cytometry analysis showed that the average fluorescence intensity of calcium in the mantle cells under long-term CO\u003csub\u003e2\u003c/sub\u003e exposure (24 hours) was 82.17%, significantly higher than the mantle cells in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG\u0026amp;H).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Dysregulation of calcium signaling pathway and SMPs in \u003cem\u003eC. virginica\u003c/em\u003e mantle cells under elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eTwo calcium signaling-related proteins, CaM (\u003cem\u003eCv-CaM\u003c/em\u003e) and CaN (\u003cem\u003eCv-CaN\u003c/em\u003e), were assessed to evaluate the cellular responses of calcium signaling pathway to increased \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e level in oyster mantle cells. Expression patterns of the \u003cem\u003eCv-CaM\u003c/em\u003e were significantly upregulated at the mRNA level by 2.47\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.31-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004) under 1.5% CO\u003csub\u003e2\u003c/sub\u003e condition compared with mantle cells with an ambient CO\u003csub\u003e2\u003c/sub\u003e environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, \u003cem\u003eCv-CaN\u003c/em\u003e, the downstream gene of \u003cem\u003eCv-CaM\u003c/em\u003e, showed a contrasting pattern of mRNA expression. The expression of \u003cem\u003eCv-CaN\u003c/em\u003e was downregulated significantly to 0.75 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.08-fold (\u003cem\u003ep\u003c/em\u003e = 0.0493) in 1.5% CO\u003csub\u003e2\u003c/sub\u003e-treated mantle cells compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Similarly, the protein expression of CaM and CaN quantified by the immunofluorescence (IF) analysis showed a pattern consistent with the transcriptional analysis. The CaM expression at the protein level increased by 112.53% in the \u003cem\u003eC. virginica\u003c/em\u003e mantle cells cultured in 1.5% atmospheric CO\u003csub\u003e2\u003c/sub\u003e exposure compared to the cells in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, p\u0026thinsp;=\u0026thinsp;0.0042). The \u003cem\u003eC. virginica\u003c/em\u003e mantle cells dramatically decreased production of CaN protein by 86.46% under the 1.5% CO\u003csub\u003e2\u003c/sub\u003e condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, p\u0026thinsp;=\u0026thinsp;0.023).\u003c/p\u003e\u003cp\u003eIn the mantle epithelial cells, the transcription of SMPs, which are well-studied for their role in mollusks shell formation, were utilized to assess the responses of shell formation to elevated CO\u003csub\u003e2\u003c/sub\u003e concentration. Despite species-specific variations in SMPs, SMP repertories are reported to share four functional domains, including von Willebrand factor type A domains (VWA), chitin-binding-2 domains (CB-2), carbonic anhydrase domains (CA), and tyrosinase domains in diverse molluscan species \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The presence of these shared domains in different SMPs suggests a conserved biomineralization toolkit across bivalve species. Therefore, we selected four conserved SMPs-encoding genes to represent the shell construction conditions at the molecular level, including Pif97 (\u003cem\u003eCv-Pif97\u003c/em\u003e, VWA and CB-2 domains)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, Tyrosinase (\u003cem\u003eCv-Tyr\u003c/em\u003e, Tyrosinase domain)\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, Nacrein (\u003cem\u003eCv-Nacrein\u003c/em\u003e, CA domain)\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, and Chitin synthase (\u003cem\u003eCv-Chit\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. The mRNA expression of the four SMPs encoding genes were all significantly upregulated under the 1.5% CO\u003csub\u003e2\u003c/sub\u003e exposure compared to the control groups. The increased mRNA expression levels were 8.70 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.51-fold (\u003cem\u003ep\u003c/em\u003e = 0.0001), 9.85 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 2.19-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0007), 10.31 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 3.49-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), and 11.98 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 3.27-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) for \u003cem\u003eCv\u003c/em\u003e-\u003cem\u003eNacrein\u003c/em\u003e, \u003cem\u003eCv\u003c/em\u003e-\u003cem\u003ePif97, Cv\u003c/em\u003e-\u003cem\u003eChits\u003c/em\u003e, and \u003cem\u003eCv-Tyr\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-H).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e disturbed early shell formation and shell organic matrix biosynthesis \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo investigate the effects of elevated \u003cem\u003ep\u003c/em\u003eCO₂ on early shell formation \u003cem\u003ein vivo\u003c/em\u003e, developmental morphology and shell composition of \u003cem\u003eC. virginica\u003c/em\u003e larvae were examined across critical early stages. With increased \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e exposure, the early shell development during the period from trochophore (12 hours old) to D-shaped (36 hours old) \u003cem\u003eC. virginica\u003c/em\u003e larvae (Supplementary Data, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC) exhibited significant morphological changes. The trochophore larvae failed to develop into a D-shape morphology and showed asymmetric shell shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026amp;B). Larval shell deformity patterns, including \u0026ldquo;concave\u0026rdquo; hinge and protruding mantle on the fringe \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e were also detected under elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e concentration (Supplementary Data, Figure S4). In addition, the combination of Calcofluor/Calcein staining was utilized to assess the production of organic matrix and CaCO\u003csub\u003e3\u003c/sub\u003e, respectively. Compared to the distribution of the calcofluor signal (blue, indicator of organic matrix) on the outer shell surface of the larvae in control group, the location of calcofluor staining in OA treated D-shaped larvae dispersedly distributed across the entire shell surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026amp;B). Notably, shell calcification also irregularly displayed a brighter calcein (green, indicator of calcium) signal around hinge areas under simulated OA conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.4 Abnormal gene expression profiles of early shell formation under elevated atmospheric\u003c/b\u003e \u003cb\u003ep\u003c/b\u003e\u003cb\u003eCO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eduring\u003c/b\u003e \u003cb\u003eC. virginica\u003c/b\u003e \u003cb\u003elarval development\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt the four primary developmental stages of \u003cem\u003eC. virginica\u003c/em\u003e larvae (Supplementary Data, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC), including trochophore (T, 12 hours), D-shape (D, 36 hours), umbonal (U, 10 days), and pediveliger (P, 20 days) stages, the transcriptional pattern of \u003cem\u003eCv-CaM\u003c/em\u003e, \u003cem\u003eCv-CaN\u003c/em\u003e, and four SMPs biomarkers encoded genes showed differential responses to the elevated CO\u003csub\u003e2\u003c/sub\u003e environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026amp;D). Although the upregulation of \u003cem\u003eCv-CaM\u003c/em\u003e by elevated pCO\u003csub\u003e2\u003c/sub\u003e condition was observed at the trochophore (3.01 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.691 fold of control, \u003cem\u003ep\u003c/em\u003e = 0.028) and the pediveliger stages (2.40 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.669 fold of control, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048), there was downregulation of the CaM expression pattern at the D-shape stages (0.84 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.09 fold of control, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033) and no significant changes in the umbonal stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). On the other hand, significant downregulation of Cv-\u003cem\u003eCaN\u003c/em\u003e was observed in all the larval development stages under the OA simulation (fold changes ranging from 0.47 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.11 to 0.86 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.13, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), except D-shape larvae, which showed similar expression pattern between control and OA treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). While the mRNA levels of \u003cem\u003eCv-Nacrein\u003c/em\u003e and \u003cem\u003eCv-Pif97\u003c/em\u003e showed no significant changes at the D-shape and the pediveliger stages, they were both significantly upregulated at the trochophore stage (Nacrein: 3.76 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.87 fold of control, \u003cem\u003ep\u003c/em\u003e = 0.016; Pif97: 3.56 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.79 fold of control, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.041) and downregulated at the umbonal stage (Nacrein: 0.36 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.08 fold of control, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008; Pif97: 0.33 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.07 fold of control, \u003cem\u003ep\u003c/em\u003e = 0.017) under the OA simulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026amp;F). Compared with the control group, expression of \u003cem\u003eCv-Tyr\u003c/em\u003e in the CO\u003csub\u003e2\u003c/sub\u003e-treated larvae only displayed significant downregulation at the umbonal stage (0.43 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.09-fold of control, \u003cem\u003ep\u003c/em\u003e = 0.004, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Chitin synthase, another SMP regulating the synthesis of extracellular matrix components, showed significant downregulation at the trochophore (0.73\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.10-fold of control, \u003cem\u003ep\u003c/em\u003e = 0.049) and umbonal stages (0.47\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.10-fold of control, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0007) for the OA treatment but no significant changes at the D-shape and pediveliger stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.5 Elevated\u003c/b\u003e \u003cb\u003ep\u003c/b\u003e\u003cb\u003eCO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003etreatment and pharmacological calcium-CaM signaling disruption led to comparable dysregulation of calcium signaling-related genes and SMPs-encoding genes\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results of elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e treatment above showed that the simulated OA condition not only dysregulated the calcium-signaling pathway but also affected shell development by changing the expression of SMPs. Thus, we assumed the modified expression of SMPs resulted from the dysregulated calcium signaling pathway under increased \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e concentration and ultimately led to shell deformation. To test this hypothesis, we assayed the relationship between the calcium signaling pathway and SMPs expression in the mantle epithelial cells by inhibiting the calcium-CaM binding site using W-7. Similar to the mantle cells under elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e exposure, the cells treated by W-7 also demonstrated significant upregulation of CaM and downregulation of CaN at both mRNA and protein levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B \u0026amp; G-H). With 25 \u0026micro;M W-7 treatment, the mRNAs of \u003cem\u003eCv-CaM\u003c/em\u003e were upregulated by 4.10\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.40-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Compared to the control group, there was 0.86 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.32-fold (\u003cem\u003ep\u003c/em\u003e = 0.045) downregulation of \u003cem\u003eCv-CaN\u003c/em\u003e under the W-7 treatment at the mRNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, the significant upregulations of \u003cem\u003eCv\u003c/em\u003e-\u003cem\u003eNacrein\u003c/em\u003e (13.75 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 5.89-fold of control, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037), \u003cem\u003eCv\u003c/em\u003e-\u003cem\u003ePif97\u003c/em\u003e (12.71 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 5.04 fold of control, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017), \u003cem\u003eCv-Tyr\u003c/em\u003e (43.99 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 28.53-fold of control, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043) and \u003cem\u003eCv\u003c/em\u003e-\u003cem\u003eChits\u003c/em\u003e (5.70 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 2.31 fold of control, \u003cem\u003ep\u003c/em\u003e = 0.046) were also detected in the mantle cells under the W-7 treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F), which is consistent with their mRNA expression pattern under the OA simulation treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, there was 110.51% higher protein production of CaM in the \u003cem\u003eC. virginica\u003c/em\u003e mantle cells under the W-7 treatment than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In contrast to the CaM protein level, the protein level of CaN under the W-7 treatment decreased by 92.98% compared with the cells under the control condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Notably, with the blockage of CaM binding site on calcium by W-7, the phosphatase enzymatic activity of CaN was significantly decreased (0.511\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e0.035-fold of control, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00208, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Likewise, the elevated environmental CO\u003csub\u003e2\u003c/sub\u003e level also reduced the phosphatase enzymatic activity of CaN to 0.534\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.0097-fold of control in the mantle cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00151, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Inhibition of calcium-CaM signaling disrupted the early shell formation of D-shape \u003cem\u003eC. virginica\u003c/em\u003e larvae\u003c/h2\u003e\u003cp\u003eWith the W-7 blockage of calcium-CaM binding, \u003cem\u003eC. virginica\u003c/em\u003e larvae also demonstrated disorganization of shell organic matrix production, similar to those larvae in the elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e experiment. After 24 hours of W-7 treatment, the organization of the organic shell matrix, indicated by the calcofluor signal (blue), exhibited a distinct pattern in the D-shape larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). In the control group, the calcofluor signal was visibly concentrated in the center of the shell field and gradually faded toward the front end of the shell edge (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Under 1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eM W-7 treatment, the calcofluor signal was more uniformly distributed across the whole shell surface and showed higher intensity in the center (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In contrast, larvae exposed to 2 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eM W-7 displayed a reduced calcofluor signal compared to the above treatments. The organic matrix appeared to have irregular patterns, with asymmetric growth of the organic matrix and lower intensity of the calcofluor signal compared to the control group. However, there was no obvious malformation of the calcification process in the oyster shell (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). A dramatic change was detected under 5 \u0026micro;M W-7 treatment, where the shell exhibited substantial calcein signal around the center. Nevertheless, the organic matrix was confined to a thin layer along the margins of the valve, with a faint calcofluor signal observed at the center (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eThe ratio between matrix-occupied and calcified areas in a single valve from each larva was measured during early larval development. Despite there being no significant difference in the average ratio of matrix to calcified areas between the larvae in the control group and 1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eM W-7 treatment groups, the ratio significantly reduced by 57.22% under 2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eM and 45.31% under 5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eM W-7 treatment compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Notably, compared with the calcofluor signal intensity on the shell surface in the control group, the average fluorescent intensity was significantly enhanced by 59.79% under 1 \u0026micro;M W-7 treatment. In contrast, the average intensity decreased substantially by 56.34% and 48.80% in the larvae exposed to 2 \u0026micro;M and 5 \u0026micro;M W-7 treatments, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.7 Rescue of calcium signaling pathway and SMP production in mantle cells under CO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003estress by CaN\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the \u003cem\u003eC.virginica\u003c/em\u003e mantle epithelial cells, both elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e and W-7 experiments displayed decreased protein production and phosphatase activity of CaN due to the dysregulated calcium signaling pathway. Based on this result, we further determined that the inhibition of CaN under the elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e concentration affected the SMPs expression using a CaN addition rescue experiment. Expression of the \u003cem\u003eCv-CaM\u003c/em\u003e and four SMPs in CO\u003csub\u003e2\u003c/sub\u003e-exposed \u003cem\u003eC. virginica\u003c/em\u003e mantle cells in the presence of CaN were consistent with the mantle cells under ambient air conditions. The mRNA expression of \u003cem\u003eCv-CaM\u003c/em\u003e in the CO\u003csub\u003e2\u003c/sub\u003e-treated mantle cells with 20U (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) and 100U (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0025) CaN addition was significantly downregulated to control levels compared to the cells without CaN treatment, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). However, \u003cem\u003eCv-CaN\u003c/em\u003e mRNA expression showed no significant difference between cells treated with elevated CO\u003csub\u003e2\u003c/sub\u003e alone and those exposed to both CO\u003csub\u003e2\u003c/sub\u003e and CaN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Compared to mantle cells exposed to increased CO\u003csub\u003e2\u003c/sub\u003e without CaN treatment, \u003cem\u003eCv-Nacrein\u003c/em\u003e mRNA expression in CO\u003csub\u003e2\u003c/sub\u003e-exposed mantle cells was significantly downregulated with 20U (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00153) and 100U (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00164) CaN addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Similarly, \u003cem\u003eCv-Pif97\u003c/em\u003e mRNA expression in CO\u003csub\u003e2\u003c/sub\u003e-exposed mantle cells treated with 20U and 100U CaN returned to control levels, significantly lower than in the mantle cells treated with elevated CO\u003csub\u003e2\u003c/sub\u003e alone (20U CaN: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00579; 100U CaN: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01577) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Under elevated CO\u003csub\u003e2\u003c/sub\u003e conditions, \u003cem\u003eCv-Tyr\u003c/em\u003e mRNA expression in mantle cells with 20U (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0495) and 100U (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0414) CaN addition was significantly lower than in mantle cells without CaN treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Furthermore, the \u003cem\u003eCv-Chits\u003c/em\u003e also displayed a significantly reduced pattern at the mRNA level in CO\u003csub\u003e2\u003c/sub\u003e-stimulated mantle cells treated with CaN compared to those exposed to CO\u003csub\u003e2\u003c/sub\u003e alone (20U CaN: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0441, 100U CaN: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0485) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). There was also no significant difference between the CO\u003csub\u003e2\u003c/sub\u003e-treated cells in the presence of CaN and the mantle cells in the control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eDespite calcium being the most abundant mineral element in the marine bivalve shell structure, its role extends beyond providing substrate resources for biomineralization, as it also regulates various physiological processes as a secondary messenger intracellularly \u003csup\u003e\u003cspan additionalcitationids=\"CR75\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Under the stress of OA, the dysregulation patterns of intracellular calcium concentration are observed in oyster hemocytes \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, suggesting that marine bivalves experience fluctuations in both intracellular and extracellular calcium levels in response to changes in carbonate chemistry in the ocean induced by the OA conditions. Recent studies highlight the crucial role of the calcium-CaM signaling pathway in vertebrate bone formation by regulating osteoblast differentiation and proliferation \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Similarly, several important calcium signaling-related genes, including CaM and CaN, are reported to be involved in shell formation in marine bivalves \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In this study, we identified an intracellular calcium influx pattern in oyster mantle epithelial cells, triggering dysregulation of the calcium-CaM signaling pathway under elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e conditions. As a result of the calcium-CaM signaling pathway disruption, dramatic morphological changes in shell shape and the shell organic matrix were observed, which are consistent with previous reports of OA effects on shell formation \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo link calcium signaling with the genetic regulation of \u003cem\u003eC. virginica\u003c/em\u003e shell formation in response to OA, we established a more efficient \u003cem\u003ein vitro\u003c/em\u003e model using oyster mantle cells. Compared to previous methods for isolating mantle cells from oysters using cell dissociation and earlier mantle tissue explant techniques \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, our method significantly reduced the populations of other cell types, such as fibroblast cells and hemocytes (Supplementary Data, Figure S3). The high-quality, long-term \u003cem\u003ein vitro\u003c/em\u003e maintenance of oyster mantle epithelial cells in this study provided a more robust system for monitoring prolonged, low-level environmental changes. Under elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e conditions, the pH of the oyster cell culture medium dropped from 7.8 to 7.4 within 48 hours of treatment (Supplementary data, Figure S5). A corresponding increase in intracellular calcium concentration in the mantle epithelial cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-H) is consistent with an increased trend of extracellular calcium in the hemolymph of adult oysters exposed to elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e for 60 days \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. These results suggest that the elevated CO\u003csub\u003e2\u003c/sub\u003e level may induce calcium influx in the oyster mantle epithelial cells as a compensatory response to maintain calcium homeostasis in fluctuating ambient conditions \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Along with CaM, several calcium-binding proteins have been shown to be upregulated in the oyster mantle under elevated CO\u003csub\u003e2\u003c/sub\u003e conditions \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Several studies indicate that the OA conditions disrupt the carbonate system and calcium level in marine bivalves \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Consequently, this study suggested that the dynamic of calcium concentrations in the intracellular and extracellular environments of oyster mantle epithelial cells triggered the differential expression of calcium-binding proteins and alteration of the calcium signaling pathway.\u003c/p\u003e\u003cp\u003eIn the calcium signaling pathways, CaM plays an essential role in various cellular processes in marine bivalves, such as the processes of environmental stress acclimation \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e, immune response \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e, and neuroendocrine regulation \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. However, most studies on the biomineralization-related function of CaM focus on its role as an SMP in organizing the structure of shell layers and the transformation of CaCO\u003csub\u003e3\u003c/sub\u003e crystals during marine bivalve shell development. Our study demonstrated the upregulation of CaM at both mRNA and protein levels in isolated mantle epithelial cells under increased \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e exposure (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). These results are consistent with previous findings that reported upregulation of several CaM isoforms in oyster mantle tissue following CO\u003csub\u003e2\u003c/sub\u003e exposure in adult oysters \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e and at the cellular level \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, in our \u003cem\u003ein vivo\u003c/em\u003e oyster larvae models, only the trochophore and pediveliger stages exhibited upregulation of CaM in response to OA conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Notably, these two stages are involved in initial shell formation and the preparation of metamorphosis for settlement \u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. Further, several CaM-like proteins have shown downregulation patterns in mantle tissue due to a potential calcium compensation strategy among the gill and mantle induced by CO\u003csub\u003e2\u003c/sub\u003e exposure \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Given the diverse cellular functions of CaM at the organism level, the \u003cem\u003ein vitro\u003c/em\u003e mantle epithelial cell model demonstrated a higher efficiency and specificity in revealing the responses of the mantle epithelium cellular processes to the stress of simulated acidification conditions.\u003c/p\u003e\u003cp\u003eAs an essential downstream effector of the calcium-CaM signaling pathway, CaN is revealed to play an important role in marine bivalve shell formation by regulating the BMP signaling pathway \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In a previous study, two CaN subunits were isolated from the pearl oyster \u003cem\u003eP. fucata\u003c/em\u003e: subunit A, which functions as the phosphatase catalysis unit, and subunit B, which serves as the calcium-binding site \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To explore the role of phosphatase activity in CaN during shell formation, we used the CaN subunit A homolog from \u003cem\u003eC. virginica\u003c/em\u003e to assess the responses of phosphatase-related genes to OA treatment (Supplementary Data, Table S3). CaN expression was downregulated at the mRNA and protein levels in oyster mantle cells with increased CO\u003csub\u003e2\u003c/sub\u003e exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026amp;D). Similarly, CaN downregulation was observed across oyster larval development stages in acidified conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), which is consistent with a previous study documenting the decreased transcription level of CaN in \u003cem\u003eM. gigas\u003c/em\u003e D-shaped larvae exposed to decreased pH stress \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. These shared patterns among the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models suggest that the dysregulation of the calcium-CaM signaling pathway is induced by elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eIn addition to the dysregulation of calcium-CaM, \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e exposure also affected the expression of four conserved SMPs associated with bivalve shell formation in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e oyster models (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The upregulation of \u003cem\u003eCv-Nacrein\u003c/em\u003e in oyster mantle epithelial cells suggests a potential reduction of oyster calcification rate \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. While \u003cem\u003ein vitro\u003c/em\u003e analysis also revealed an upregulation pattern of \u003cem\u003eCv-Nacrein\u003c/em\u003e during initial shell formation at the trochophore stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), these findings contrasted with previous studies that significant nacrein upregulation was only detected after the pediveliger stage \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e,\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. The unexpected early upregulation was potentially related to a disruption of crystal formation during shell construction \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. Since Pif97 regulates shell formation by inhibiting calcite crystal growth and amorphous CaCO\u003csub\u003e3\u003c/sub\u003e stabilization \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e, the increased expression level of this gene in the mantel epithelial cells suggested an OA-induced disruption of shell framework construction. The consistent upregulation of \u003cem\u003eCv-Pif97\u003c/em\u003e during trochophore development under \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e exposure indicated a negative impact of OA on the transformation and crystallization of CaCO\u003csub\u003e3\u003c/sub\u003e for early shell formation \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e,\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCompared to the upregulation of chitin synthase in the \u003cem\u003eC. virginica\u003c/em\u003e mantle epithelial cells under OA conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), the \u003cem\u003eCv-Chits\u003c/em\u003e at both trochophore and umbonal stages exposed to elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e exhibited the opposite expression pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Previous studies find chitin decomposition during early shell formation in acidified-treated trochophore oysters \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e. Given the evolutionary diversity of chitin synthase isoforms among mollusks, the contrasting \u003cem\u003eCv-Chits\u003c/em\u003e expression between the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models under OA suggests alternative biological functions of chitin synthetase beyond the biomineralization process in adult \u003cem\u003eC. virginica\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e. Similarly, the tyrosinase (\u003cem\u003eCv-Tyr\u003c/em\u003e) also showed contrasting expression patterns between \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models. While there was no significant impact of elevated CO\u003csub\u003e2\u003c/sub\u003e stress on the expression of \u003cem\u003eCv-Tyr\u003c/em\u003e at the trochophore stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), the upregulation of \u003cem\u003eCv-Tyr\u003c/em\u003e in the mantle cells under the OA stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) is in accordance with the increased mRNA expression of tyrosinase at the trochophore stage in \u003cem\u003eCrassostrea angulata\u003c/em\u003e responding to OA \u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e. The upregulation of \u003cem\u003eCv-Tyr\u003c/em\u003e revealed a potential disruption of the shell matrix production of oysters \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. The reversed expression pattern of \u003cem\u003eCv-Tyr\u003c/em\u003e in larval \u003cem\u003eC. virginica\u003c/em\u003e at the umbonal stage indicated a potential deformation of periostracum, the initial shell layer during the oyster\u0026rsquo;s early development \u003csup\u003e\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e,\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e, which suggests potentially different shell formation mechanisms compared to adult oysters. Collectively, these findings indicate that elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e disrupts multiple aspects of shell biomineralization. The variable transcriptional level of the SMPs during \u003cem\u003eC. virginica\u003c/em\u003e larval development also implied potentially different acclimation strategies for the shell formation process across larval stages due to the different forms of mineralization related to the developmental phases of oyster larvae \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e,\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study also illustrated a potential connection between the dysregulated calcium-CaM signaling pathway and the abnormal expression of biomineralization-related SMPs in oysters under OA conditions. By blocking the calcium-binding sites on CaM, the CaM antagonist W-7 induced mantel epithelial cells to produce CaM while suppressing CaN protein production. The W-7 exposure functioned similarly to OA stress (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026amp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In addition, decreased phosphatase activity of CaN in the \u003cem\u003eC. virginica\u003c/em\u003e mantle epithelial cells was also detected under both the elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e and the W-7 treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results suggested that dysregulation of the calcium-CaM signaling pathway inhibited CaN activity. Although no direct evidence linked CaM to the expression of CaN, a possible explanation involves the upregulation of calcium/CaM dependent protein kinase II (CaMKII), which was detected in both CO\u003csub\u003e2\u003c/sub\u003e-exposed and W-7 treated \u003cem\u003eC. virginica\u003c/em\u003e mantle cells (Supplementary Data, Figure S6). CaMKII activation is found to inhibit the production of the Nuclear Factor of Activated T-cells (NFAT), which is identified as a transcription factor of CaN expression \u003csup\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. Interestingly, the activity of NFAT is also triggered by the dephosphorylation through CaN \u003csup\u003e\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAs the hypothesized downstream products of the calcium signaling pathway, the four SMPs in mantle epithelial cells were also regulated consistently between the treatments of the W-7 and increased \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e. The anomalous changes in the organic matrix were attributed to the abnormal production of SMPs, which organize the further calcification step by transforming and organizing CaCO\u003csub\u003e3\u003c/sub\u003e deposition \u003csup\u003e\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. The presence of the chitin-binding domains in the Pif97 \u003csup\u003e13\u003c/sup\u003e and tyrosinase \u003csup\u003e\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e and a calcium-binding domain in nacrein \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e further suggests the role for these SMPs in oyster shell construction. Collectively, these results highlight the negative impact of the dysregulated calcium-CaM pathway on the production of SMPs and the organization of the organic matrix framework on the oyster shell surface.\u003c/p\u003e\u003cp\u003eThe CaN rescue experiments demonstrated that elevated CaN levels in the oyster mantle cells resulted in upregulated SMP expression in the CO\u003csub\u003e2\u003c/sub\u003e-treated cells returning to the control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-F). The reduced CaN activity under OA conditions was linked in this study to the transcription of SMP encoding genes, including \u003cem\u003eCv-Nacrein\u003c/em\u003e, \u003cem\u003eCv-Pif97\u003c/em\u003e, \u003cem\u003eCv-Tyr\u003c/em\u003e, and \u003cem\u003eCv-Chits\u003c/em\u003e in \u003cem\u003eC. virginica\u003c/em\u003e. As mentioned above, the decreased CaN production was possibly caused by the suppression of NFAT activity \u003csup\u003e\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e,\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u003c/sup\u003e, which interferes with several biomineralization-related signaling pathways, such as the BMP/Smads \u003csup\u003e\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, Wnt \u003csup\u003e\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e\u003c/sup\u003e, and NF-κB signaling pathways \u003csup\u003e\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e. For example, previous studies show that Rel/ NF-κB mediates the transcription of nacrein in \u003cem\u003eP. fucata\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The transcription of Pif97 is demonstrated to be related to the BMP/Smads signaling pathway \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e\u003c/sup\u003e. The inhibition of CaN is found to decrease the biosynthesis of dopamine \u003csup\u003e\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e\u003c/sup\u003e and subsequently affect the synthesis of tyrosinase and chitin \u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e. Surprisingly, the transcription levels of \u003cem\u003eCv-CaN\u003c/em\u003e in the CO\u003csub\u003e2\u003c/sub\u003e-treated mantle cells, following the addition of CaN, displayed a similar expression pattern to mantle cells without CaN treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). One possible explanation for the reduced CaM expression in the CO\u003csub\u003e2\u003c/sub\u003e -exposed mantle cells treated with CaN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) is that the external introduction of CaN was activated by binding to calcium-bound CaM. This binding likely decreased the availability of elevated free calcium, which in turn led to lower CaM gene expression.\u003c/p\u003e\u003cp\u003eBased on the results of this study, we propose a model of the calcium-CaM signaling pathway regulating SMP production to explain the impact of future OA conditions on the biomineralization process in \u003cem\u003eC. virginica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Under OA stress, the dissolution of shell in marine bivalves leads to increased extracellular calcium levels and carbonate ions in the extrapallial fluid to support calcification \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. To maintain the calcium homeostasis between the intra- and extracellular environment, the mantle epithelial cells of marine bivalves release large amounts of calcium intracellularly and enhance the calcium transportation from the ambient environment through the cell membrane (Wang et al., 2017; Chandra Rajan et al., 2021; Li et al., 2023). To counteract shell dissolution under long-term OA stress, oysters are shown to increase extracellular calcium (serum level) and upregulate CaM production, which plays a critical role in calcium deposition and transportation \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. However, at the same time, this persistent upregulation of CaM may dysregulate the calcium-CaM signaling pathway by inhibiting the activities of CaN-NFAT and triggering the CaMKII-CREB pathway due to elevated concentrations of the calcium-bound CaM complex. Similar effects are suggested in human cardiac myocytes \u003csup\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. Finally, the decreased CaN activity results in the abnormal expression pattern of several SMPs by interrupting relevant signaling pathways, including the BMP signaling pathway (Pif97, Zhao et al., 2014; Shi et al., 2020), NF-κB signaling pathway (nacrein, Sun et al., 2015), and TGF-\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e signaling pathway (chitin synthase and tyrosinase, Liu et al., 2020).\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, our results identified increased intracellular calcium and the dysregulation of calcium-CaM in the \u003cem\u003eC. virginica\u003c/em\u003e mantle epithelial cells under a simulated OA environment as a strategy to maintain calcium homeostasis. The persistent calcium regulation, driven by the upregulation of CaM, reduced CaN protein production and phosphatase activity in the oyster mantle epithelial cells. The reduced CaN activity induced by elevated CO\u003csub\u003e2\u003c/sub\u003e exposure disrupted the production of various SMPs involved in the shell formation process and, consequently, disorganized the shell organic matrix framework and the shell construction process. This study clarifies the role of the calcium signaling pathway in marine bivalve shell formation. These findings, especially the essential role of CaN in regulating oyster SMP production and shell organic matrix organization, serve as valuable data for understanding the cellular response to OA conditions in marine bivalves and for developing potential strategies for mitigation.\u003c/p\u003e"},{"header":"5. Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e5.1 Oyster mantle cell culture\u003c/h2\u003e\u003cp\u003eAdult \u003cem\u003eC. virginica\u003c/em\u003e oysters of south Texas origin were obtained from the Texas A\u0026amp;M AgriLife Research Mariculture Facility (Corpus Christi, TX) and acclimated for two weeks in a recirculating system with 1 \u0026micro;m-filtered seawater from Corpus Christi Bay, Texas (~\u0026thinsp;30ppt salinity) at 22\u0026thinsp;~\u0026thinsp;24 \u003csup\u003e◦\u003c/sup\u003eC. Before primary cell culture, oysters were incubated overnight in sterilized calcium and magnesium free artificial seawater solution (CMFSS) (Supplementary Data, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, supplemented with 100units/ml of penicillin and 100\u0026micro;g/ml of streptomycin (CMFSS-Pen/Strep, pH\u0026thinsp;=\u0026thinsp;7.7).\u003c/p\u003e\u003cp\u003ePre-treated oysters were dissected, and connective tissues along the mantle pallial were collected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Mantle tissues were rinsed with CMFSS-Pen/Strep, then filtered with a 100 \u0026micro;m cell strainer. The rinsed mantle pieces were digested with 0.2% collagenase Type I (Thermo Fisher Scientific) in oyster cell culture basal medium (Supplementary Data, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) to separate fibroblast cells from mantle explants. After 3 hours of digestion with collagenase I at 37\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e, the tissues were collected and minced into 1\u0026ndash;2 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e explants using a sterilized scalpel. Four to five explants were placed in a 25 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e tissue culture flask (Thermo Fisher Scientific) for 45 minutes to adhere. The explants were then covered with 1 mL oyster cell culture medium supplemented with 10% Fetal Bovine Serum (FBS, Avantor, US), 100 units/ml of penicillin, and 100 \u0026micro;g/mL of streptomycin (oyster cell culture working medium). Primary cultures were incubated at 28\u0026deg;C in a humidified incubator without CO\u003csub\u003e2\u003c/sub\u003e for 48 hours to allow cell migration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e5.2 Stimulation of oyster mantle cells with increased environmental CO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003e\u003cem\u003eC. virginica\u003c/em\u003e mantle cells were observed under a microscope on the 2nd day post-explantation (dpe) to confirm attachment. On the 3rd dpe, the medium was replaced with 1.5 ml fresh oyster cell culture medium\u0026thinsp;+\u0026thinsp;+\u0026thinsp;before the elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e treatment. Cells from explants were exposed to ambient air (Control) or 1.5% CO\u003csub\u003e2\u003c/sub\u003e on the 5th dpe. After 48-hour exposure, mantle cells were harvested for RNA isolation using TRIzol Reagent (Molecular Research Center, Cincinnati) or fixed in 4% paraformaldehyde (PFA) (Thermo Fisher Scientific) for immunofluorescent staining. The explants were removed by filtering through 40 \u0026micro;m cell strainers before RNA isolation in TRIzol. Mantle cells from eight \u003cem\u003eC. virginica\u003c/em\u003e oysters were used for the control and the CO\u003csub\u003e2\u003c/sub\u003e exposure groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e5.3 Intracellular calcium level measurement in oyster mantle cells under elevated pCO\u003csub\u003e2\u003c/sub\u003e treatment\u003c/h2\u003e\u003cp\u003eShort-term (1 hour) and long-term (24 hours) CO\u003csub\u003e2\u003c/sub\u003e exposure experiments were conducted to evaluate intracellular calcium changes under elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e in \u003cem\u003eC. virginica\u003c/em\u003e mantle cells. Intracellular calcium levels were measured using Fluo-4 AM (Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s protocol. Briefly, a 2:1 ratio of 1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eM Fluo-4 AM and 1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eM Polyethylenimine (PEI) mixture was added to the oyster cell culture working medium and incubated for 30 minutes before staining.\u003c/p\u003e\u003cp\u003eFor short-term exposure, the calcium dye was incubated with isolated mantle cells for 1 hour at 28\u0026deg;C on the 5th dpe. Then, the Fluo-4 AM stained mantle cells were treated with 1.5% CO\u003csub\u003e2\u003c/sub\u003e for 1 hour. The fluorescent intensity in the stained cells was quantified before and after the CO\u003csub\u003e2\u003c/sub\u003e exposure under a fluorescent microscope (Olympus CKX53). At least five 10x magnified images derived from one oyster were captured for calcium measurement. The fluorescent signal of intracellular calcium was measured by calculating the mean grey value of the cells in each image via ImageJ \u003csup\u003e\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor long-term CO\u003csub\u003e2\u003c/sub\u003e exposure, untreated and 24-hour 1.5% CO\u003csub\u003e2\u003c/sub\u003e-exposed mantle cells were incubated with Fluo-4 AM in oyster cell culture medium for 1 hour following the staining protocol above. The oyster mantle cells were harvested with 0.05% trypsin (Corning Inc.), and explants were removed using 40 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003em cell strainers. Trypsinized mantle cells were rinsed and resuspended with Dulbecco's phosphate-buffered saline (DPBS, Corning Inc.). The calcium fluorescence in each cell was subsequently quantified by flow cytometry (BD Accuri\u0026trade; C6 Plus) with excitation wavelength at 495 nm and emission wavelength at 518 nm, analyzing 10000 events. Mantle cells isolated from four \u003cem\u003eC. virginica\u003c/em\u003e oysters were used in short- and long-term CO\u003csub\u003e2\u003c/sub\u003e exposure experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e5.4 Assay of oyster shell formation process under W-7 treatment in mantle cells\u003c/h2\u003e\u003cp\u003eN-(6-aminohexyl)-5-chloro-naphthalene sulfonilamide hydrochloride (W-7), a specific CaM antagonist inhibiting calcium-CaM binding site on its target proteins CaN \u003csup\u003e\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u003c/sup\u003e, was used to investigate the role calcium-CaM signaling pathway in \u003cem\u003eC. virginica\u003c/em\u003e mantle cells. The mantle cells were treated with 25 \u0026micro;M W-7 (EMD Millipore Corp, IC₅₀ = 28 \u0026micro;M for calcium-CaM-dependent phosphodiesterase inhibition) on the 5th dpe for 24 hours. A 100 mM stock solution of W-7 was prepared by dissolving the W-7 powder in dimethyl sulfoxide (DMSO) and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. The working concentration of 25 \u0026micro;M W-7 was prepared by diluting the stock solution in oyster cell culture working medium. Treated mantle cells were harvested for RNA isolation or immunofluorescent staining, as described in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e. The mantle cells from eight \u003cem\u003eC. virginica\u003c/em\u003e oysters were used in the W-7 treatment group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e5.5 Immunofluorescence analysis of CaM and CaN in oyster mantle cells\u003c/h2\u003e\u003cp\u003eImmunofluorescence (IF) was used to visualize and quantify the expression of CaM and CaN at the protein level of \u003cem\u003eC. virginica\u003c/em\u003e mantle cells in the control and treatment groups. After the CO\u003csub\u003e2\u003c/sub\u003e exposure or W-7 treatment, the mantle cells were rinsed with phosphate-buffered saline (PBS) and fixed in 4% PFA for 1 hour. The fixed samples were then blocked with 10% lamb serum in 1% Triton-PBS (PBST) for 2 hours at room temperature. Primary antibodies of CaM and CaN (purchased from the Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA) diluted in PBST (1:100) were applied to cover the samples in each culture flask, respectively and incubated at 4\u0026deg;C overnight. The primary antibodies used in this study contained mouse-anti-fungus CaM and mouse-anti-human CaN. After removing the primary antibodies and rinsing with PBS, the secondary antibody (goat anti-mouse) conjugated with Alexa flour TM 594 (red) dye (Thermo Fisher Scientific) was diluted in PBST (1: 400) and incubated with the samples for 2 hours in a dark area at room temperature. A blue nuclear dye, 4\u0026prime;,6-diamidino-2-phenylindole (DAPI, Biotium), was used to fluorescently stain the nuclear DNA to localize the individual cells with 1:5000 dilution in PBST. The samples were monitored under the Olympus BX53 fluorescent microscope after IF staining. Three pictures were captured for each culture flask under 20\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e magnification. The exposure time of the image was adjusted to 50 ms under the UV channel and 250 ms under the Texas Red channel. The same exposure time was used on all images of each antibody. Pictures were also taken from secondary antibodies-stained samples without the primary antibodies as a negative control to avoid the disturbance of autofluorescence.\u003c/p\u003e\u003cp\u003eThe total fluorescent signal of CaM and CaN in each cell was measured using ImageJ \u003csup\u003e\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e. The protein expression level was calculated as the corrected total cell fluorescence (CTCF), which was calculated as\u003c/p\u003e\u003cp\u003e\u003cem\u003eIntegrated Density \u0026ndash; (Area of selected cell\u003c/em\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e \u003cem\u003eMean fluorescence of background readings).\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e5.6 CaN phosphatase activity assay in \u003cem\u003eC. virginica\u003c/em\u003e mantle cells\u003c/h2\u003e\u003cp\u003ePost-treatment \u003cem\u003eC. virginica\u003c/em\u003e mantle cells from sections \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e and 2.4 were harvested in 1 mL of 0.05% trypsin (Corning Inc.), centrifuged to form a pellet, and lysed in 50 \u0026micro;L of calcineurin assay buffer (Enzo) for protein isolation. Calcineurin phosphatase activity was measured using the calcineurin phosphatase assay system (BML-AK904, Enzo) with 3 \u0026micro;g of protein from mantle cells, following the manufacturer's instructions. The activity was quantified spectrophotometrically by measuring the release of free phosphate from the calcineurin-specific RII phosphopeptide substrate using the BIOTEK Cytation 5 image reader (Agilent).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e5.7 Larval oysters production and simulated acidification treatments\u003c/h2\u003e\u003cp\u003eSouth Texas \u003cem\u003eC. virginica\u003c/em\u003e oyster larvae were collected from the Texas A\u0026amp;M AgriLife Mariculture Center, following the center\u0026rsquo;s spawning and culture protocols. Four early life stages of oysters, including trochophore, D-shaped, umbonal, and pediveliger stages, were collected respectively in 12 hours, 48 hours, 10 days, and 20 days post-fertilization. A light microscope was used to ensure that \u0026gt;\u0026thinsp;70% of the larvae had reached the particular development stages (Supplementary Data, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOyster larvae were exposed to two different \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e conditions, including current \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e (425 ppm) and predicted \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e (1000 ppm) levels based on IPCC \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Each experimental trial involved larvae placed in 1-liter plastic beakers with 700mL of filtered seawater. A simulated OA environment was set up in a glovebox filled with CO\u003csub\u003e2\u003c/sub\u003e gas to mimic the elevated CO\u003csub\u003e2\u003c/sub\u003e in the atmosphere (Supplementary Data, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A programmable CO\u003csub\u003e2\u003c/sub\u003e controller (ICC-500T, INKBIRD) was utilized to maintain a consistent atmospheric CO\u003csub\u003e2\u003c/sub\u003e concentration of 1000 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 50 ppm. Four replicates of larvae were treated with ambient air and elevated CO\u003csub\u003e2\u003c/sub\u003e conditions, respectively. The larval samples were allocated to different groups for 24 hours of treatment during the trochophore stage or 48 hours treatment at later larval stages, to ensure adequate CO\u003csub\u003e2\u003c/sub\u003e dissolution. The number of larvae in each replicate was optimized based on the guidelines from the Mariculture Center (Supplementary Data, Table S2). After a certain period of CO\u003csub\u003e2\u003c/sub\u003e exposure, the larvae from each treatment replicate were harvested in 7 ml TRIzol Reagent for RNA isolation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e5.8 Evaluation of shell organic matrix synthesis by larval oysters under CO\u003csub\u003e2\u003c/sub\u003e exposure\u003c/h2\u003e\u003cp\u003eShell formation in 12-hour-old trochophores under the CO\u003csub\u003e2\u003c/sub\u003e exposure was observed after 24-hour treatment. Early D-shape larvae were evaluated for the synthesis of shell organic matrix materials and calcification of the shell using fluorescent dyes calcein and calcofluor, respectively \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Calcein (MP Biomedicals), a calcium-dependent fluorescent signal (UV channel, Exc: 408 nm/Em: 450\u0026ndash;490 nm), was utilized for CaCO\u003csub\u003e3\u003c/sub\u003e staining of the calcified shell (final concentration 10 mM in 0.01% DMSO). Calcofluor White Stain (Sigma-Aldrich), a chitin-staining fluorescent signal (FITC channel, Exc: 488 nm/Em: 520\u0026ndash;560 nm), was used to visualize the shell organic matrix (final concentration 0.02 mM in 0.01% DMSO). Before staining, overnight fixed larvae were washed with DPBS to remove the 4% PFA. Calcein was applied to the rinsed larvae for 2 hours. After the DPBS rinsing to remove the excess Calcein dye, the larval samples were stained with the Calcofluor White Stain. After 30 minutes of Calcofluor staining, the larvae were rinsed again with DPBS to remove Calcofluor and immediately imaged with a fluorescent microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e5.9 CaM-inhibition assay for larval oyster shell development\u003c/h2\u003e\u003cp\u003eW-7 was used to assess the function of the calcium-CaM signaling pathway in the biomineralization process during the early development of \u003cem\u003eC. virginica\u003c/em\u003e larvae. Approximately 100 early D-shape larvae (24 hours old) were separately treated with 2 mL of different concentrations of W-7 (1 \u0026micro;M, 2 \u0026micro;M, and 5 \u0026micro;M) diluted in filtered seawater for 24 hours in the dark at room temperature. After treatment, 10 larvae per group were fixed in 4% PFA at 4\u0026deg;C overnight for shell biogenesis analysis. The early shell development was assessed using Calcein and Calcofluor White Stain, as described in section 2.8. The areas occupied by calcein and calcofluor on a single valve of each larva were manually outlined in ImageJ to determine the ratio of calcein to calcofluor as an indicator of shell biogenesis \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. In addition, the fluorescence intensity of calcofluor in larval samples was quantified to estimate the production of shell matrix materials.\u003c/p\u003e\u003cp\u003e\u003cb\u003e5.10 Recovery experiment of CaN on regulating SMPs expression in\u003c/b\u003e \u003cb\u003eC. virginica\u003c/b\u003e \u003cb\u003emantle cells under elevated CO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. virginica\u003c/em\u003e mantle cells treated with CaN during elevated CO\u003csub\u003e2\u003c/sub\u003e exposure were used to investigate the involvement of CaN in regulating the expression of SMPs under simulated OA conditions. A 5000U human CaN stock solution (BML-SE163, Enzo), where 1U of calcineurin catalyzes the dephosphorylation of 1 mM substrate per minute, was mixed with 10 \u0026micro;M PEI (1:1 ratio) for 30 minutes incubation to facilitate cell delivery. The mixture was then diluted with oyster cell culture working medium to prepare 20U and 100U CaN-supplemented cell culture medium. On the 5th dpe, the mantle cells were exposed to 1.5% CO\u003csub\u003e2\u003c/sub\u003e exposure in CaN- supplemented media for 48 hours. Treated cells were harvested for RNA isolation, as described in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e. The mantle cells from four \u003cem\u003eC. virginica\u003c/em\u003e oysters were used in both the 20U and 100U CaN treatment groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e5.11 Quantification of gene expression in \u003cem\u003eC. virginica\u003c/em\u003e mantle cells and \u003cem\u003eC. virginica\u003c/em\u003e larvae\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from \u003cem\u003eC. virginica\u003c/em\u003e mantle cells across different treatment groups using a standard Trizol-chloroform protocol \u003csup\u003e\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e\u003c/sup\u003e, followed by DNA removal with the Ambion TURBO DNA-free Kit (Thermo Fisher Scientific). SuperScript IV Reverse Transcriptase (Invitrogen) was used to synthesize cDNA. The cDNA samples were used for SYBR Green qPCR analysis on a QuantStudio 3 Real-Time PCR System (Applied Biosystems). The qPCR program was run at 50\u0026deg;C for 2 min, 94\u0026deg;C for 2 min, followed by 40 cycles at 94\u0026deg;C for 30 s, 55\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s. The 2\u003csup\u003e\u0026minus; ΔΔCt\u003c/sup\u003e method was used to analyze the gene expression data.\u003c/p\u003e\u003cp\u003eThe mRNA expression levels of CaM, CaN, and four SMP-encoding genes, were measured under different treatment groups. Candidate genes previously characterized from molluscan species, including \u003cem\u003eP. fucata\u003c/em\u003e, \u003cem\u003eM. gigas\u003c/em\u003e, and \u003cem\u003eAtrina rigida\u003c/em\u003e were used to perform BLASTp analysis against the whole genome derived from \u003cem\u003eC. virginica\u003c/em\u003e to identify the homologous mRNA sequences of CaM (\u003cem\u003eCv\u003c/em\u003e-\u003cem\u003eCaM\u003c/em\u003e), CaN (\u003cem\u003eCv\u003c/em\u003e-\u003cem\u003eCaN\u003c/em\u003e), Pif97 (\u003cem\u003eCv\u003c/em\u003e-\u003cem\u003ePif97\u003c/em\u003e), Nacrein (\u003cem\u003eCv\u003c/em\u003e-\u003cem\u003eNacrein\u003c/em\u003e), Tyrosinase (\u003cem\u003eCv-Tyr\u003c/em\u003e), and Chitin synthase (\u003cem\u003eCv\u003c/em\u003e-\u003cem\u003eChits\u003c/em\u003e) (Supplementary Data, Table S3). The targeted genes for qPCR analysis were identified based on their amino acid similarity from the BLASTp results. The specificities of the primers designed for the qPCR analysis by Integrated DNA Technologies (IDT) were confirmed by running the amplicons on a 7% acrylamide TBE gel (Supplementary Data, Figure S2). All the Ct values of the biomarkers were normalized by those of the housekeeping gene \u003cem\u003eElongation factor 1α\u003c/em\u003e gene (\u003cem\u003eCv-ef1α\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe Trizol-stored larval samples from sections 2.7 and 2.9 were homogenized using a microtube homogenizer (Bertin technologies). The homogenized samples were utilized for qPCR analysis to evaluate the relative expression levels of the biomineralization-related biomarkers (Table S3) following the procedures detailed above.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e5.12 Statistical analysis\u003c/h2\u003e\u003cp\u003eComparisons of intracellular calcium fluorescent signal, gene expressions of calcium signaling pathway related genes and SMPs at mRNA level, IF quantification of \u003cem\u003eCv\u003c/em\u003e-CaM and \u003cem\u003eCv\u003c/em\u003e-CaN, organic matrix/shell area ratio, Calcofluor fluorescent intensity, and CaN phosphatase activity were all conducted between the control and treatment groups using one-way ANOVA in R version 3.6.2 \u003csup\u003e112,113\u003c/sup\u003e. All quantitative data were expressed as mean \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e standard error (SE). In all the statistical evaluations, P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Dr. Michael Wetz\u0026rsquo;s group at the TAMU-Corpus Christi, Harte Research Institute for assisting us with the flow cytometry work. This study was supported by the National Science Foundation CAREER Award (2046049).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRies, J.B., Cohen, A.L., McCorkle, D.C.: Marine calcifiers exhibit mixed responses to CO2-induced ocean acidification. 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(2013)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biomineralization, Calcium Signaling, Shell Matrix Proteins, Ocean Acidification, Primary Mantel Cell Culture, Oyster","lastPublishedDoi":"10.21203/rs.3.rs-7180529/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7180529/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnthropogenically increased atmospheric carbon dioxide (\u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e) leads to ocean acidification, disrupting calcification in marine calcifiers by reducing the saturation state of calcium carbonate. Calcium is not only a crucial component in the shell and skeleton structure but also serves as an essential second messenger for regulating biomineralization across many species. Ocean acidification is well-studied as causing shell dissolution in a diversity of bivalve species by disordering calcium deposition. However, it remains unclear whether the calcium-mediated signaling pathway regulating biomineralization is also affected. This study assessed eastern oyster (\u003cem\u003eCrassostrea virginica\u003c/em\u003e) to determine how calcium signaling responds to elevated \u003cem\u003ep\u003c/em\u003eCO₂ and influences shell formation. Under elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e, increased intracellular calcium concentration was found in primary epithelial cell cultures from oyster mantle. Meanwhile, we observed upregulation of calmodulin, a primary sensor of intracellular calcium, while its downstream effector, calcineurin, was downregulated. In addition, four conserved shell matrix proteins (SMPs), representing shell construction conditions, were significantly upregulated in the CO\u003csub\u003e2\u003c/sub\u003e-exposed mantle cells. \u003cem\u003eIn vivo\u003c/em\u003e, larval \u003cem\u003eC. virginica\u003c/em\u003e exhibited developmental stage-dependent alterations in calcium signaling and SMPs disarrangement stimulated by \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e. We hypothesize that dysregulation of calcium signaling disrupts the expressions of SMPs and causes oyster shell deformation. Pharmaceutical blockage of the calcium-calmodulin binding induced abnormal expression of related genes and shell matrix changes consistent with those caused by elevated \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Importantly, calcineurin restored SMPs expression in CO\u003csub\u003e2\u003c/sub\u003e-treated mantle cells. These findings suggest that shell deformities under ocean acidification are related to disruption of the calcium-calmodulin signaling pathway, inhibiting calcineurin activity and affecting SMPs production.\u003c/p\u003e","manuscriptTitle":"Ocean acidification disrupts the biomineralization process in the oyster Crassostrea virginica via intracellular calcium signaling dysregulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 13:23:15","doi":"10.21203/rs.3.rs-7180529/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"49918a3d-e8a8-43ef-9c12-5ee8fd7d2680","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52960609,"name":"Biological sciences/Developmental biology/Experimental organisms/Non-model organisms"},{"id":52960610,"name":"Biological sciences/Evolution/Evolutionary developmental biology"}],"tags":[],"updatedAt":"2026-05-06T07:11:04+00:00","versionOfRecord":{"articleIdentity":"rs-7180529","link":"https://doi.org/10.1038/s42003-026-09861-y","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2026-03-17 04:00:00","publishedOnDateReadable":"March 17th, 2026"},"versionCreatedAt":"2025-08-19 13:23:15","video":"","vorDoi":"10.1038/s42003-026-09861-y","vorDoiUrl":"https://doi.org/10.1038/s42003-026-09861-y","workflowStages":[]},"version":"v1","identity":"rs-7180529","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7180529","identity":"rs-7180529","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}