Coral calcification resistance to acidification is physiologically linked with complex intracellular calcium ion dynamics between host and symbiont cells

Coral calcification resistance to acidification is physiologically linked with complex intracellular calcium ion dynamics between host and symbiont cells | 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 Coral calcification resistance to acidification is physiologically linked with complex intracellular calcium ion dynamics between host and symbiont cells David Armstrong, Christopher Hollenbeck, Conall McNicholl, Keisha Bahr This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7402868/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Coral calcification is a highly complex process with numerous caveats regarding the mechanisms that dictate productivity and function. Ion homeostasis, however, is the foremost physiological process unanimously shared among Scleractinia and essential for calcification. Consequently, changes to the seawater environment may elicit adverse effects on ion homeostasis. With increasing climate shifts, the physicochemical regime of our global ocean is changing rapidly. Responses of coral calcification to physicochemical change prevail in having little uniformity on an unambiguous mechanism of resistance. Therefore, this study chose a relatively tolerant Hawaiian coral, Montipora capitata to focus efforts on understanding ion homeostasis under chemical seawater manipulation designed to limit calcification. Results indicate a physiological hormesis (two-phase adaptive response) of overall coral host gene expression that was not shared with algal symbionts and decoupled from calcification rates. The sole ion homeostatic mechanism shown was calcium ion regulation by both the host and symbiont cells. Calcium ion homeostasis was also found to be mechanistically different between winter and summer seasons. Thus, potentially indicating complex interactions between host and symbiont cells, as well as the ability for M. capitata to promote calcification under stress. Putatively synthesized here are the physiological cascades and mechanisms of resistance to environmental triggers of acidosis and seasonal change. This work provides insight into linking calcium ion homeostasis with coral resistance and aims to suggest this mechanism as biomolecular indicator used in future assessments to compare tolerance. Biological sciences/Molecular biology/Transcriptomics Biological sciences/Physiology Earth and environmental sciences/Ocean sciences/Marine biology Aragonite saturation state (Ω) RNA-seq carbonate chemistry ocean acidification gene expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Tropical coral reefs are constructed by marine calcifiers (i.e., corals, mollusks, and calcifying algae), providing unique habitats and supporting global oceanic biodiversity 1 , 2 . Corals and other calcifiers synthesize calcium carbonate (CaCO 3 ) polymorphs (i.e., aragonite, calcite, etc.) through the biogenic process of calcification 3 , 4 . Marine calcifiers, specifically corals, are at the forefront as bioindicators of future climate change 5 . Changes in the physicochemical environment driving coral calcification are a primary concern as the climate shifts to warmer and more acidic seas 5 , 6 . Ca 2+ + DIC ⇄ CaCO 3 + 2H + (1) The process of calcification involves the reaction of substrates, calcium (Ca 2+ ), and dissolved inorganic carbon (DIC) to synthesize CaCO 3 and two hydrogen ions in excess (H + ; Eq. 1; Fig. 1 ) 7 . The primary source of calcium for calcification originates from seawater, whereas DIC, consisting of the species CO 2(aq) , bicarbonate (HCO 3 − ), and carbonate (CO 3 2− ), may be partially metabolically derived (CO 2(m) ) 8 , 9 . In tropical scleractinian corals, this reaction occurs in the extracellular calcifying fluid (ECF), mostly isolated from the seawater (Fig. 1 ) 10 . The ECF’s ionic composition relies greatly on physiological control via ion transport mechanisms that connect this space with the seawater 3 , 4 , 10 , 11 . Because diffusive CO 2(m) supply may persist through metabolism, calcium supply is most reliant on ion regulatory mechansims 8 , 12 , 13 . Although HCO 3 − transport also relies on anion transporters 14 , Hohn and Merico 8 modeled the most likely pathway for reactant supply as nearly 1:1 calcium and CO 2(m) , with minimal contributions of HCO 3 − to calcification (Fig. 1 ). Nevertheless, calcification depends on physiologically concentrating reactants, such as calcium and DIC, and diffusing hydrogen ions to promote the precipitation of skeletal aragonite 15 . The greatest chemical threat to calcification is ocean acidification (OA) 16 , 17 , and aside from ocean warming, OA persists as the highest global risk to reduce calcification in corals 6 , 18 . Ocean acidification is a detriment to some, but the directionality of response is not unanimously negative among corals and other marine calcifiers 5 , 19 , 20 . Hence, several underlying factors have been identified to determine the scleractinian coral calcification response, i.e., life stage 21 , growth rate 22 , heterotrophy 23 , and metabolism 24 . However, current conclusions lack a clear understanding of the unambiguously shared mechanisms that underpin calcification resistance to OA 25 . Therefore, this study aims to address this knowledge gap by highlighting the physiological demands of ion acquisition and homeostasis—the foremost basal calcification process unambiguously consistent in all tropical scleractinian corals 4 . Functional gene expression of ion homeostasis mechanisms was compared against a sensitivity gradient that incrementally reduces reactants ([DIC]) and increases waste products ([H + ]) in seawater. In contrast to singular OA studies (solely increased pCO 2 ), this comparison evaluates the biological control of ion acquisition in light of changes in seawater chemistry to the proportion of increasing limiting factors for calcification (Eq. 1) 26 . Therefore, this study is motivated by OA but aims to achieve a deeper understanding of the physiological drivers for calcification in comparison to the physicochemical seawater environment. In this study, the examination of the Hawaiian rice coral Montipora capitata was chosen due to its well-documented history of resistance and resilience to environmental disturbance 27 – 32 . Other works have thus far measured its unique tolerance through ecologically derived 28 , genomic 33 , and transcriptomic 32 assays. The transcriptomics approach is particularly valuable for understanding gene expression patterns determining individual physiological function and short-term adaptation 30 , 34 , 35 . With the onset of recent high-throughput ribonucleic acid sequencing (RNA-seq) 34 , subsequent studies have reached new depths in understanding the complex molecular pathways involved in coral response to acidification 36 – 40 . An emerging physiological trend is identified as impacts on ion transport 37 , 39 , 40 . The work presented here is the first to correlate the physiology of ion acquisition with predetermined seawater chemistry ratios explicitly designed to limit calcification. This assessment hopes to illuminate the inner biological framework that drives a tolerant coral to maintain calcification despite ecologically relevant chemical stressors. Results Physicochemical environment and physiological function Throughout the 31-day exposure period, summer (11-Jul-23 to 08-Aug-23) temperatures ranged from 26.9 to 27.1°C while winter (11-Jan-24 to 08-Feb-24) temperatures ranged from 23.9 to 24.0°C (two-way ANOVA p = 2e − 16 , 0.01, df = 1, F = 3916.0), with a Δ between the two seasons of ~ 3°C (Table S2; Fig. 2 b). Natural light intensity and day length also differed between seasons (Fig. 2 a). The pH T and TA were consistently different between treatments (two-way ANOVA p = 2e − 16 , df = 3, F = 682.3 and p = 2e − 16 0.01, df = 3, F = 529.0 respectively; Fig. 2 c), which was, in the majority, reflected in the calculated carbonate parameters (i.e., pCO 2 , DIC, and Ω arag ; Fig. 2 c). There were also differences in seasons for pH T and TA (two-way ANOVA p = 0.001, df = 3, F = 11.03 and p = 1.49e − 7 , df = 3, F = 30.5, respectively), which is expected with the methodology of Δ -0.30 pH T and Δ -100 µmol TA compared to ambient conditions (Table S2). Calcification Rate The calcification rate (CaCO 3 g d − 1 ) in treatment corals was relatively more stable in the summer season (slope = 0.286; Fig. 2 d) and showed slightly more sensitivity to a lower [DIC]:[H + ] ratio (higher [H + ] compared to [DIC]) in the winter season (slope = 0.676; Fig. 2 e). However, the calcification rate was not well explained by treatment seawater chemistry (summer R 2 = 0.02 and winter R 2 = 0.19). The calcification rate was also not significantly different among treatments (two-way ANOVA p = 0.694, df = 3, F = 0.491) in the summer and winter seasons or between seasons (two-way ANOVA p = 0.612, df = 3, F = 0.616), confirming suspected tolerance of M. capitata to [DIC]:[H + ] change. Differential Gene Expression of the Coral Host Cells The count data output showed 22,545 unique gene transcript counts for the samples. After filtering (pp ≥ 95; excluding those assigned to the null model) for differentially expressed genes (DEGs), 511 for the summer and 194 for the winter were found (Fig. 2 f). Among the DEGs, 15 were shared between seasons, and 496 were found only in summer and 179 in winter (Fig. 2 f). Log 2 fold changes (LFC) were compared among all the DEGs, and distributions were plotted (Fig. 2 g). The physiological analysis used top trends in annotation clusters generated by the program DAVID 42 , 43 . The summer and winter DEGs resulted in 24 clusters with 129 matching Uniprot Protein Accession terms and 15 clusters with 145 matching terms, respectively. Gene expression patterns The relationship in response to treatment was visualized by both a ridgeline plot (Fig. 1 g) and heatmaps (Fig. 2 h, i) of LFC per treatment comparison, respectively, of season. Patterns in the ridgeline plot show reduced less differential gene expression when increasing [H + ] and decreasing [DIC] in the summer (Fig. 2 g). The summer LFC of Tmt 1 was significantly different from Tmt 3 (one-way ANOVA p = 2.39e − 7 , df = 2, F = 10.25) and Tmt 2 was also shown to be significantly different from Tmt 3 (one-way ANOVA p = 0.014, df = 2, F = 10.25; Fig. 2 g). However, gene expression in the winter was less variable compared to the summer months, coupled with the indication of fewer DEGs (Fig. 2 f, g). The LFC of winter corals was not significant among treatments (one-way ANOVA, p = 0.372, df = 2, F = 0.991; Fig. 2 g). The summer heatmap also shows a clear response of muted gene expression to Tmt 3 compared to the Amb. treatment via the cluster groupings (Fig. 1 h). Both Tmt 1 and Tmt 2 are clustered with the Amb. treatment, and similarly with Tmt 3. The cluster group of Tmt 1 and Tmt 2 shows a similar response to both treatments. The treatment relationships were more variable in the winter (Fig. 2 i). Overall, exposure to levels at Tmt 2 created a larger LFC compared to all other treatments in the winter. Calcium Ion Homeostasis The only identifiable mechanisms of ion acquisition that were differentially expressed by M. capitata were mechanisms of calcium ion homeostasis and signaling. A principal component analysis (PCA) revealed a similar trend of overall gene expression patterns, where PC1 showed significant treatment effects (one-way ANOVA p = 5.42e − 4 , df = 3, F = 19.25). The physiological function of maintaining calcium homeostasis revealed four separate DEGs associated with several related functions in the summer (Fig. 2 b, c) and only one DEG with an associated function found in the winter (Table S3). CHRNA7 was found to control calcium channel activity (GO:0005262; GO:0006816) and was responsible for intracellular calcium homeostasis (GO:0006874). The localization of CHRNA7 shows its related proteins are found in the (apical) plasma membrane (GO:0016324). Three DEGs (UMOD, CASR, and HMCN1) were found to regulate proteins associated with calcium ion binding (GO:0005509; Fig. 2 b). UMOD was simultaneously shown to control cell adhesion and maintenance of the ECM utilizing calcium (Table S3). CASR is involved in calcium ion binding, import (GO:0070509), overall transport (GO:0006816), extracellular sensing (IPR000068), and intracellular homeostasis. The proteins controlled by CASR are also localized to the (apical) plasma membrane. The gene HMCN1, similar to UMOD, was only found in regulating calcium ion binding (Table S3). However, HMCN1 was found to be highly involved in cell adhesion and other functions utilizing calcium (Table S3). The only ion regulation DEG was found in the winter experiment, CALU, was shown to be related to calcium ion binding (GO:0005509), and localized to the sarcoplasmic (endoplasmic) reticulum (GO:0033018; GO:0005788) and Golgi apparatus (GO:0005794; Table S3). Holobiont Ion Homeostasis In the symbiont samples, 16 DEGs for the summer season and three for the winter season were found. Functional annotations for 15 summer DEGs were available, and only one gene in the winter had functional annotation information. There were four DEGs related to calcium and potassium ion homeostasis found in the samples (Fig. 3 b). The gene 345622 was found related to functions in ion channel activity (GO:0015267), particularly the potassium ion (GO:0006813; Table S3). The gene 353148 was found to regulate voltage-gated calcium channel activity (GO:005245) involved in calcium transport (Table S3). Gene 353558 was associated with cellular calcium ion homeostasis (GO:0006874) and regulation of calcium ion transport (GO:0051928), along with potassium channel regulation (GO:0015459; Table S3). Gene 355892 was found to regulate potassium ion transport (GO:0043268) among other functions (Table S3). Discussion In this study, the coral M. capitata was exposed to a uniquely developed sensitivity gradient for the reactants and waste products created to chemically limit coral calcification (decreasing [DIC]:[H + ]). These experiments showed a relative tolerance to calcification in the coral M. capitata in response to this chemical gradient. However, several hundred significantly differentially expressed genes related to the chemical disturbance were identified, and emerging trends point toward complex relationships of ion homeostasis between the host and symbiont to maintain calcification. This study chose to mainly examine ion transport, due to the ubiquitous use of this physiological mechanism in corals and other marine calcifiers. Seasonality Patterns of gene expression differed by season, with a higher abundance of DEGs in the summer than in winter. In Bahr et al. 28 , M. capitata showed seasonally distinct calcification responses to light and temperature but was not impacted by acidification. Furthermore, seasonal calcification in Acropora digitifera (sister taxa) has also shown no differences under acidification 44 . In this study, seasonality of experiments naturally altered the physical environment (i.e., water temperature, light levels, and day length). Few gene expression studies have considered seasonality in this context, and in this study, ion homeostasis mechanisms were shown here to be impacted by these physicochemical shifts. A functional response in ion transport mechanisms is an emerging topic in temperature manipulation studies 45 , 46 . Bernardet et al. 47 found elevated temperature and light to correlate with increased diversity of gene expression related to ion transport mechanisms (i.e., Ca 2+ , DIC, and H + ). In this study, M. capitata primarily differentially expressed ion regulatory genes related to calcium ion homeostasis, with mechanistic differences influenced by season. Physiological Hormesis Gene expression exhibited physiological hormesis 48 (two-phase adaptive response) in the host cells, decoupled with calcification. The lowest [DIC]:[H + ] treatment (Tmt 3) showed the lowest proportion of |LFC| in the summer and winter (though more variable in the winter). Results in Comeau et al. 49 found no indication of a calcification tipping point in several marine calcifiers, including corals, when exposed to increasing pCO 2 . Cornwall et al. 50 , 51 have also reached similar conclusions determining calcification linearly responds to OA. The calcification data showed that M. capitata maintained calcification despite the chemical seawater manipulation. However, from the physiological perspective, internal mechanisms that may sustain calcification did exhibit hermetic trends on several levels. This finding could be indicative of alternative mechanisms outside of the host’s cellular control that are involved in maintaining calcification. Though unclear, the coral holobiont could have aided in maintaining calcification at the lowest [DIC]:[H + ] levels. Barreto et al. 52 have found strong responses to OA in the active microbial communities of the coral S. pistillata . We did not see hormesis in summer symbiont gene expression, indicative of potential holobiont contributions to sustaining calcification. Changes in the holobiont gene expression coincided with reduced change in host cells at Tmt 3, suggesting that the holobiont may be compensating for host cells. Future work could benefit from coupling gene expression with in-depth characterization of the holobiont to identify potential phenotypic plasticity that could aid calcification. Ion Homeostasis Calcium ion transport and binding were the leading functional responses for maintaining ionic homeostasis related to calcification, with no indication of impacts to HCO 3 − and H + ion transporters or carbonic anhydrases (CAs). The coral host and symbiont cells both showed differential expression of genes related to calcium ion maintenance (Fig. 4 ). Coral calcium ion regulation has been consistently highlighted in molecular studies following exposure to acidification 37 , 39 , 40 , 53 and thermal stress 46 , 47 . Calcium ion dynamics related to coral tolerance are also conceptually backed by quantitative assessments 54 and recently reviewed in detail by Armstrong and Bahr 55 . Calculations from Hohn and Merico 8 show calcium as the highest transcellularly transported substrate for calcification. Also, found in Reymond and Hohn 56 , calcification increased three times more following an increase in [Ca 2+ ]:[CO 3 2− ] ECF (higher [Ca 2+ ]) within a simulated ECF environment. Though in high chemical abundance, corals likely use the most energy to concentrate calcium at the site of calcification. Thus, the physiological function of calcium homeostasis could potentially control calcification tolerance in corals under acidification stress 55 . The mechanism of paracellular leakage from the ECF may threaten the ability of corals to maintain ionic homeostasis, particularly concerning [Ca 2+ ] and [H + ]. Hohn and Merico 8 model paracellular leakage as a threat to coral calcification in OA, and calcium is highly susceptible to paracellular leakage from the ECF. Indeed, the present study observed several mechanisms of cell adhesion mediated by calcium ion binding impacted by decreasing [DIC]:[H + ], similarly found by Radice et al. 40 . The upregulation of calcium ion transporters in Tmt 1 could indicate a demand for maintaining concentrations in the extracellular calcifying fluid (ECF). Interestingly, there was no differential regulation of calcium-ATPase, previously reported in Porites astereoides 40 and Acropora austera 37 . Instead, only the upregulation of calcium channel activity was observed (GO:0005262), which also exhibited hormetic trends in the lowest [DIC]:[H + ], where LFC decreased to near zero. Simultaneously, this study shows the repression of calcium channel activity in symbionts by -55% in the lowest [DIC]:[H + ]. Symbiont calcium and potassium ion transport gene expression was repressed along the decreasing [DIC]:[H + ] gradient, and this is the first study to show this response in coral-algal symbionts. All 16 DEGs found in the summer were repressed by increasing [H + ] relative to [DIC], and the only clear response shared between the animal and symbiont was the mechanism of calcium ion homeostasis. It is unknown how the symbionts use calcium channels and the implications this may have for the animal host. Perhaps calcium uptake by symbionts was repressed to supplement calcification in the host. However, this interpretation may be immensely oversimplified. Calcium is highly toxic to animal cells 57 , 58 , though it is impossible to name a physiological process that does not rely on calcium 57 . A symbiont’s obligate relationship with the coral could be similar to the obligate symbiosis of animal cell mitochondria. Calcium pulses intracellularly from organelles (i.e., mitochondria, sarcoplasmic reticulum, and endoplasmic reticulum) are hypothesized to have a crucial role in early endosymbiosis and the formation of the eukaryotic cell 57 , 58 . However, it is unclear how the repression of calcium channels in symbionts relates to maintaining calcification or symbiosis in the face of seawater chemistry stress. Early work has reported that lower [Ca 2+ ] sw can negatively affect coral calcification 59 . Evidence does support the link between calcification and photosynthesis 14 , 60 , 61 and light-enhanced calcification 11 , 62 . Interestingly, the increased repression of calcium transporters was followed directly by the reduced repression of genes coding for calcium ion binding in the animal, one of which consists of proteins interacting in the ECM (Fig. 4 a). Calcium pulses from coral symbionts have not been considered a link to calcification, and more work needs to be done to determine the relative importance of this dynamic. Particularly considering the toxicity of calcium to animal cells and the potential independent use of calcium by the symbionts to regulate animal function. In the winter season, there were no interactions with the symbionts and calcium homeostasis via ion transporters (Fig. 4 b). Instead, gene expression patterns revealed the potential use of intracellular stores in host cells to supplement calcium to the ECM. Here, an upregulation of a gene that regulates calcium ion binding proteins, localized in the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER), is secreted from the intracellular environment. The animal cell SR and ER are involved in calcium ion storage and release 63 . The upregulation of this gene was observed solely in Tmt 2 and not differentially expressed in Tmt 1 or 3. In winter, the metabolic demand for sustaining calcium through ion transporters may be less metabolically favorable than in summer when photosynthesis is highest. However, the molecular mechanisms of calcium ion homeostasis are highly complex. Indirect Processes Influencing Calcification Aside from direct impacts on calcification through physiological control of ion homeostasis, a large body of DEGs corresponded to metabolism, electron transport chain, protein synthesis, methylation, DNA/histone regulation, and immune responses for both seasons (see Appendix A, B). The high degree of functional methylation and transcription factors could have implications for future work assessing the epigenetic impacts of OA on M. capitata 64 , 65 . In the coral S. pistillata , known for its tolerance to OA, Liew et al. 66 showed epigenetic factors fine-tuning the expression of specific genes, which may contribute to calcification responses. However, results from Putnam et al. 67 showed M. capitata to exhibit little DNA methylation when exposed to OA, during experiments conducted between March and April. Only shown is methylation activity primarily in the summer treatments (July-Aug) and only regulation of histones in winter one month prior to Putnam et al. 67 (Appendices A, B). The concept that phenotypic trait heritability is dictated only by genetic means has met scrutiny through emerging trends in epigenetics 68 , 69 . An indication here that some degree of methylation could occur in M. capitata raises potential questions on what processes could be prioritized and if those traits have been passed on trans-generationally. More work is needed to identify the adaptive capacity of M. capitata , but certainly, this species has a robust response to environmental change 30 . The physiological dynamics here are undoubtedly complex but likely working in conjunction with ion transport to maintain calcification. Considering the wide range of indirect responses, it is likely that ion transport is not the sole mechanism that allows M. capitata to continue calcification under OA. However, this mechanism is the baseline and most direct pathway for future experimentation, accessible to disciplines outside of functional genomics. Conclusions and The Road Ahead This study revealed complex interactions of ion homeostasis present in the tolerant coral M. capitata with mechanisms that differ seasonally. Calcification builds reefs, foundationally supporting marine biodiversity 70 , and global ocean change (i.e., warming and acidification) 18 could threaten this process in corals 71 , 72 . The mechanisms for coral calcification are highly complex 3 . However, it is clear that utilizing ion transport combining reactants calcium and DIC in concert with H + diffusion must co-occur to precipitate CaCO 3 73 . Ocean acidification will chemically alter [DIC] and [H + ] while [Ca 2+ ] remains unaffected 17 . This study shows that M. capitata host and symbiont interactions functionally regulated calcium under chemically stressful seawater conditions. It is unclear how rising OA and the disruption of the chemical environment drive lower calcification in some corals but not others 74 . This work proposes that functional calcium regulation plays a partial role in tolerance in M. capitata . The information presented here is important for initiatives exploring the physiological resilience of corals to future environmental change using predictive bioindicators 47 . Climate stress mitigation tactics implementing assisted evolution have been proposed as an intervention strategy conceptually based on physiological resilience. However, assisted evolution hosts diverse costs and benefits 75 , and some argue that the practicality of impactful restoration efforts is low 76 . Nevertheless, despite the limitations of human intervention strategies, understanding the adaptive capacity and transgenerational plasticity of corals is essential for conservation 47 , 77 . Studies like the one here will benefit future efforts by developing a deeper understanding of the underlying biological processes involved in physiological resilience to environmental change. The future of coral reefs will depend on the culmination of adaptation through generations to interacting locally and globally derived stressors. This study did not introduce any interacting environmental stressors (i.e., thermal, sediment, and pollution) that corals may experience. Future work should investigate the introduction of interacting stressors, but it is suggested from the results to implement targeted approaches assessing calcium homeostasis (i.e., proteomics, confocal or RAMAN spectroscopy, and single-cell RNA analysis) when addressing impacts on calcification. Methods Coral Collection and Mesocosm Environment Colonies of M. capitata were collected approximately 600 m from the shore of the Hawaiʻi Institute of Marine Biology in Kāneʻohe Bay, Hawaiʻi (21° 26’ 43” N, 157° 47’ 46” W; SAP 2022–2023 and 2023–2024). The coral colonies were acclimated to a flow-through seawater mesocosm environment (an area of 1.37 m 2 and SW volume ~ 50 m 3 ) for seven days. Mesocosms each contained 20 colonies of M. capitata and 20 colonies of Pocillopora acuta. Treatments were set up by row, and there were four mesocosm rows, each with three tanks per row. Mesocosms received seawater directly from the bay, < 1km from the collection site, with a tank residence time of one hour, and were environmentally controlled—exposed to natural light (shaded to 50% - PAR 800 µmol s − 1 ). Seawater Chemistry The seawater chemistry manipulation techniques and physical mesocosm environments were identical between seasons. Mesocosm row header mixing chambers received additions of 1M HCl in the Tmt 1 treatment and were monitored by adjusting seawater flow rates or drip speed to achieve a Δ TA of ~ 100 µmol compared to the control (Table S2). In the Tmt 2 treatment, an air + CO 2 mixture was bubbled directly to each mesocosm and adjusted rates based on the pH STAT methodology to a Δ pH of ~ 0.30 compared to the control 78 . Then, a combined Tmt 3 treatment was implemented via both methods to the third row of mesocosms (Table S2). The control mesocosm seawater chemistry was not manipulated, but all other aspects were identical to the treatment conditions. Other seawater parameters were environmentally controlled and not manipulated. Carbonate chemistry parameters (i.e., [DIC], pCO 2 , and Ω arag ) were calculated utilizing the R package ‘ seacarb ’, inputting direct measures of temperature, salinity, pH T , and TA (Table S2). Calcification Rate Calculation Coral colonies were weighed in seawater at the beginning of each experiment and at the end of the 31-day exposure period to determine calcification in the exposure. This study followed the buoyant weight technique 41 procedures, along with Eq. 2, to determine the rate of CaCO 3 precipitation in the coral colonies. G = 1.54 × ( W f – W i )/ d (2) Equation 2 calculates G, or the calcification rate (CaCO 3 g d − 1 ), by the final buoyant weight ( W f ) subtracted from the initial buoyant weight ( W i ) over time in days ( d ), all multiplied by the seawater and aragonite density constant (1.54) to achieve the dry weight of aragonite 41 . RNA-seq Subsampling Coral colonies of M. capitata were subsampled on the 31st day of exposure from 12:00 to 14:00 for both summer and winter seasons. Colonies were transported in seawater to the lab sampling space (< 10 m from mesocosms). The lab bench was continuously sterilized with a 1 M NaOH distilled water solution before and between subsampling. Five sterilized (autoclaved at 121°C for 30 minutes) 1.5 mL Eppendorf tubes filled with 1 mL of RNA Later for each coral colony. Medical grade bone cutters sterilized by dipping (sequentially) in seawater, deionized water, 10% bleach, 1 M NaOH, and finally Ethanol, which was burned off, were used to subsample coral colonies. We sterilized bone cutters via this sequential dipping process between each coral colony but only dipped in the colony’s respective seawater when taking multiple samples from the same individual. We took quintuplicate samples per individual and focused localization only at the tip of branching colonies covering < 1 cm down from the tip. Fragments were placed directly in the RNA Later Eppendorf tubes, capped, and snap-frozen in liquid nitrogen for five seconds. The samples were then chilled on ice for two hours and placed in a -80°C freezer until shipment for sequencing. Library Prep and RNA-seq Raw tissue samples were sent directly to the AZENTA Life Sciences labs, where they conducted extraction, library prep, and sequencing following their protocols in the ‘Standard RNA-seq bundle with PolyA selection’. Samples were sequenced at 50 million paired-end reads per sample utilizing the Illumina, 2x150bp sequencing configuration. This generated ~ 1.4 billion reads over 24 samples, with a mean quality score of 38.92 and 94.64% bases ≥ 30. Bioinformatic Pipeline The bioinformatics pipeline used here was adopted from Chille et al. 79 , and Radice et al. 40 . Raw Fastq files were quality checked utilizing Fastqc v0.11.9 and Multiqc v1.25.1 platforms. Raw reads were trimmed using Fastp v0.23.4, focused on removing the overrepresented sequence and cleaned reads were confirmed via a second Multiqc report. The most current M. capitata reference genome 80 was indexed ( hisat2-build ), and cleaned reads were aligned using HISAT2 v2.2.1 (-- dta ). No individual aligned greater than 60% for both summer and winter to the reference genome, and thus, we chose to conduct a de novo transcriptome assembly using Trinity v2.15.2 (-- normalize_max_read_cov 200). The longest isoforms were extracted from the assembly via a Python v3.9 script (Biopython v1.79) with a minimum threshold of ≥ 500 bp. The assembly was compared via BLASTn to the SILVA rRNA LSU and SSU NR99 database (downloaded January 2, 2025), and matches at a minimum of 1e-5, 78% at 100bp (- qcov_hsp_perc 100) were removed. The de novo assembly was compared via BLASTn (1e-5, 70% identity at 100bp) to two databases, cnidarian and Symbiodiniaceae (NCBI Nucleotide Database). The assemblies were separated into host and symbiont-specific assemblies, resulting in a final host assembly with 34,048 contigs and a symbiont assembly with 759 contigs. Assemblies were annotated via BLASTx, 1e-4 (- max_target_seqs 10) using NCBI (nr protein; downloaded January 10, 2025) and hierarchically uniprot sprot (downloaded January 11, 2025) along with uniprot TrEMBL (downloaded January 11, 2025) databases. All contigs were matched to the NCBI nr db, then all contigs were again matched with the well-curated sprot db. Contigs that did not match with the sprot db were then matched with the TrEMBL db. Due to the resulting small size, the symbiont de novo assembly was not used. Read alignment was conducted for the host de novo assembly using Bowtie2 v2.5.4 (-- local ), however, the symbiont assembly was created aligning with a reference Cladocopium goreaui genome. Finally, RNA quantification per sample was conducted using Salmon v1.10.3 (-l A, -- validateMappings ). All differential gene expression analysis and plotting were conducted in R Studio 81 . The package EBSeq 82 , 83 was used to identify significant differentially expressed genes (DEGs) in the samples (maxround = 50 host and 5 symbiont, AllParti = Table S1, pp > 0.95). The biological patterns used were the smallest number of relationships without changing the number of DEGs and remaining relevant to hypothesis testing (Table S1). The null model (H o ) assigned DEGs pp < 0.95 were removed, and analysis proceeded forward with DEGs assigned to H 1 -H 5 . Annotated filtered DEGs with only Uniprot protein accession numbers were uploaded to the web program DAIVD 42 , 43 for functional characterization. Functional annotations indicating a relationship to ion regulatory mechanisms and cellular properties were extracted from the top clusters, which were then traced to DEGs. Data Availability Raw sequence reads can be found at NCBI under Bioproject PRJNA1307607 (uploaded following review). 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The extracellular calcifying fluid is shown as (ECF) and dissolved in organic carbon as (DIC). Ion transport mechanisms are shown as black squares at the interface of cell membranes.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7402868/v1/50916dadd973b44439da676e.png"},{"id":92209243,"identity":"16355f87-90b8-49cd-a04a-9114191d67b3","added_by":"auto","created_at":"2025-09-25 19:41:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":292003,"visible":true,"origin":"","legend":"\u003cp\u003eDiel continuous environmental data collected by the Hawaiʻi Institute of Marine Biology weather station (https://www.pacioos.hawaii.edu/weather/obs-mokuoloe/) for the duration of experimental summer and winter periods (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e). Summer (orange) measurements were between the dates 11-Jul-23 and 08-Aug-23. Winter (purple) measurements were between the dates 11-Jan-24 and 08-Feb-24. Hourly mean photosynthetic active radiation (PAR) values in µmol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e were collected by the weather station and halved as the mesocosms were shaded 50% (\u003cstrong\u003ea\u003c/strong\u003e). Hourly mean bay water temperature (°C) was collected by \u003cem\u003ein situ \u003c/em\u003eprobes 2 m water depth, \u0026lt; 10 m from experimental mesocosms (\u003cstrong\u003eb\u003c/strong\u003e). Also, the calculated mean log converted ratio of the concentration of dissolved inorganic carbon ([DIC]) to hydrogen ions ([H\u003csup\u003e+\u003c/sup\u003e]) for each mesocosm treatment (n = 3; \u003cstrong\u003ec\u003c/strong\u003e). Treatments were separated by Tmt 1 through Tmt 3 and are indicative of individual manipulation techniques. Error bars are ±SD of the mean for all plots (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e). Calcification rates (CaCO\u003csub\u003e3\u003c/sub\u003e g d\u003csup\u003e-1\u003c/sup\u003e) were\u003cem\u003e \u003c/em\u003emeasured via the buoyant weight technique\u003csup\u003e41\u003c/sup\u003e in \u003cem\u003eMontipora capitata \u003c/em\u003eusing initial and final weights from the experimental period of 31 days. Seawater chemistry ratios ([DIC]/[H+]) were used to fit a linear model for both summer (\u003cstrong\u003ed\u003c/strong\u003e) and winter (\u003cstrong\u003ee\u003c/strong\u003e) seasons, with a 95% confidence interval (CI) error. A ridgeline plot (\u003cstrong\u003ef\u003c/strong\u003e) with log\u003csub\u003e2\u003c/sub\u003e fold change (LFC) comparisons within treatment conditions and a Venn plot (\u003cstrong\u003eg\u003c/strong\u003e) with unique and shared differentially expressed genes (DEG) counts per season. The groups in the ridgeline plot show LFC respective of treatment, and ridges are colored by season (orange = summer and purple = winter). Ridge height is thus a proportion of DEGs that fall under the respective LFC on the x-axis. A zero LFC means no response in a specific treatment comparison. For clarity, only the highest density clamping tails to a LFC ≤ |5| is shown, but some LFCs extended much further. Two heat maps, summer (\u003cstrong\u003eh\u003c/strong\u003e) and winter (\u003cstrong\u003ei\u003c/strong\u003e), are shown with interacting clusters among treatments, with colors representing the degree of LFC. Comparison groups are separated by an em dash (—) to indicate the LFC of group 1 over group 2.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7402868/v1/944ea098af508566ddd64b7f.png"},{"id":92209253,"identity":"4e623292-dab0-4d59-b1c5-3d9003dae74b","added_by":"auto","created_at":"2025-09-25 19:41:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":381988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2.\u003c/strong\u003e A principal component analysis (\u003cstrong\u003ea\u003c/strong\u003e) of transcript counts bootstrapped (N = 500), ellipses are set at CI = 0.5. Color represents treatment and large solid points represent original samples whereas smaller surrounding points are bootstrapped values. Chord diagram (\u003cstrong\u003eb\u003c/strong\u003e) with gene symbols following the highest BLASTx hits to coral genes (right) and connections to functional information (left). A line plot (\u003cstrong\u003ec\u003c/strong\u003e) that shows gene expression in LFC changes over the course of the treatments for the four differentially expressed genes related to calcium ion homeostasis. Treatments are abbreviated by Tmt 1-3, where Tmt 3 is the lowest [DIC]:[H\u003csup\u003e+\u003c/sup\u003e]. This figure only shows a summer comparison.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7402868/v1/5023310507c7db908196dbe3.png"},{"id":92209248,"identity":"581630b9-9a1b-4847-8423-46e16ddf701a","added_by":"auto","created_at":"2025-09-25 19:41:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":152069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 3. \u003c/strong\u003eA stacked barplot (\u003cstrong\u003ea\u003c/strong\u003e) showing the mean ion homeostasis regulatory mechanism gene expression in Log Fold Change (LFC) of symbiont in green, and host cells in orange. A line plot of four differentially expressed genes (DEGs) related to ion homeostasis in symbiont cells (\u003cstrong\u003eb\u003c/strong\u003e) expression over the treatments. Overall gene expression in the 16 DEGs found in symbiont cells (\u003cstrong\u003ec\u003c/strong\u003e) is shown through a ridgeline plot where ridges show the density of LFC values per the DEGs. Treatments are abbreviated by Tmt 1-3, where Tmt 3 is the lowest [DIC]:[H\u003csup\u003e+\u003c/sup\u003e].\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7402868/v1/e42746f1f9b7cf36bd7990c2.png"},{"id":92209250,"identity":"d356a409-9cfe-446c-bca7-142ff5d408ff","added_by":"auto","created_at":"2025-09-25 19:41:50","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1366223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4.\u003c/strong\u003e A putative physiological cascade of summer (\u003cstrong\u003ea\u003c/strong\u003e) and winter (\u003cstrong\u003eb\u003c/strong\u003e) functional responses to tissue acidosis. See figure legends and cascade roman numeral identifiers for in-depth descriptions.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7402868/v1/43fc5a7f4dcd9ec9c40b3119.jpeg"},{"id":92210120,"identity":"1220c8a6-89bc-4184-aae3-d3a458e1e66d","added_by":"auto","created_at":"2025-09-25 20:05:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3417789,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7402868/v1/f3d8eef3-dc86-43fa-9fdf-a024b04b088f.pdf"},{"id":92209241,"identity":"a25090fa-c860-44b3-97bd-015f270d93d6","added_by":"auto","created_at":"2025-09-25 19:41:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":47581,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7402868/v1/257469e7e5acb88d3bdf88c1.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Coral calcification resistance to acidification is physiologically linked with complex intracellular calcium ion dynamics between host and symbiont cells","fulltext":[{"header":"Background","content":"\u003cp\u003eTropical coral reefs are constructed by marine calcifiers (i.e., corals, mollusks, and calcifying algae), providing unique habitats and supporting global oceanic biodiversity\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Corals and other calcifiers synthesize calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) polymorphs (i.e., aragonite, calcite, etc.) through the biogenic process of calcification\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Marine calcifiers, specifically corals, are at the forefront as bioindicators of future climate change\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Changes in the physicochemical environment driving coral calcification are a primary concern as the climate shifts to warmer and more acidic seas\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e + DIC ⇄ CaCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2H\u003csup\u003e+\u003c/sup\u003e (1)\u003c/p\u003e\u003cp\u003eThe process of calcification involves the reaction of substrates, calcium (Ca\u003csup\u003e2+\u003c/sup\u003e), and dissolved inorganic carbon (DIC) to synthesize CaCO\u003csub\u003e3\u003c/sub\u003e and two hydrogen ions in excess (H\u003csup\u003e+\u003c/sup\u003e; Eq.\u0026nbsp;1; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The primary source of calcium for calcification originates from seawater, whereas DIC, consisting of the species CO\u003csub\u003e2(aq)\u003c/sub\u003e, bicarbonate (HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), and carbonate (CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e), may be partially metabolically derived (CO\u003csub\u003e2(m)\u003c/sub\u003e)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In tropical scleractinian corals, this reaction occurs in the extracellular calcifying fluid (ECF), mostly isolated from the seawater (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The ECF\u0026rsquo;s ionic composition relies greatly on physiological control via ion transport mechanisms that connect this space with the seawater\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Because diffusive CO\u003csub\u003e2(m)\u003c/sub\u003e supply may persist through metabolism, calcium supply is most reliant on ion regulatory mechansims\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Although HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e transport also relies on anion transporters\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, Hohn and Merico\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e modeled the most likely pathway for reactant supply as nearly 1:1 calcium and CO\u003csub\u003e2(m)\u003c/sub\u003e, with minimal contributions of HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e to calcification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Nevertheless, calcification depends on physiologically concentrating reactants, such as calcium and DIC, and diffusing hydrogen ions to promote the precipitation of skeletal aragonite\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe greatest chemical threat to calcification is ocean acidification (OA)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and aside from ocean warming, OA persists as the highest global risk to reduce calcification in corals\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Ocean acidification is a detriment to some, but the directionality of response is not unanimously negative among corals and other marine calcifiers\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Hence, several underlying factors have been identified to determine the scleractinian coral calcification response, i.e., life stage\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, growth rate\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, heterotrophy\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and metabolism\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, current conclusions lack a clear understanding of the unambiguously shared mechanisms that underpin calcification resistance to OA\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, this study aims to address this knowledge gap by highlighting the physiological demands of ion acquisition and homeostasis\u0026mdash;the foremost basal calcification process unambiguously consistent in all tropical scleractinian corals\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Functional gene expression of ion homeostasis mechanisms was compared against a sensitivity gradient that incrementally reduces reactants ([DIC]) and increases waste products ([H\u003csup\u003e+\u003c/sup\u003e]) in seawater. In contrast to singular OA studies (solely increased pCO\u003csub\u003e2\u003c/sub\u003e), this comparison evaluates the biological control of ion acquisition in light of changes in seawater chemistry to the proportion of increasing limiting factors for calcification (Eq.\u0026nbsp;1)\u003csup\u003e26\u003c/sup\u003e. Therefore, this study is motivated by OA but aims to achieve a deeper understanding of the physiological drivers for calcification in comparison to the physicochemical seawater environment.\u003c/p\u003e\u003cp\u003eIn this study, the examination of the Hawaiian rice coral \u003cem\u003eMontipora capitata\u003c/em\u003e was chosen due to its well-documented history of resistance and resilience to environmental disturbance\u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Other works have thus far measured its unique tolerance through ecologically derived\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, genomic\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and transcriptomic\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e assays. The transcriptomics approach is particularly valuable for understanding gene expression patterns determining individual physiological function and short-term adaptation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. With the onset of recent high-throughput ribonucleic acid sequencing (RNA-seq)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, subsequent studies have reached new depths in understanding the complex molecular pathways involved in coral response to acidification\u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38 CR39\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. An emerging physiological trend is identified as impacts on ion transport\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The work presented here is the first to correlate the physiology of ion acquisition with predetermined seawater chemistry ratios explicitly designed to limit calcification. This assessment hopes to illuminate the inner biological framework that drives a tolerant coral to maintain calcification despite ecologically relevant chemical stressors.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePhysicochemical environment and physiological function\u003c/h2\u003e\n \u003cp\u003eThroughout the 31-day exposure period, summer (11-Jul-23 to 08-Aug-23) temperatures ranged from 26.9 to 27.1\u0026deg;C while winter (11-Jan-24 to 08-Feb-24) temperatures ranged from 23.9 to 24.0\u0026deg;C (two-way ANOVA p\u0026thinsp;=\u0026thinsp;2e\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e, 0.01, df\u0026thinsp;=\u0026thinsp;1, F\u0026thinsp;=\u0026thinsp;3916.0), with a \u0026Delta; between the two seasons of ~\u0026thinsp;3\u0026deg;C (Table S2; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). Natural light intensity and day length also differed between seasons (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). The pH\u003csub\u003eT\u003c/sub\u003e and TA were consistently different between treatments (two-way ANOVA p\u0026thinsp;=\u0026thinsp;2e\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e, df\u0026thinsp;=\u0026thinsp;3, F\u0026thinsp;=\u0026thinsp;682.3 and p\u0026thinsp;=\u0026thinsp;2e\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e 0.01, df\u0026thinsp;=\u0026thinsp;3, F\u0026thinsp;=\u0026thinsp;529.0 respectively; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), which was, in the majority, reflected in the calculated carbonate parameters (i.e., pCO\u003csub\u003e2\u003c/sub\u003e, DIC, and Ω\u003csub\u003earag\u003c/sub\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). There were also differences in seasons for pH\u003csub\u003eT\u003c/sub\u003e and TA (two-way ANOVA p\u0026thinsp;=\u0026thinsp;0.001, df\u0026thinsp;=\u0026thinsp;3, F\u0026thinsp;=\u0026thinsp;11.03 and p\u0026thinsp;=\u0026thinsp;1.49e\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e, df\u0026thinsp;=\u0026thinsp;3, F\u0026thinsp;=\u0026thinsp;30.5, respectively), which is expected with the methodology of \u0026Delta; -0.30 pH\u003csub\u003eT\u003c/sub\u003e and \u0026Delta; -100 \u0026micro;mol TA compared to ambient conditions (Table S2).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCalcification Rate\u003c/h3\u003e\n\u003cp\u003eThe calcification rate (CaCO\u003csub\u003e3\u003c/sub\u003e g d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in treatment corals was relatively more stable in the summer season (slope\u0026thinsp;=\u0026thinsp;0.286; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed) and showed slightly more sensitivity to a lower [DIC]:[H\u003csup\u003e+\u003c/sup\u003e] ratio (higher [H\u003csup\u003e+\u003c/sup\u003e] compared to [DIC]) in the winter season (slope\u0026thinsp;=\u0026thinsp;0.676; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee). However, the calcification rate was not well explained by treatment seawater chemistry (summer R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.02 and winter R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.19). The calcification rate was also not significantly different among treatments (two-way ANOVA p\u0026thinsp;=\u0026thinsp;0.694, df\u0026thinsp;=\u0026thinsp;3, F\u0026thinsp;=\u0026thinsp;0.491) in the summer and winter seasons or between seasons (two-way ANOVA p\u0026thinsp;=\u0026thinsp;0.612, df\u0026thinsp;=\u0026thinsp;3, F\u0026thinsp;=\u0026thinsp;0.616), confirming suspected tolerance of \u003cem\u003eM. capitata\u003c/em\u003e to [DIC]:[H\u003csup\u003e+\u003c/sup\u003e] change.\u003c/p\u003e\n\u003ch3\u003eDifferential Gene Expression of the Coral Host Cells\u003c/h3\u003e\n\u003cp\u003eThe count data output showed 22,545 unique gene transcript counts for the samples. After filtering (pp\u0026thinsp;\u0026ge;\u0026thinsp;95; excluding those assigned to the null model) for differentially expressed genes (DEGs), 511 for the summer and 194 for the winter were found (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef). Among the DEGs, 15 were shared between seasons, and 496 were found only in summer and 179 in winter (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef). Log\u003csub\u003e2\u003c/sub\u003e fold changes (LFC) were compared among all the DEGs, and distributions were plotted (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg). The physiological analysis used top trends in annotation clusters generated by the program DAVID\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The summer and winter DEGs resulted in 24 clusters with 129 matching Uniprot Protein Accession terms and 15 clusters with 145 matching terms, respectively.\u003c/p\u003e\n\u003ch3\u003eGene expression patterns\u003c/h3\u003e\n\u003cp\u003eThe relationship in response to treatment was visualized by both a ridgeline plot (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg) and heatmaps (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eh, i) of LFC per treatment comparison, respectively, of season. Patterns in the ridgeline plot show reduced less differential gene expression when increasing [H\u003csup\u003e+\u003c/sup\u003e] and decreasing [DIC] in the summer (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg). The summer LFC of Tmt 1 was significantly different from Tmt 3 (one-way ANOVA p\u0026thinsp;=\u0026thinsp;2.39e\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e, df\u0026thinsp;=\u0026thinsp;2, F\u0026thinsp;=\u0026thinsp;10.25) and Tmt 2 was also shown to be significantly different from Tmt 3 (one-way ANOVA p\u0026thinsp;=\u0026thinsp;0.014, df\u0026thinsp;=\u0026thinsp;2, F\u0026thinsp;=\u0026thinsp;10.25; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg). However, gene expression in the winter was less variable compared to the summer months, coupled with the indication of fewer DEGs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef, g). The LFC of winter corals was not significant among treatments (one-way ANOVA, p\u0026thinsp;=\u0026thinsp;0.372, df\u0026thinsp;=\u0026thinsp;2, F\u0026thinsp;=\u0026thinsp;0.991; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg). The summer heatmap also shows a clear response of muted gene expression to Tmt 3 compared to the Amb. treatment via the cluster groupings (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eh). Both Tmt 1 and Tmt 2 are clustered with the Amb. treatment, and similarly with Tmt 3. The cluster group of Tmt 1 and Tmt 2 shows a similar response to both treatments. The treatment relationships were more variable in the winter (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ei). Overall, exposure to levels at Tmt 2 created a larger LFC compared to all other treatments in the winter.\u003c/p\u003e\n\u003ch3\u003eCalcium Ion Homeostasis\u003c/h3\u003e\n\u003cp\u003eThe only identifiable mechanisms of ion acquisition that were differentially expressed by \u003cem\u003eM. capitata\u003c/em\u003e were mechanisms of calcium ion homeostasis and signaling. A principal component analysis (PCA) revealed a similar trend of overall gene expression patterns, where PC1 showed significant treatment effects (one-way ANOVA p\u0026thinsp;=\u0026thinsp;5.42e\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, df\u0026thinsp;=\u0026thinsp;3, F\u0026thinsp;=\u0026thinsp;19.25). The physiological function of maintaining calcium homeostasis revealed four separate DEGs associated with several related functions in the summer (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, c) and only one DEG with an associated function found in the winter (Table S3). CHRNA7 was found to control calcium channel activity (GO:0005262; GO:0006816) and was responsible for intracellular calcium homeostasis (GO:0006874). The localization of CHRNA7 shows its related proteins are found in the (apical) plasma membrane (GO:0016324). Three DEGs (UMOD, CASR, and HMCN1) were found to regulate proteins associated with calcium ion binding (GO:0005509; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). UMOD was simultaneously shown to control cell adhesion and maintenance of the ECM utilizing calcium (Table S3). CASR is involved in calcium ion binding, import (GO:0070509), overall transport (GO:0006816), extracellular sensing (IPR000068), and intracellular homeostasis. The proteins controlled by CASR are also localized to the (apical) plasma membrane. The gene HMCN1, similar to UMOD, was only found in regulating calcium ion binding (Table S3). However, HMCN1 was found to be highly involved in cell adhesion and other functions utilizing calcium (Table S3). The only ion regulation DEG was found in the winter experiment, CALU, was shown to be related to calcium ion binding (GO:0005509), and localized to the sarcoplasmic (endoplasmic) reticulum (GO:0033018; GO:0005788) and Golgi apparatus (GO:0005794; Table S3).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eHolobiont Ion Homeostasis\u003c/h2\u003e\n \u003cp\u003eIn the symbiont samples, 16 DEGs for the summer season and three for the winter season were found. Functional annotations for 15 summer DEGs were available, and only one gene in the winter had functional annotation information. There were four DEGs related to calcium and potassium ion homeostasis found in the samples (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). The gene 345622 was found related to functions in ion channel activity (GO:0015267), particularly the potassium ion (GO:0006813; Table S3). The gene 353148 was found to regulate voltage-gated calcium channel activity (GO:005245) involved in calcium transport (Table S3). Gene 353558 was associated with cellular calcium ion homeostasis (GO:0006874) and regulation of calcium ion transport (GO:0051928), along with potassium channel regulation (GO:0015459; Table S3). Gene 355892 was found to regulate potassium ion transport (GO:0043268) among other functions (Table S3).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the coral \u003cem\u003eM. capitata\u003c/em\u003e was exposed to a uniquely developed sensitivity gradient for the reactants and waste products created to chemically limit coral calcification (decreasing [DIC]:[H\u003csup\u003e+\u003c/sup\u003e]). These experiments showed a relative tolerance to calcification in the coral \u003cem\u003eM. capitata\u003c/em\u003e in response to this chemical gradient. However, several hundred significantly differentially expressed genes related to the chemical disturbance were identified, and emerging trends point toward complex relationships of ion homeostasis between the host and symbiont to maintain calcification. This study chose to mainly examine ion transport, due to the ubiquitous use of this physiological mechanism in corals and other marine calcifiers.\u003c/p\u003e\n\u003ch3\u003eSeasonality\u003c/h3\u003e\n\u003cp\u003ePatterns of gene expression differed by season, with a higher abundance of DEGs in the summer than in winter. In Bahr et al.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eM. capitata\u003c/em\u003e showed seasonally distinct calcification responses to light and temperature but was not impacted by acidification. Furthermore, seasonal calcification in \u003cem\u003eAcropora digitifera\u003c/em\u003e (sister taxa) has also shown no differences under acidification\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In this study, seasonality of experiments naturally altered the physical environment (i.e., water temperature, light levels, and day length). Few gene expression studies have considered seasonality in this context, and in this study, ion homeostasis mechanisms were shown here to be impacted by these physicochemical shifts. A functional response in ion transport mechanisms is an emerging topic in temperature manipulation studies\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Bernardet et al.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e found elevated temperature and light to correlate with increased diversity of gene expression related to ion transport mechanisms (i.e., Ca\u003csup\u003e2+\u003c/sup\u003e, DIC, and H\u003csup\u003e+\u003c/sup\u003e). In this study, \u003cem\u003eM. capitata\u003c/em\u003e primarily differentially expressed ion regulatory genes related to calcium ion homeostasis, with mechanistic differences influenced by season.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePhysiological Hormesis\u003c/h2\u003e\u003cp\u003eGene expression exhibited physiological hormesis\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e (two-phase adaptive response) in the host cells, decoupled with calcification. The lowest [DIC]:[H\u003csup\u003e+\u003c/sup\u003e] treatment (Tmt 3) showed the lowest proportion of |LFC| in the summer and winter (though more variable in the winter). Results in Comeau et al.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e found no indication of a calcification tipping point in several marine calcifiers, including corals, when exposed to increasing pCO\u003csub\u003e2\u003c/sub\u003e. Cornwall et al.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e have also reached similar conclusions determining calcification linearly responds to OA. The calcification data showed that \u003cem\u003eM. capitata\u003c/em\u003e maintained calcification despite the chemical seawater manipulation. However, from the physiological perspective, internal mechanisms that may sustain calcification did exhibit hermetic trends on several levels. This finding could be indicative of alternative mechanisms outside of the host’s cellular control that are involved in maintaining calcification. Though unclear, the coral holobiont could have aided in maintaining calcification at the lowest [DIC]:[H\u003csup\u003e+\u003c/sup\u003e] levels. Barreto et al.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e have found strong responses to OA in the active microbial communities of the coral \u003cem\u003eS. pistillata\u003c/em\u003e. We did not see hormesis in summer symbiont gene expression, indicative of potential holobiont contributions to sustaining calcification. Changes in the holobiont gene expression coincided with reduced change in host cells at Tmt 3, suggesting that the holobiont may be compensating for host cells. Future work could benefit from coupling gene expression with in-depth characterization of the holobiont to identify potential phenotypic plasticity that could aid calcification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIon Homeostasis\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCalcium ion transport and binding were the leading functional responses for maintaining ionic homeostasis related to calcification, with no indication of impacts to HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e and H\u003csup\u003e+\u003c/sup\u003e ion transporters or carbonic anhydrases (CAs). The coral host and symbiont cells both showed differential expression of genes related to calcium ion maintenance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Coral calcium ion regulation has been consistently highlighted in molecular studies following exposure to acidification\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e and thermal stress\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Calcium ion dynamics related to coral tolerance are also conceptually backed by quantitative assessments\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e and recently reviewed in detail by Armstrong and Bahr\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Calculations from Hohn and Merico\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e show calcium as the highest transcellularly transported substrate for calcification. Also, found in Reymond and Hohn\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, calcification increased three times more following an increase in [Ca\u003csup\u003e2+\u003c/sup\u003e]:[CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2−\u003c/sup\u003e]\u003csub\u003eECF\u003c/sub\u003e (higher [Ca\u003csup\u003e2+\u003c/sup\u003e]) within a simulated ECF environment. Though in high chemical abundance, corals likely use the most energy to concentrate calcium at the site of calcification. Thus, the physiological function of calcium homeostasis could potentially control calcification tolerance in corals under acidification stress\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe mechanism of paracellular leakage from the ECF may threaten the ability of corals to maintain ionic homeostasis, particularly concerning [Ca\u003csup\u003e2+\u003c/sup\u003e] and [H\u003csup\u003e+\u003c/sup\u003e]. Hohn and Merico\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e model paracellular leakage as a threat to coral calcification in OA, and calcium is highly susceptible to paracellular leakage from the ECF. Indeed, the present study observed several mechanisms of cell adhesion mediated by calcium ion binding impacted by decreasing [DIC]:[H\u003csup\u003e+\u003c/sup\u003e], similarly found by Radice et al.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The upregulation of calcium ion transporters in Tmt 1 could indicate a demand for maintaining concentrations in the extracellular calcifying fluid (ECF). Interestingly, there was no differential regulation of calcium-ATPase, previously reported in \u003cem\u003ePorites astereoides\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eAcropora austera\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Instead, only the upregulation of calcium channel activity was observed (GO:0005262), which also exhibited hormetic trends in the lowest [DIC]:[H\u003csup\u003e+\u003c/sup\u003e], where LFC decreased to near zero. Simultaneously, this study shows the repression of calcium channel activity in symbionts by -55% in the lowest [DIC]:[H\u003csup\u003e+\u003c/sup\u003e].\u003c/p\u003e\u003cp\u003eSymbiont calcium and potassium ion transport gene expression was repressed along the decreasing [DIC]:[H\u003csup\u003e+\u003c/sup\u003e] gradient, and this is the first study to show this response in coral-algal symbionts. All 16 DEGs found in the summer were repressed by increasing [H\u003csup\u003e+\u003c/sup\u003e] relative to [DIC], and the only clear response shared between the animal and symbiont was the mechanism of calcium ion homeostasis. It is unknown how the symbionts use calcium channels and the implications this may have for the animal host. Perhaps calcium uptake by symbionts was repressed to supplement calcification in the host. However, this interpretation may be immensely oversimplified. Calcium is highly toxic to animal cells\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, though it is impossible to name a physiological process that does not rely on calcium\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. A symbiont’s obligate relationship with the coral could be similar to the obligate symbiosis of animal cell mitochondria. Calcium pulses intracellularly from organelles (i.e., mitochondria, sarcoplasmic reticulum, and endoplasmic reticulum) are hypothesized to have a crucial role in early endosymbiosis and the formation of the eukaryotic cell\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. However, it is unclear how the repression of calcium channels in symbionts relates to maintaining calcification or symbiosis in the face of seawater chemistry stress.\u003c/p\u003e\u003cp\u003eEarly work has reported that lower [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003esw\u003c/sub\u003e can negatively affect coral calcification\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Evidence does support the link between calcification and photosynthesis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e and light-enhanced calcification\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Interestingly, the increased repression of calcium transporters was followed directly by the reduced repression of genes coding for calcium ion binding in the animal, one of which consists of proteins interacting in the ECM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Calcium pulses from coral symbionts have not been considered a link to calcification, and more work needs to be done to determine the relative importance of this dynamic. Particularly considering the toxicity of calcium to animal cells and the potential independent use of calcium by the symbionts to regulate animal function.\u003c/p\u003e\u003cp\u003eIn the winter season, there were no interactions with the symbionts and calcium homeostasis via ion transporters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Instead, gene expression patterns revealed the potential use of intracellular stores in host cells to supplement calcium to the ECM. Here, an upregulation of a gene that regulates calcium ion binding proteins, localized in the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER), is secreted from the intracellular environment. The animal cell SR and ER are involved in calcium ion storage and release\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The upregulation of this gene was observed solely in Tmt 2 and not differentially expressed in Tmt 1 or 3. In winter, the metabolic demand for sustaining calcium through ion transporters may be less metabolically favorable than in summer when photosynthesis is highest. However, the molecular mechanisms of calcium ion homeostasis are highly complex.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eIndirect Processes Influencing Calcification\u003c/h2\u003e\u003cp\u003eAside from direct impacts on calcification through physiological control of ion homeostasis, a large body of DEGs corresponded to metabolism, electron transport chain, protein synthesis, methylation, DNA/histone regulation, and immune responses for both seasons (see Appendix A, B). The high degree of functional methylation and transcription factors could have implications for future work assessing the epigenetic impacts of OA on \u003cem\u003eM. capitata\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. In the coral \u003cem\u003eS. pistillata\u003c/em\u003e, known for its tolerance to OA, Liew et al.\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e showed epigenetic factors fine-tuning the expression of specific genes, which may contribute to calcification responses. However, results from Putnam et al.\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e showed \u003cem\u003eM. capitata\u003c/em\u003e to exhibit little DNA methylation when exposed to OA, during experiments conducted between March and April. Only shown is methylation activity primarily in the summer treatments (July-Aug) and only regulation of histones in winter one month prior to Putnam et al.\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e (Appendices A, B).\u003c/p\u003e\u003cp\u003eThe concept that phenotypic trait heritability is dictated only by genetic means has met scrutiny through emerging trends in epigenetics\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. An indication here that some degree of methylation could occur in \u003cem\u003eM. capitata\u003c/em\u003e raises potential questions on what processes could be prioritized and if those traits have been passed on trans-generationally. More work is needed to identify the adaptive capacity of \u003cem\u003eM. capitata\u003c/em\u003e, but certainly, this species has a robust response to environmental change\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The physiological dynamics here are undoubtedly complex but likely working in conjunction with ion transport to maintain calcification. Considering the wide range of indirect responses, it is likely that ion transport is not the sole mechanism that allows \u003cem\u003eM. capitata\u003c/em\u003e to continue calcification under OA. However, this mechanism is the baseline and most direct pathway for future experimentation, accessible to disciplines outside of functional genomics.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions and The Road Ahead","content":"\u003cp\u003eThis study revealed complex interactions of ion homeostasis present in the tolerant coral \u003cem\u003eM. capitata\u003c/em\u003e with mechanisms that differ seasonally. Calcification builds reefs, foundationally supporting marine biodiversity\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, and global ocean change (i.e., warming and acidification)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e could threaten this process in corals\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. The mechanisms for coral calcification are highly complex\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, it is clear that utilizing ion transport combining reactants calcium and DIC in concert with H\u003csup\u003e+\u003c/sup\u003e diffusion must co-occur to precipitate CaCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e73\u003c/sup\u003e. Ocean acidification will chemically alter [DIC] and [H\u003csup\u003e+\u003c/sup\u003e] while [Ca\u003csup\u003e2+\u003c/sup\u003e] remains unaffected\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This study shows that \u003cem\u003eM. capitata\u003c/em\u003e host and symbiont interactions functionally regulated calcium under chemically stressful seawater conditions. It is unclear how rising OA and the disruption of the chemical environment drive lower calcification in some corals but not others\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. This work proposes that functional calcium regulation plays a partial role in tolerance in \u003cem\u003eM. capitata\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe information presented here is important for initiatives exploring the physiological resilience of corals to future environmental change using predictive bioindicators\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Climate stress mitigation tactics implementing assisted evolution have been proposed as an intervention strategy conceptually based on physiological resilience. However, assisted evolution hosts diverse costs and benefits\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e, and some argue that the practicality of impactful restoration efforts is low\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Nevertheless, despite the limitations of human intervention strategies, understanding the adaptive capacity and transgenerational plasticity of corals is essential for conservation\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Studies like the one here will benefit future efforts by developing a deeper understanding of the underlying biological processes involved in physiological resilience to environmental change. The future of coral reefs will depend on the culmination of adaptation through generations to interacting locally and globally derived stressors. This study did not introduce any interacting environmental stressors (i.e., thermal, sediment, and pollution) that corals may experience. Future work should investigate the introduction of interacting stressors, but it is suggested from the results to implement targeted approaches assessing calcium homeostasis (i.e., proteomics, confocal or RAMAN spectroscopy, and single-cell RNA analysis) when addressing impacts on calcification.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eCoral Collection and Mesocosm Environment\u003c/h2\u003e\u003cp\u003eColonies of \u003cem\u003eM. capitata\u003c/em\u003e were collected approximately 600 m from the shore of the Hawaiʻi Institute of Marine Biology in Kāneʻohe Bay, Hawaiʻi (21° 26’ 43” N, 157° 47’ 46” W; SAP 2022–2023 and 2023–2024). The coral colonies were acclimated to a flow-through seawater mesocosm environment (an area of 1.37 m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and SW volume ~ 50 m\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) for seven days. Mesocosms each contained 20 colonies of \u003cem\u003eM. capitata\u003c/em\u003e and 20 colonies of \u003cem\u003ePocillopora acuta.\u003c/em\u003e Treatments were set up by row, and there were four mesocosm rows, each with three tanks per row. Mesocosms received seawater directly from the bay, \u0026lt; 1km from the collection site, with a tank residence time of one hour, and were environmentally controlled—exposed to natural light (shaded to 50% - PAR 800 µmol s\u003csup\u003e− 1\u003c/sup\u003e).\u003c/p\u003e\u003ch2\u003eSeawater Chemistry\u003c/h2\u003e\u003cp\u003eThe seawater chemistry manipulation techniques and physical mesocosm environments were identical between seasons. Mesocosm row header mixing chambers received additions of 1M HCl in the Tmt 1 treatment and were monitored by adjusting seawater flow rates or drip speed to achieve a Δ TA of ~ 100 µmol compared to the control (Table S2). In the Tmt 2 treatment, an air + CO\u003csub\u003e2\u003c/sub\u003e mixture was bubbled directly to each mesocosm and adjusted rates based on the pH\u003csub\u003eSTAT\u003c/sub\u003e methodology to a Δ pH of ~ 0.30 compared to the control\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Then, a combined Tmt 3 treatment was implemented via both methods to the third row of mesocosms (Table S2). The control mesocosm seawater chemistry was not manipulated, but all other aspects were identical to the treatment conditions. Other seawater parameters were environmentally controlled and not manipulated. Carbonate chemistry parameters (i.e., [DIC], pCO\u003csub\u003e2\u003c/sub\u003e, and Ω\u003csub\u003earag\u003c/sub\u003e) were calculated utilizing the R package ‘\u003cem\u003eseacarb\u003c/em\u003e’, inputting direct measures of temperature, salinity, pH\u003csub\u003eT\u003c/sub\u003e, and TA (Table S2).\u003c/p\u003e\u003ch2\u003eCalcification Rate Calculation\u003c/h2\u003e\u003cp\u003eCoral colonies were weighed in seawater at the beginning of each experiment and at the end of the 31-day exposure period to determine calcification in the exposure. This study followed the buoyant weight technique\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e procedures, along with Eq.\u0026nbsp;2, to determine the rate of CaCO\u003csub\u003e3\u003c/sub\u003e precipitation in the coral colonies.\u003c/p\u003e\u003cp\u003eG = 1.54 × (\u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e – \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e)/ \u003cem\u003ed\u003c/em\u003e (2)\u003c/p\u003e\u003cp\u003eEquation 2 calculates G, or the calcification rate (CaCO\u003csub\u003e3\u003c/sub\u003e g d\u003csup\u003e− 1\u003c/sup\u003e), by the final buoyant weight (\u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e) subtracted from the initial buoyant weight (\u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e) over time in days (\u003cem\u003ed\u003c/em\u003e), all multiplied by the seawater and aragonite density constant (1.54) to achieve the dry weight of aragonite\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eRNA-seq Subsampling\u003c/h2\u003e\u003cp\u003eCoral colonies of \u003cem\u003eM. capitata\u003c/em\u003e were subsampled on the 31st day of exposure from 12:00 to 14:00 for both summer and winter seasons. Colonies were transported in seawater to the lab sampling space (\u0026lt; 10 m from mesocosms). The lab bench was continuously sterilized with a 1 M NaOH distilled water solution before and between subsampling. Five sterilized (autoclaved at 121°C for 30 minutes) 1.5 mL Eppendorf tubes filled with 1 mL of RNA Later for each coral colony. Medical grade bone cutters sterilized by dipping (sequentially) in seawater, deionized water, 10% bleach, 1 M NaOH, and finally Ethanol, which was burned off, were used to subsample coral colonies. We sterilized bone cutters via this sequential dipping process between each coral colony but only dipped in the colony’s respective seawater when taking multiple samples from the same individual. We took quintuplicate samples per individual and focused localization only at the tip of branching colonies covering \u0026lt; 1 cm down from the tip. Fragments were placed directly in the RNA Later Eppendorf tubes, capped, and snap-frozen in liquid nitrogen for five seconds. The samples were then chilled on ice for two hours and placed in a -80°C freezer until shipment for sequencing.\u003c/p\u003e\u003ch2\u003eLibrary Prep and RNA-seq\u003c/h2\u003e\u003cp\u003eRaw tissue samples were sent directly to the AZENTA Life Sciences labs, where they conducted extraction, library prep, and sequencing following their protocols in the ‘Standard RNA-seq bundle with PolyA selection’. Samples were sequenced at 50\u0026nbsp;million paired-end reads per sample utilizing the Illumina, 2x150bp sequencing configuration. This generated ~ 1.4\u0026nbsp;billion reads over 24 samples, with a mean quality score of 38.92 and 94.64% bases ≥ 30.\u003c/p\u003e\u003ch2\u003eBioinformatic Pipeline\u003c/h2\u003e\u003cp\u003eThe bioinformatics pipeline used here was adopted from Chille et al.\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, and Radice et al.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Raw Fastq files were quality checked utilizing \u003cem\u003eFastqc\u003c/em\u003e v0.11.9 and \u003cem\u003eMultiqc\u003c/em\u003e v1.25.1 platforms. Raw reads were trimmed using \u003cem\u003eFastp\u003c/em\u003e v0.23.4, focused on removing the overrepresented sequence and cleaned reads were confirmed via a second \u003cem\u003eMultiqc\u003c/em\u003e report. The most current \u003cem\u003eM. capitata\u003c/em\u003e reference genome \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e was indexed (\u003cem\u003ehisat2-build\u003c/em\u003e), and cleaned reads were aligned using \u003cem\u003eHISAT2\u003c/em\u003e v2.2.1 (--\u003cem\u003edta\u003c/em\u003e). No individual aligned greater than 60% for both summer and winter to the reference genome, and thus, we chose to conduct a \u003cem\u003ede novo\u003c/em\u003e transcriptome assembly using \u003cem\u003eTrinity\u003c/em\u003e v2.15.2 (--\u003cem\u003enormalize_max_read_cov\u003c/em\u003e 200). The longest isoforms were extracted from the assembly via a Python v3.9 script (Biopython v1.79) with a minimum threshold of ≥ 500 bp. The assembly was compared via BLASTn to the SILVA rRNA LSU and SSU NR99 database (downloaded January 2, 2025), and matches at a minimum of 1e-5, 78% at 100bp (-\u003cem\u003eqcov_hsp_perc\u003c/em\u003e 100) were removed. The \u003cem\u003ede novo\u003c/em\u003e assembly was compared via BLASTn (1e-5, 70% identity at 100bp) to two databases, cnidarian and Symbiodiniaceae (NCBI Nucleotide Database). The assemblies were separated into host and symbiont-specific assemblies, resulting in a final host assembly with 34,048 contigs and a symbiont assembly with 759 contigs. Assemblies were annotated via BLASTx, 1e-4 (-\u003cem\u003emax_target_seqs\u003c/em\u003e 10) using NCBI (nr protein; downloaded January 10, 2025) and hierarchically uniprot sprot (downloaded January 11, 2025) along with uniprot TrEMBL (downloaded January 11, 2025) databases. All contigs were matched to the NCBI nr db, then all contigs were again matched with the well-curated sprot db. Contigs that did not match with the sprot db were then matched with the TrEMBL db.\u003c/p\u003e\u003cp\u003eDue to the resulting small size, the symbiont de novo assembly was not used. Read alignment was conducted for the host \u003cem\u003ede novo\u003c/em\u003e assembly using \u003cem\u003eBowtie2\u003c/em\u003e v2.5.4 (--\u003cem\u003elocal\u003c/em\u003e), however, the symbiont assembly was created aligning with a reference \u003cem\u003eCladocopium goreaui\u003c/em\u003e genome. Finally, RNA quantification per sample was conducted using \u003cem\u003eSalmon\u003c/em\u003e v1.10.3 (-l A, --\u003cem\u003evalidateMappings\u003c/em\u003e). All differential gene expression analysis and plotting were conducted in R Studio\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. The package \u003cem\u003eEBSeq\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e,\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e was used to identify significant differentially expressed genes (DEGs) in the samples (maxround = 50 host and 5 symbiont, AllParti = Table S1, pp \u0026gt; 0.95). The biological patterns used were the smallest number of relationships without changing the number of DEGs and remaining relevant to hypothesis testing (Table S1). The null model (H\u003csub\u003eo\u003c/sub\u003e) assigned DEGs pp \u0026lt; 0.95 were removed, and analysis proceeded forward with DEGs assigned to H\u003csub\u003e1\u003c/sub\u003e-H\u003csub\u003e5\u003c/sub\u003e. Annotated filtered DEGs with only Uniprot protein accession numbers were uploaded to the web program DAIVD\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e for functional characterization. Functional annotations indicating a relationship to ion regulatory mechanisms and cellular properties were extracted from the top clusters, which were then traced to DEGs.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eRaw sequence reads can be found at NCBI under Bioproject PRJNA1307607 (uploaded following review). This study used data from NCBI and UniProt databases. Code used here is available at github.com/CROH-Lab/Mcap-Carb-Chem-RNA-Seq (will be uploaded following review).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGraham NAJ, Nash KL (2013) The importance of structural complexity in coral reef ecosystems. Coral Reefs 32:315\u0026ndash;326\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoegh-Guldberg O, Poloczanska ES, Skirving W, Dove S (2017) Coral Reef Ecosystems under Climate Change and Ocean Acidification. Front Mar Sci 4\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Horani FA (2015) Physiology of Skeletogenesis in Scleractinian Coral. in \u003cem\u003eDiseases of Coral\u003c/em\u003e (eds. Woodley, C. M., Downs, C. A., Bruckner, A. W., Porter, J. W. \u0026amp; Galloway, S. 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Bioinformatics 29:1035\u0026ndash;1043\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":false,"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":"Aragonite saturation state (Ω), RNA-seq, carbonate chemistry, ocean acidification, gene expression","lastPublishedDoi":"10.21203/rs.3.rs-7402868/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7402868/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCoral calcification is a highly complex process with numerous caveats regarding the mechanisms that dictate productivity and function. Ion homeostasis, however, is the foremost physiological process unanimously shared among Scleractinia and essential for calcification. Consequently, changes to the seawater environment may elicit adverse effects on ion homeostasis. With increasing climate shifts, the physicochemical regime of our global ocean is changing rapidly. Responses of coral calcification to physicochemical change prevail in having little uniformity on an unambiguous mechanism of resistance. Therefore, this study chose a relatively tolerant Hawaiian coral, \u003cem\u003eMontipora capitata\u003c/em\u003e to focus efforts on understanding ion homeostasis under chemical seawater manipulation designed to limit calcification. Results indicate a physiological hormesis (two-phase adaptive response) of overall coral host gene expression that was not shared with algal symbionts and decoupled from calcification rates. The sole ion homeostatic mechanism shown was calcium ion regulation by both the host and symbiont cells. Calcium ion homeostasis was also found to be mechanistically different between winter and summer seasons. Thus, potentially indicating complex interactions between host and symbiont cells, as well as the ability for \u003cem\u003eM. capitata\u003c/em\u003e to promote calcification under stress. Putatively synthesized here are the physiological cascades and mechanisms of resistance to environmental triggers of acidosis and seasonal change. This work provides insight into linking calcium ion homeostasis with coral resistance and aims to suggest this mechanism as biomolecular indicator used in future assessments to compare tolerance.\u003c/p\u003e","manuscriptTitle":"Coral calcification resistance to acidification is physiologically linked with complex intracellular calcium ion dynamics between host and symbiont cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 19:41:46","doi":"10.21203/rs.3.rs-7402868/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":"cbc07f19-fdd4-4ae1-9e0e-d96cfad68311","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54021000,"name":"Biological sciences/Molecular biology/Transcriptomics"},{"id":54021001,"name":"Biological sciences/Physiology"},{"id":54021002,"name":"Earth and environmental sciences/Ocean sciences/Marine biology"}],"tags":[],"updatedAt":"2025-09-25T19:41:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 19:41:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7402868","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7402868","identity":"rs-7402868","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}