Combined Effects of Low pH and High Water Temperature Conditions on Oxidative Stress and Cell Damage in Juvenile Olive Flounder Paralichthys Olivaceus

Combined Effects of Low pH and High Water Temperature Conditions on Oxidative Stress and Cell Damage in Juvenile Olive Flounder Paralichthys Olivaceus | 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 Research Article Combined Effects of Low pH and High Water Temperature Conditions on Oxidative Stress and Cell Damage in Juvenile Olive Flounder Paralichthys Olivaceus Dae-Won Lee, Jin Ah Song, Heung Sik Park, Cheol Young Choi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-902059/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract The use of fossil fuels by anthropogenic activities causes ocean acidification and warming, and the combined conditions of these two environments can negatively affect fish metabolism, growth, and survival. Additionally, it can affect the antioxidant enzyme activity and cell damage caused by lipid peroxidation. In this study, we explores the adaptive potential for future marine conditions by investigating the stress and antioxidant enzyme activity, and apoptosis of juvenile olive flounder in an environment where ocean acidification and warming coexist. We found that juvenile olive flounder had increased oxidative stress and apoptosis under both warming and combined warming, and acidification conditions. Additionally, the effect on acidification under warming conditions does not seem to exceed the effect of high temperature conditions, and it is considered that the effect of acidification at low water temperatures is greater. General Biochemistry Molecular Genetics Molecular Biology abiotic stress cell damage high water temperature low pH olive flounder Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The use of fossil fuels for anthropogenic and industrial activities is accompanied by the emission of carbon dioxide (CO 2 ) into the atmosphere. Additionally, the emitted CO 2 plays a role in raising the surface water temperature of the ocean along with causing acidification that lowers the pH of the ocean (IPCC, 2014 ). Therefore, if the emission of CO 2 continues with the current trend without efforts to reduce its emissions, then the surface water temperature of the ocean will rise by about 4°C within 100 years and the pH will decrease by 0.3–0.4 units from the current level (about pH 8.1). It is predicted that the pH of the ocean will decrease by 0.7–0.8 units within the next 300 years (Caldeira and Wickett, 2003 ; IPCC, 2014 ). Based on such predictions, it is believed that ocean warming and acidification will inevitably occur simultaneously. Therefore, conducting ocean acidification related research is necessary to reveal aspects of the complex effects of at least two environmental change factors (pH and water temperature) on marine life, rather than just one environmental factor. Changes in ocean pH concentration and water temperature are known to negatively affect the survival, growth, morphology, and behaviour of marine organisms (Kroeker et al., 2013 ; Little et al., 2020 ). These environmental stressors negatively affect various physiological mechanisms, which include neuronal signal transduction and osmoregulation, cellular homeostasis, and oxidative stress in marine organisms (Heuer and Grosell, 2014 ). Meanwhile, stress induces redox reactions in the body and the production of reactive oxygen species (ROS), which cause oxidative stress in cells and tissues. With the production of ROS, cell damage and an increase in antioxidant enzyme activity are usually induced by lipid peroxidation. At this stage, one of ROS and highly reactive superoxide radical (O 2 − ) is converted into hydrogen peroxide (H 2 O 2 ) by superoxide dismutase (SOD) and the action of antioxidant enzymes to remove oxidative stress. H 2 O 2 converted by SOD is finally converted by catalase (CAT) into non-toxic water and oxygen, thereby, preventing O 2 − from accumulating in cells and tissues (Lesser, 2006 ). Generally, fish are sensitive to changes in water temperature, and hence, a high-temperature environment induces oxidative stress and increases apoptosis in fish (Kim et al., 2016 ). However, when the antioxidant response due to the change of water temperature reaches its peak, there are cases where the antioxidant enzyme is inactivated due to protein denaturation and decomposition in the body (Madeira et al., 2013 ). Heat shock protein 70 (HSP70) plays an important role in cellular thermotolerance, apoptosis, and heat shock stress. Additionally, it can be used as an ideal biomarker to confirm the effect of environmental stress because it is induced when cells are exposed to noxious stimuli (Kregel, 2002 ). Cortisol is also a major stress hormone and is used as an indicator of environmental stress caused by pollution or climate change (Sadoul and Geffroy, 2019 ). It is reported that along with the single effect of ocean acidification (pH decrease) on fish, the combined effect of ocean warming (high water temperature) and acidification environment can also negatively affect fish survival (Rosa et al., 2016 and Araújo et al., 2018 ). The complex environment of combined ocean warming and acidification negatively affects fish metabolism, heat tolerance, growth, skeletal development, and survival (Pimentel et al., 2014 ). Additionally, it is known to negatively affect the activity of antioxidant enzymes and cause cell damage by lipid peroxidation (Pimentel et al., 2015 ). The aforementioned complex changes in the ocean environment can change the surrounding food ecosystems due to habitat changes (vertical migration) of marine upper predators (jumbo squid, Dosidicus gigas ) (Rosa and Seibel, 2008 ). Additionally, it is reported that in the case of Atlantic cod, a major commercial fish, it can affect fishery production due to habitat movement (Hänsel et al., 2020 ). As ocean warming and acidification by CO 2 emitted into the atmosphere are simultaneous phenomena, it is necessary to investigate the physiological responses of fish through the combined effects of these environments to accurately understand their impact on marine species. Therefore, in this study, the stress response through changes in cortisol and HSP70 and the antioxidant response through changes in SOD and CAT were investigated in a complex environment in which ocean warming (increase in water temperature) and acidification (reduction in pH) coexisted. Additionally, the level of apoptosis was investigated through caspase 3 (CASP3) activity and terminal transferase dUTP nick end labelling (TUNEL) assay. Through the above analyses, we attempted to understand the adaptation potential of olive flounder to future ocean conditions, which has high industrial value in Korea, China, and Japan. 2. Materials And Methods 2.1. Animals and experimental condition Olive flounder Paralichthys olivaceus (length 17.3 ± 0.9 cm; weight 40.1 ± 5.8 g) were purchased from a commercial hatchery and acclimated for 4 weeks in six 350-L recirculating filtration tank system, consisting of four mini tanks (40 cm × 40 cm × 40 cm, 60 L) and a sump tank. Sufficient feed was provided to all fish twice a day, both during the experiment and acclimation period. In the environment setting for the experiment, warming conditions were set to 20 (control), 25, and 30°C, at pH 8.1 and combined warming and acidification conditions (CWA) were set to 20, 25 and 30°C, at pH 7.5. All environmental factors were measured daily twice using a pH meter (WM-32EP, DKK-TOA Corporation, Tokyo, Japan) for pH and temperature and using a Pro2030 (YSI Inc., USA) for salinity. Photoperiod conditions were set to 12 h light and 12 h dark. Fish were anesthetized with 2-phenoxyethanol at 200 ppm for 3 min, and plasma was collected using a heparin-coated syringe. Collected plasma was centrifuged at 1500 × g , 4°C for 20 min and immediately stored at ˗80°C until subsequent analysis. The liver tissues were collected from the fish, and stored at -80°C until the analysis of immunohistochemistry (IHC). 2.2. Seawater CO 2 manipulation Seawater pH was adjusted using a pH-controller (Tunze smart controller 7000, Aquarientechnik GmbH, Germany). pH sensors and ceramic CO 2 diffusers were installed in sump tanks to achieve the target pH level 7.5 by the pH-controller. The pH sensor was calibrated using an NBS (National Bureau of Standards) buffer. The p CO 2 was checked three times in a day using a pump-aspirated sampling-type CO 2 meter (GM70, Vaisala, Finland). Seawater carbonate chemistry was calculated by CO2SYS using the constants of Dickson and Millero (Lewis and Wallace, 1998 ), as described in Table 1 . Table 1 Carbonate chemistry at each condition of seawater Desired condition Salinity (psu) Temperature (°C) pH NBS p CO2 (µatm) A T (µmol kg − 1 ) pH 8.1 20℃ 34.51 ± 0.05 20.2 ± 0.1 8.08 ± 0.01 446 ± 4 1779 ± 41 25℃ 34.47 ± 0.04 25.2 ± 0.1 8.08 ± 0.01 441 ± 3 1905 ± 43 30℃ 34.43 ± 0.04 30.1 ± 0.1 8.11 ± 0.01 454 ± 3 2090 ± 55 pH 7.5 20℃ 34.47 ± 0.10 20.2 ± 0.1 7.48 ± 0.02 1443 ± 38 1390 ± 45 25℃ 34.41 ± 0.06 25.1 ± 0.1 7.51 ± 0.02 1363 ± 31 1342 ± 34 30℃ 34.43 ± 0.07 29.9 ± 0.1 7.47 ± 0.01 1481 ± 38 1307 ± 27 Salinity, Temperature, pH NBS , p CO 2 were measured by equipment directly and total alkalinity (A T ) was calculated in CO 2 SYS (Lewis and Wallace, 1998 ). All data values represent mean ± standard deviation. 2.3. Analysis of plasma parameters Cortisol, SOD, CAT, and CASP3 were analyzed from plasma samples using an enzyme-linked immunosorbent assay (ELISA, #MBS704055 for cortisol, #MBS 705758 for SOD, #MBS038818 for CAT, and #MBS012786 for CASP3; MyBioSource Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Absorbance was measured using a microplate reader (Spark, TECAN Trading AG, Switzerland), and the concentration was calculated by plotting a standard curve. 2.4. IHC IHC was performed using hsp70 antibody (#OASE00332, AVIVA) to assess the heat stress response of fish liver cells to CO 2 and high water temperature exposure. The analysis procedure is briefly described as follows: First, deparaffin was treated with fresh xylene 3 times for 7 min. Then rehydration was sequentially performed in ethanol of 100% (2 times in 3 min), 95% (2 times in 3 min), 80% (1 time in 3 min), and 70% (1 time in 3 min). This was followed by treating the samples in 3% H 2 O 2 and performing antigen retrieval for 15 min each, respectively. After treatment in 4% bovine serum albumin for 30 min, the primary and secondary antibodies were treated for 60 min and 30 min, respectively, and after 7 min of treatment with 3,3´- diaminobenzidine at 24°C, the samples were treated with Mayer’s hematoxylin at 24°C for 3 min. Finally, after dehydration, mounting was performed. Sides were observed under a microscope (Axio Imager A2, Germany). 2.5. TUNEL assay TUNEL analysis was performed using commercially available ApopTag peroxidase In Situ Apoptosis Detection Kit (catalogue number, S7100, Chemicon, Germany) as per the manufacturer’s instructions to assess the apoptosis response of fish liver cells to elevated CO 2 and low-salinity seawater exposure. Tissue section was washed with xylene and ethanol, and fresh diluted proteinase K was applied to the pre-treated tissue specimen for 15 min at room temperature. Then equilibration buffer and working strength terminal deoxynucleotidyl transferase enzyme were applied. This was followed by applying anti-digoxigenen conjugate and incubating in a humidified chamber for 30 min at room temperature. At the end of this process, it was washed with phosphate buffered saline. To determine the optimal staining duration, the slides were observed under a microscope and the color development in peroxidase substrate was monitored. The specimens were mounted silanized glass slides and observed under a microscope (Axio Imager A2, Germany). The brown cells indicated apoptosis. 2.6. Statistical analysis All the data were analysed using the SPSS software (version 27.0; IBM, USA). The independent samples t-test was used to determine the difference between pH levels under the same temperature and exposure days. A one-way analysis of variance (ANOVA) test followed by Tukey’s post-hoc test was used to compare the differences between temperatures on the same exposure day. Two-way ANOVA test was employed to determine the interaction effects of ocean acidification and warming as described in Table 2 . The values are expressed as means ± standard deviation, and P -values lower than 0.05 were considered statistically significant. Table 2 Interaction effects of water temperature and pH level. Cortisol SOD CAT CASP3 D0 F 0.000 0.000 0.000 0.000 P 1.000 1.000 1.000 1.000 D3 F 1.851 1.649 2.662 9.959 P 0.179 0.213 0.090 0.001* D7 F 7.236 7.061 18.666 4.747 P 0.003* 0.004* < 0.001* 0.018* D14 F 26.575 28.389 2.425 19.046 P < 0.001* < 0.001* 0.110 < 0.001* D21 F 8.511 3.764 6.780 17.454 P 0.002* 0.038* 0.005* < 0.001* D28 F 1.069 0.423 11.134 5.646 P 0.395 0.660 < 0.001* 0.010* The symbol “*” indicates a significant difference. 3. Results 3.1. Changes in the activity of plasma parameters 3.1.1. Cortisol Cortisol showed higher activity as the water temperature increased until the 7th day of the experiment in both warming (25, 30°C) and CWA conditions (Fig. 1 , P < 0.05). Additionally, the overall cortisol activity was significantly higher in the CWA condition than in the warming condition. However, on the 14th day, cortisol activity was higher as the water temperature increased (25°C and 30°C) under the warming condition (Fig. 1 , P < 0.05). Furthermore, higher cortisol activity was observed at lower water temperature (20°C) under the CWA condition. On days 21 and 28, the higher the water temperature, the higher the cortisol activity in both warming and CWA conditions was observed (Fig. 1 , P < 0.05). Except for the 28th day at 20°C, all cortisol activities were higher under the acidifying condition than under the warming condition (Fig. 1 , P < 0.05). In the cortisol activity, the effect of the interaction of acidification with warming was found to have significant differences at the 7, 14, and 21 days, respectively (Table 2 ). 3.1.2. SOD Overall, SOD showed higher activity as the water temperature increased under the warming conditions. SOD activity in the CWA condition was significantly lower compared to that in the warming condition at 25°C on the 3rd day (Fig. 2 , P < 0.05). On the 7th and 14th day, the activity of SOD at 20°C was significantly higher in the CWA condition than in the warming condition, and it showed a high activity similar to that at 25°C (Fig. 2 , P < 0.05). In the SOD activity, the effect of the interaction of acidification with warming was found to have significant differences at the 7th, 14th, and 21st day (Table 2 ). 3.1.3. CAT CAT activity was significantly higher as the overall water temperature increased in both the warming and the CWA conditions. However, the CAT activity showed a low activity similar to that at 20°C and 30°C on the 7th day of CWA conditions (Fig. 3 , P < 0.05). CAT activity showed significantly higher activity (30°C on day 14, all water temperature conditions on day 21, 20°C and 30°C on day 28) compared to the warming condition even after the 7th day (Fig. 3 , P < 0.05). The effect of the interaction of acidification with warming in CAT activity was found to have a significant difference on the 7th, 21st, and 28th days (Table 2 ). 3.1.4. CASP3 CASP3 showed significantly higher activity as the water temperature increased until the 7th day of the experiment under the warming conditions. In the CWA condition, high CASP3 activity was observed at 20°C and 25°C on the 3rd and 7th day, respectively, and in particular, the highest activity was observed in the CWA condition on the 3rd day (Fig. 4 , P < 0.05). At 20°C, CASP3 activity was significantly higher in the CWA than in the warming condition, except for the 28th day (Fig. 4 , P < 0.05). The effect of the interaction of acidification with warming in CASP3 activity was found to have a significant difference for all experimental period (Table 2 ). 3.2. IHC of HSP70 in liver tissue As a result of investigating IHC in liver tissue of olive flounder juvenile on days 14 and 28 under warming and CWA conditions, it was found that the higher the water temperature, the more active the HSP70 antibody response in the liver tissue on days 14 and 28 (Fig. 5 ). However, on the 14th day, an active HSP70 antibody response similar to the warming and CWA conditions were observed in the acidification condition at 20°C (Fig. 5 ). 3.3. TUNEL assay From investigating the presence of apoptotic cells in the liver tissue of olive flounder juvenile on days 14 and 28 under warming and CWA conditions using the TUNEL assay, in both warming and CWA conditions, the higher the water temperature, the higher the frequency of apoptotic cells appeared in the liver tissue on both the 14th and 28th days (Fig. 6 , B). However, on the 14th day, a high frequency of apoptotic cells similar to the warming and CWA conditions were observed in the acidification condition at 20°C (Fig. 6 , B). 4. Discussion Excessive water temperature rise increases the energy consumption of the fish and thus requires additional energy supply, thereby increasing the cost of growth in fish (Little et al., 2020 ). The formation of ROS and the increase in metabolic demand under the rise of CO 2 in seawater as well as water temperature generally lead to damage caused by peroxidation and increased activity of antioxidant enzymes, and additional energy consumption is unavoidable (Rosa et al. 2016 ). Contrarily, in the case of a species with high adaptability (resilient) to environmental stressors, it also prevents cell damage (lipid peroxidation) by upregulating the action of antioxidant enzymes to cope with oxidative stress caused by warming (Sampaio et al. 2018 ). The complex environment of ocean acidification and warming generates ROS and antioxidant enzymes act to remove ROS, but excessive production of ROS exceeds the action capacity of antioxidant enzymes, so it can have a negative effect on fish. Araújo et al. ( 2018 ) found that the effects of ocean acidification and the combined effects of two complex environments (ocean warming and acidification) have negative synergistic effects such as increased energy demand, anaerobic metabolism, and impaired proteolysis in sea bream Sparus aurata . It is reported to induce cytotoxic effects and pose a serious threat to sea bream populations. Rosa et al. ( 2016 ) reported that when juvenile bamboo sharks Chiloscyllium punctatum were exposed to a combined environment of ocean warming and acidification, antioxidant enzymes detoxified the ROS. However, upregulation of these antioxidant enzymes was insufficient to minimize the increase in cholinergic neurotransmitters caused by peroxidative damage and stress response in the brain. In our study, it was found that under the warming conditions (higher water temperature), the stress response and the activity of antioxidant enzymes were high, and the appearance of apoptosis was also high. This findings are consistent with that of a similar study by Madeira et al. ( 2016 ) who reported that excessive oxidative stress induced in the muscles, liver, and brain of Gilt-head bream S parus aurata at high water temperature (30°C) increased the activity of antioxidant enzymes (SOD, CAT, GST) to counteract the effect of increasing ROS. Additionally, it was similar to the results of Kim et al. ( 2016 ) who reported that a high water temperature environment increased the antioxidant enzyme activity of olive flounder and induced apoptosis in liver tissue by increasing the activity of CASP3. Additionally, in our study, the stress response, the antioxidant enzyme activity, and the appearance of apoptosis were found to be high in the combined warming (high water temperature) and acidification (pH decrease) conditions, similar to the overall warming conditions. This result was confirmed in a study by Rosa et al. ( 2016 ) and Araújo et al. ( 2018 ) who state that the combined effect of the two environments appears to act negatively to induce cytotoxicity, which results in increased emergence of apoptosis. Specifically, this implies that the antioxidant enzyme to remove ROS acted not just in a high temperature environment but also in a complex environment with warming (high water temperature) and acidification (pH decrease), although the appearance of apoptosis was not inhibited because the action capacity of the antioxidant enzyme exceeded due to a large amount of ROS. The activity of CAT in our study was consistently high under the conditions in which warming and acidification were combined. This is described in Carney Almroth et al. ( 2019 ) who state that Atlantic halibut Hippoglossus maintained a significant correlation of high SOD and CAT activity in a warming (high water temperature) environment but not in a high CO 2 concentration (low pH level) environment. Fish may have increased amounts of hydrogen ions (H + ) in their body to normalize the pH levels around blood cells by increasing HCO 3 − in an environment exposed to CO 2 (Heuer and Grosell, 2014 ). This excess presence of H + in the blood can combine with H 2 O to form H 2 O 2 (Sampaio et al., 2018 ), which is believed to induce a sustained increase in CAT, the key enzyme that generally scavenges H 2 O 2 . The results of this study under the combined environmental conditions of warming and acidification, stress and antioxidant enzyme activity, and apoptosis all tend to be high at 20°C. Grans et al. ( 2014 ) found that warming and acidification conditions increased oxygen demand and cardiac performance in Atlantic halibut Hippoglossus hippoglossus , although these increases were not interpreted as growth improvements. Instead, it was reported that Atlantic halibut was more affected by acidification under lower water temperature conditions than the optimum growth water temperature, and hence the growth decreased. When cells are exposed to oxidative stress, ROS is also generated in the electron transport chain of mitochondria, but considering the generation of ROS due to increased respiration in a high-temperature environment, in our study, the effects of acidification under the combined warming and acidification conditions do not exceed the effects of warming. Rather, the effect of acidification appears to be greater in low-temperature than in high-temperature conditions. Summarizing the results of our study on juvenile olive flounder, 1) Oxidative stress and apoptosis tend to increase under both the warming (high water temperature), combined warming (high water temperature), and acidification (pH decrease) conditions. 2) Considering the increase in ROS due to oxidative stress as well as the increase in ROS generation due to increased respiration in the high water temperature condition, the effect on acidification in the combined warming and acidification condition does not seem to exceed the effect of warming. 3) It is implied that the effect on acidification is greater at a relatively low water temperature (20°C) than at a high temperature condition. Excessive induction of oxidative stress due to warming and acidification may lead to a decrease in the growth of fish, induce disease due to reduced immunity, and shorten their lifespan. Therefore, understanding the physiological responses of marine organisms in combination with the prediction of future changes in the marine environment is important for predicting the direct impact of climate change on marine organisms and their vulnerability. The data thus obtained can also be very important for predicting the sustainability of the fishery and aquaculture industries. Additionally, in the mid-latitude regions where Korea is located, seawater temperature fluctuates greatly in many cases due to seasonal changes. Therefore, we suggest that additional research on oxidative stress and antioxidant responses of marine organisms is needed in an environment in which low water temperature and acidification conditions coexist in winter, which cannot guarantee sufficient nutritional status due to poor food intake. Abbreviations ANOVA,analysis of variance;CASP3, caspase 3; CAT, catalase; CO 2 , carbon dioxide; CWA, combined warming and acidification conditions; H 2 O 2 , hydrogen peroxide; HSP70, heat shock protein 70; IHC, immunohistochemistry; NBS, National Bureau of Standards; O 2 - ,superoxide radical; ROS, reactive oxygen species; SOD,superoxide dismutase; TUNEL,Terminal transferase dUTP nick end labelling. Declarations - Funding This work was supported by the Ministry of Oceans and Fisheries, Korea [PE99922]. - Conflicts of interest/Competing interests The authors declare that they have no conflict of interests. - Availability of data and material The datasets and materials used during the current study are available from the corresponding author on reasonable request. - Code availability Not applicable. - Authors' contributions Dae-Won Lee: Data curation, Writing - original draft. Jin Ah Song: Data curation, Writing - editing Heung-Sik Park: Funding acquisition,Data curation, Formal analysis, Review & editing. Cheol Young Choi: Conceptualization, Project administration, Writing - review & editing. - Ethics approval All experiments were conducted in accordance with the guidelines and ethical principles of the Institutional Animal Care and Use Committee operation established by ministry of agriculture, food and rural affairs of Korea. - Consent to participate Not applicable. - Consent for publication Not applicable. References Araújo, J.E., Madeira, D., Vitorino, R., Repolho, T., Rosa, R., Diniz, M., 2018. Negative synergistic impacts of ocean warming and acidification on the survival and proteome of the commercial sea bream, Sparus aurata . J. Sea. Res. 139, 50-61. Caldeira, K., Wickett, M.E., 2003. Oceanography: anthropogenic carbon and ocean pH. Nat. 425, 365. Carney Almroth, B., Bresolin De Souza, K., Jönsson, E., Sturve, J., 2019. 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Sampaio, E., Lopes, A.R., Francisco, S., Paula, J.R., Pimentel, M., Maulvault, A.L., Repolho, T., Grilo, T.F., Pousao-Ferreira, P., Marques, A., Rosa, R., 2018. Ocean acidification dampens physiological stress response to warming and contamination in a commercially-important fish ( Argyrosomus regius ). Sci. Total Environ, 618, 388-398. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 27 Sep, 2021 Editor invited by journal 19 Sep, 2021 Editor assigned by journal 14 Sep, 2021 First submitted to journal 13 Sep, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-902059","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":54716135,"identity":"688c4634-f50c-494e-8ef8-8806fc032e5e","order_by":0,"name":"Dae-Won Lee","email":"","orcid":"","institution":"Korea Institute of Ocean Science \u0026 Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dae-Won","middleName":"","lastName":"Lee","suffix":""},{"id":54716136,"identity":"a220e3dd-0b77-4111-a046-307c911e05b7","order_by":1,"name":"Jin Ah Song","email":"","orcid":"","institution":"Korea Maritime and Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jin","middleName":"Ah","lastName":"Song","suffix":""},{"id":54716137,"identity":"628d85f7-e7db-48c5-a644-a8edd15c6c6b","order_by":2,"name":"Heung Sik Park","email":"","orcid":"","institution":"Korea Institute of Ocean Science \u0026 Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Heung","middleName":"Sik","lastName":"Park","suffix":""},{"id":54716138,"identity":"3bf724cb-2c72-4bea-bc57-e81a64c66aa5","order_by":3,"name":"Cheol Young Choi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYDACCQaGAwwVBxIgvANEazlDqhYGxjZStPDPbj544Oe8O3kGB5gffmA4c48IS+4cSzjYu+1ZscEBNmMJhhvFhLUYSOQYHGbcdjhxwwEGMwaGDwnEapkD0sL+jRQtDSAtPEBbbhChReJGWsLBnmOHE2ce5imWSDhDhBb+GcmHP/yoOZzYd7x944cPx4jQggDMQEyShlEwCkbBKBgFuAEA+glCvsc7mpkAAAAASUVORK5CYII=","orcid":"","institution":"Korea Maritime University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Cheol","middleName":"Young","lastName":"Choi","suffix":""}],"badges":[],"createdAt":"2021-09-13 20:12:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-902059/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-902059/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":14206239,"identity":"f78103da-e922-461c-952c-c4239661cb1b","added_by":"auto","created_at":"2021-10-01 21:37:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4026665,"visible":true,"origin":"","legend":"Bar plots depicting the comparison of cortisol activity in the plasma of olive flounder exposed to elevated CO2 and water temperature treatment. The letters indicate a significant difference according to the water temperature variation at the same day interval (P \u003c 0.05). The symbol “*” indicates a significant difference between the pH level at the same day interval (P \u003c 0.05). All values are means ± standard deviation (n = 5).","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-902059/v1/ca493da27b4e857ed88f07ef.png"},{"id":14206238,"identity":"14cffd51-8f1f-4d7c-9f0f-dcc1c84058c3","added_by":"auto","created_at":"2021-10-01 21:37:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2988540,"visible":true,"origin":"","legend":"Bar plots showing the comparison of superoxide dismutase (SOD) activity in the plasma of olive flounder exposed to elevated CO2 and water temperature treatment. The letters indicate a significant difference according to the water temperature variation at the same day interval (P \u003c 0.05). The symbol “*” indicates a significant difference between the pH level at the same day interval (P \u003c 0.05). All values are means ± standard deviation (n = 5).","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-902059/v1/9c281fbae2ac3c63d475588e.png"},{"id":14206241,"identity":"44fc2e37-7baa-4230-8f07-2e759b382c3a","added_by":"auto","created_at":"2021-10-01 21:37:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3370551,"visible":true,"origin":"","legend":"Bar plots depicting the comparison of catalase (CAT) activity in the plasma of olive flounder exposed to elevated CO2 and water temperature treatment. The letters indicate a significant difference according to the water temperature variation at the same day interval (P \u003c 0.05). The symbol “*” indicates a significant difference between the pH level at the same day interval (P \u003c 0.05). All values are means ± standard deviation (n = 5).","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-902059/v1/8f5122429f1c4c9b2f61ff88.png"},{"id":14206240,"identity":"7f2c57f1-7d17-42b0-996d-4b0db37aac96","added_by":"auto","created_at":"2021-10-01 21:37:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3290752,"visible":true,"origin":"","legend":"Bar plots depicting the comparison of caspase 3 (CASP3) activity in the plasma of olive flounder exposed to elevated CO2 and water temperature treatment. The letters indicate a significant difference according to the water temperature variation at the same day interval (P \u003c 0.05). The symbol “*” indicates a significant difference between the pH level at the same day interval (P \u003c 0.05). All values are means ± standard deviation (n = 5).","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-902059/v1/dc2ca43a217914ae85c445ba.png"},{"id":14206243,"identity":"c556ae26-f07b-4451-a090-e9a172722842","added_by":"auto","created_at":"2021-10-01 21:37:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":23581529,"visible":true,"origin":"","legend":"Images from immunohistochemistry on the liver tissue of olive flounder exposed to elevated CO2 and water temperature treatment. pH 8.1 (a = control) and pH 7.5 (b) at 20 °C on day 0; pH 8.1 (c-e) and pH 7.5 (f-h) at 20, 25, and 30 °C on day 14; pH 8.1 (i-k) and pH 7.5 (l-n) at 20, 25, and 30 °C on day 28. Scale bars = 50 µm. Brown cells represent immunostained hepatocytes and are indicated by arrows.","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-902059/v1/32f294e51c8ef3b0be31f383.png"},{"id":14206242,"identity":"d2c7d0ab-2693-4c66-ab92-97a1f3acf48b","added_by":"auto","created_at":"2021-10-01 21:37:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13009645,"visible":true,"origin":"","legend":"Images from terminal transferase dUTP nick end labelling assay on the liver tissue of olive flounder exposed to elevated CO2 and water temperature treatment. (A): pH 8.1 (a = control) and pH 7.5 (b) at 20 °C on day 0; pH 8.1 (c-e) and pH 7.5 (f-h) at 20, 25, and 30 °C on day 14; pH 8.1 (i-k) and pH 7.5 (l-n) at 20, 25, and 30 °C on day 28. Scale bars = 50 µm. The brown cells indicate apoptosis. The letters indicate a significant difference according to the water temperature variation at the same day interval (P \u003c 0.05), and the symbol “*” indicates a significant difference between the pH level at the same day interval (P \u003c 0.05) at (B). Bar plots illustrating all values are means ± standard deviation (n = 5). ","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-902059/v1/2bfeed5b9563644949e738c2.png"}],"financialInterests":"","formattedTitle":"\u003cp\u003eCombined Effects of Low pH and High Water Temperature Conditions on Oxidative Stress and Cell Damage in Juvenile Olive Flounder \u003cem\u003eParalichthys Olivaceus\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe use of fossil fuels for anthropogenic and industrial activities is accompanied by the emission of carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) into the atmosphere. Additionally, the emitted CO\u003csub\u003e2\u003c/sub\u003e plays a role in raising the surface water temperature of the ocean along with causing acidification that lowers the pH of the ocean (IPCC, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, if the emission of CO\u003csub\u003e2\u003c/sub\u003e continues with the current trend without efforts to reduce its emissions, then the surface water temperature of the ocean will rise by about 4\u0026deg;C within 100 years and the pH will decrease by 0.3\u0026ndash;0.4 units from the current level (about pH 8.1). It is predicted that the pH of the ocean will decrease by 0.7\u0026ndash;0.8 units within the next 300 years (Caldeira and Wickett, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; IPCC, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Based on such predictions, it is believed that ocean warming and acidification will inevitably occur simultaneously. Therefore, conducting ocean acidification related research is necessary to reveal aspects of the complex effects of at least two environmental change factors (pH and water temperature) on marine life, rather than just one environmental factor.\u003c/p\u003e \u003cp\u003eChanges in ocean pH concentration and water temperature are known to negatively affect the survival, growth, morphology, and behaviour of marine organisms (Kroeker et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Little et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These environmental stressors negatively affect various physiological mechanisms, which include neuronal signal transduction and osmoregulation, cellular homeostasis, and oxidative stress in marine organisms (Heuer and Grosell, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Meanwhile, stress induces redox reactions in the body and the production of reactive oxygen species (ROS), which cause oxidative stress in cells and tissues. With the production of ROS, cell damage and an increase in antioxidant enzyme activity are usually induced by lipid peroxidation. At this stage, one of ROS and highly reactive superoxide radical (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) is converted into hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) by superoxide dismutase (SOD) and the action of antioxidant enzymes to remove oxidative stress. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e converted by SOD is finally converted by catalase (CAT) into non-toxic water and oxygen, thereby, preventing O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e from accumulating in cells and tissues (Lesser, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Generally, fish are sensitive to changes in water temperature, and hence, a high-temperature environment induces oxidative stress and increases apoptosis in fish (Kim et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, when the antioxidant response due to the change of water temperature reaches its peak, there are cases where the antioxidant enzyme is inactivated due to protein denaturation and decomposition in the body (Madeira et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Heat shock protein 70 (HSP70) plays an important role in cellular thermotolerance, apoptosis, and heat shock stress. Additionally, it can be used as an ideal biomarker to confirm the effect of environmental stress because it is induced when cells are exposed to noxious stimuli (Kregel, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Cortisol is also a major stress hormone and is used as an indicator of environmental stress caused by pollution or climate change (Sadoul and Geffroy, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is reported that along with the single effect of ocean acidification (pH decrease) on fish, the combined effect of ocean warming (high water temperature) and acidification environment can also negatively affect fish survival (Rosa et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e and Ara\u0026uacute;jo et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The complex environment of combined ocean warming and acidification negatively affects fish metabolism, heat tolerance, growth, skeletal development, and survival (Pimentel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, it is known to negatively affect the activity of antioxidant enzymes and cause cell damage by lipid peroxidation (Pimentel et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The aforementioned complex changes in the ocean environment can change the surrounding food ecosystems due to habitat changes (vertical migration) of marine upper predators (jumbo squid, \u003cem\u003eDosidicus gigas\u003c/em\u003e) (Rosa and Seibel, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Additionally, it is reported that in the case of Atlantic cod, a major commercial fish, it can affect fishery production due to habitat movement (H\u0026auml;nsel et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs ocean warming and acidification by CO\u003csub\u003e2\u003c/sub\u003e emitted into the atmosphere are simultaneous phenomena, it is necessary to investigate the physiological responses of fish through the combined effects of these environments to accurately understand their impact on marine species. Therefore, in this study, the stress response through changes in cortisol and HSP70 and the antioxidant response through changes in SOD and CAT were investigated in a complex environment in which ocean warming (increase in water temperature) and acidification (reduction in pH) coexisted. Additionally, the level of apoptosis was investigated through caspase 3 (CASP3) activity and terminal transferase dUTP nick end labelling (TUNEL) assay. Through the above analyses, we attempted to understand the adaptation potential of olive flounder to future ocean conditions, which has high industrial value in Korea, China, and Japan.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals and experimental condition\u003c/h2\u003e \u003cp\u003eOlive flounder \u003cem\u003eParalichthys olivaceus\u003c/em\u003e (length 17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 cm; weight 40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 g) were purchased from a commercial hatchery and acclimated for 4 weeks in six 350-L recirculating filtration tank system, consisting of four mini tanks (40 cm \u0026times; 40 cm \u0026times; 40 cm, 60 L) and a sump tank. Sufficient feed was provided to all fish twice a day, both during the experiment and acclimation period. In the environment setting for the experiment, warming conditions were set to 20 (control), 25, and 30\u0026deg;C, at pH 8.1 and combined warming and acidification conditions (CWA) were set to 20, 25 and 30\u0026deg;C, at pH 7.5. All environmental factors were measured daily twice using a pH meter (WM-32EP, DKK-TOA Corporation, Tokyo, Japan) for pH and temperature and using a Pro2030 (YSI Inc., USA) for salinity. Photoperiod conditions were set to 12 h light and 12 h dark. Fish were anesthetized with 2-phenoxyethanol at 200 ppm for 3 min, and plasma was collected using a heparin-coated syringe. Collected plasma was centrifuged at 1500 \u0026times; \u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C for 20 min and immediately stored at ˗80\u0026deg;C until subsequent analysis. The liver tissues were collected from the fish, and stored at -80\u0026deg;C until the analysis of immunohistochemistry (IHC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Seawater CO\u003csub\u003e2\u003c/sub\u003e manipulation\u003c/h2\u003e \u003cp\u003eSeawater pH was adjusted using a pH-controller (Tunze smart controller 7000, Aquarientechnik GmbH, Germany). pH sensors and ceramic CO\u003csub\u003e2\u003c/sub\u003e diffusers were installed in sump tanks to achieve the target pH level 7.5 by the pH-controller. The pH sensor was calibrated using an NBS (National Bureau of Standards) buffer. The \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e was checked three times in a day using a pump-aspirated sampling-type CO\u003csub\u003e2\u003c/sub\u003e meter (GM70, Vaisala, Finland). Seawater carbonate chemistry was calculated by CO2SYS using the constants of Dickson and Millero (Lewis and Wallace, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), as described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCarbonate chemistry at each condition of seawater\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"17\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eDesired\u003c/p\u003e \u003cp\u003econdition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eSalinity\u003c/p\u003e \u003cp\u003e(psu)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003cp\u003e(\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003epH\u003csub\u003eNBS\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c14\" namest=\"c12\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003eCO2\u003c/p\u003e \u003cp\u003e(\u0026micro;atm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c17\" namest=\"c15\"\u003e \u003cp\u003eA\u003csub\u003eT\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;mol kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003epH 8.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e20℃\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003e20.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e8.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e \u003cp\u003e446\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003e1779\u0026thinsp;\u0026plusmn;\u0026thinsp;41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e25℃\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003e25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e8.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e \u003cp\u003e441\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003e1905\u0026thinsp;\u0026plusmn;\u0026thinsp;43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e30℃\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003e30.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e8.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e \u003cp\u003e454\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003e2090\u0026thinsp;\u0026plusmn;\u0026thinsp;55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003epH 7.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e20℃\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e34.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e7.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c12\" namest=\"c10\"\u003e \u003cp\u003e1443\u0026thinsp;\u0026plusmn;\u0026thinsp;38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c15\" namest=\"c13\"\u003e \u003cp\u003e1390\u0026thinsp;\u0026plusmn;\u0026thinsp;45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e25℃\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e34.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e7.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c12\" namest=\"c10\"\u003e \u003cp\u003e1363\u0026thinsp;\u0026plusmn;\u0026thinsp;31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c15\" namest=\"c13\"\u003e \u003cp\u003e1342\u0026thinsp;\u0026plusmn;\u0026thinsp;34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e30℃\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e34.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e7.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c12\" namest=\"c10\"\u003e \u003cp\u003e1481\u0026thinsp;\u0026plusmn;\u0026thinsp;38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c15\" namest=\"c13\"\u003e \u003cp\u003e1307\u0026thinsp;\u0026plusmn;\u0026thinsp;27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"17\"\u003eSalinity, Temperature, pH\u003csub\u003eNBS\u003c/sub\u003e, \u003cem\u003ep\u003c/em\u003eCO\u003csub\u003e2\u003c/sub\u003e were measured by equipment directly and total alkalinity (A\u003csub\u003eT\u003c/sub\u003e) was calculated in CO\u003csub\u003e2\u003c/sub\u003eSYS (Lewis and Wallace, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). All data values represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Analysis of plasma parameters\u003c/h2\u003e \u003cp\u003eCortisol, SOD, CAT, and CASP3 were analyzed from plasma samples using an enzyme-linked immunosorbent assay (ELISA, #MBS704055 for cortisol, #MBS 705758 for SOD, #MBS038818 for CAT, and #MBS012786 for CASP3; MyBioSource Inc., San Diego, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured using a microplate reader (Spark, TECAN Trading AG, Switzerland), and the concentration was calculated by plotting a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. IHC\u003c/h2\u003e \u003cp\u003eIHC was performed using hsp70 antibody (#OASE00332, AVIVA) to assess the heat stress response of fish liver cells to CO\u003csub\u003e2\u003c/sub\u003e and high water temperature exposure. The analysis procedure is briefly described as follows: First, deparaffin was treated with fresh xylene 3 times for 7 min. Then rehydration was sequentially performed in ethanol of 100% (2 times in 3 min), 95% (2 times in 3 min), 80% (1 time in 3 min), and 70% (1 time in 3 min). This was followed by treating the samples in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and performing antigen retrieval for 15 min each, respectively. After treatment in 4% bovine serum albumin for 30 min, the primary and secondary antibodies were treated for 60 min and 30 min, respectively, and after 7 min of treatment with 3,3\u0026acute;- diaminobenzidine at 24\u0026deg;C, the samples were treated with Mayer\u0026rsquo;s hematoxylin at 24\u0026deg;C for 3 min. Finally, after dehydration, mounting was performed. Sides were observed under a microscope (Axio Imager A2, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. TUNEL assay\u003c/h2\u003e \u003cp\u003eTUNEL analysis was performed using commercially available ApopTag peroxidase In Situ Apoptosis Detection Kit (catalogue number, S7100, Chemicon, Germany) as per the manufacturer\u0026rsquo;s instructions to assess the apoptosis response of fish liver cells to elevated CO\u003csub\u003e2\u003c/sub\u003e and low-salinity seawater exposure. Tissue section was washed with xylene and ethanol, and fresh diluted proteinase K was applied to the pre-treated tissue specimen for 15 min at room temperature. Then equilibration buffer and working strength terminal deoxynucleotidyl transferase enzyme were applied. This was followed by applying anti-digoxigenen conjugate and incubating in a humidified chamber for 30 min at room temperature. At the end of this process, it was washed with phosphate buffered saline. To determine the optimal staining duration, the slides were observed under a microscope and the color development in peroxidase substrate was monitored. The specimens were mounted silanized glass slides and observed under a microscope (Axio Imager A2, Germany). The brown cells indicated apoptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the data were analysed using the SPSS software (version 27.0; IBM, USA). The independent samples t-test was used to determine the difference between pH levels under the same temperature and exposure days. A one-way analysis of variance (ANOVA) test followed by Tukey\u0026rsquo;s post-hoc test was used to compare the differences between temperatures on the same exposure day. Two-way ANOVA test was employed to determine the interaction effects of ocean acidification and warming as described in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The values are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and \u003cem\u003eP\u003c/em\u003e-values lower than 0.05 were considered statistically significant.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInteraction effects of water temperature and pH level.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCortisol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSOD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCAT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCASP3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eD0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.959\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eD7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.747\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.003*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.004*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.018*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eD14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.425\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.046\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eD21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.780\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.454\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.002*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.038*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.005*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eD28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.069\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.423\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.646\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.395\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.010*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eThe symbol \u0026ldquo;*\u0026rdquo; indicates a significant difference.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Changes in the activity of plasma parameters\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. Cortisol\u003c/h2\u003e \u003cp\u003eCortisol showed higher activity as the water temperature increased until the 7th day of the experiment in both warming (25, 30\u0026deg;C) and CWA conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the overall cortisol activity was significantly higher in the CWA condition than in the warming condition. However, on the 14th day, cortisol activity was higher as the water temperature increased (25\u0026deg;C and 30\u0026deg;C) under the warming condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, higher cortisol activity was observed at lower water temperature (20\u0026deg;C) under the CWA condition. On days 21 and 28, the higher the water temperature, the higher the cortisol activity in both warming and CWA conditions was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExcept for the 28th day at 20\u0026deg;C, all cortisol activities were higher under the acidifying condition than under the warming condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the cortisol activity, the effect of the interaction of acidification with warming was found to have significant differences at the 7, 14, and 21 days, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. SOD\u003c/h2\u003e \u003cp\u003eOverall, SOD showed higher activity as the water temperature increased under the warming conditions. SOD activity in the CWA condition was significantly lower compared to that in the warming condition at 25\u0026deg;C on the 3rd day (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the 7th and 14th day, the activity of SOD at 20\u0026deg;C was significantly higher in the CWA condition than in the warming condition, and it showed a high activity similar to that at 25\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the SOD activity, the effect of the interaction of acidification with warming was found to have significant differences at the 7th, 14th, and 21st day (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3. CAT\u003c/h2\u003e \u003cp\u003eCAT activity was significantly higher as the overall water temperature increased in both the warming and the CWA conditions. However, the CAT activity showed a low activity similar to that at 20\u0026deg;C and 30\u0026deg;C on the 7th day of CWA conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CAT activity showed significantly higher activity (30\u0026deg;C on day 14, all water temperature conditions on day 21, 20\u0026deg;C and 30\u0026deg;C on day 28) compared to the warming condition even after the 7th day (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The effect of the interaction of acidification with warming in CAT activity was found to have a significant difference on the 7th, 21st, and 28th days (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4. CASP3\u003c/h2\u003e \u003cp\u003eCASP3 showed significantly higher activity as the water temperature increased until the 7th day of the experiment under the warming conditions. In the CWA condition, high CASP3 activity was observed at 20\u0026deg;C and 25\u0026deg;C on the 3rd and 7th day, respectively, and in particular, the highest activity was observed in the CWA condition on the 3rd day (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 20\u0026deg;C, CASP3 activity was significantly higher in the CWA than in the warming condition, except for the 28th day (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The effect of the interaction of acidification with warming in CASP3 activity was found to have a significant difference for all experimental period (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. IHC of HSP70 in liver tissue\u003c/h2\u003e \u003cp\u003eAs a result of investigating IHC in liver tissue of olive flounder juvenile on days 14 and 28 under warming and CWA conditions, it was found that the higher the water temperature, the more active the HSP70 antibody response in the liver tissue on days 14 and 28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, on the 14th day, an active HSP70 antibody response similar to the warming and CWA conditions were observed in the acidification condition at 20\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. TUNEL assay\u003c/h2\u003e \u003cp\u003eFrom investigating the presence of apoptotic cells in the liver tissue of olive flounder juvenile on days 14 and 28 under warming and CWA conditions using the TUNEL assay, in both warming and CWA conditions, the higher the water temperature, the higher the frequency of apoptotic cells appeared in the liver tissue on both the 14th and 28th days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, B). However, on the 14th day, a high frequency of apoptotic cells similar to the warming and CWA conditions were observed in the acidification condition at 20\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eExcessive water temperature rise increases the energy consumption of the fish and thus requires additional energy supply, thereby increasing the cost of growth in fish (Little et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe formation of ROS and the increase in metabolic demand under the rise of CO\u003csub\u003e2\u003c/sub\u003e in seawater as well as water temperature generally lead to damage caused by peroxidation and increased activity of antioxidant enzymes, and additional energy consumption is unavoidable (Rosa et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Contrarily, in the case of a species with high adaptability (resilient) to environmental stressors, it also prevents cell damage (lipid peroxidation) by upregulating the action of antioxidant enzymes to cope with oxidative stress caused by warming (Sampaio et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe complex environment of ocean acidification and warming generates ROS and antioxidant enzymes act to remove ROS, but excessive production of ROS exceeds the action capacity of antioxidant enzymes, so it can have a negative effect on fish. Ara\u0026uacute;jo et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that the effects of ocean acidification and the combined effects of two complex environments (ocean warming and acidification) have negative synergistic effects such as increased energy demand, anaerobic metabolism, and impaired proteolysis in sea bream \u003cem\u003eSparus aurata\u003c/em\u003e. It is reported to induce cytotoxic effects and pose a serious threat to sea bream populations. Rosa et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported that when juvenile bamboo sharks \u003cem\u003eChiloscyllium punctatum\u003c/em\u003e were exposed to a combined environment of ocean warming and acidification, antioxidant enzymes detoxified the ROS. However, upregulation of these antioxidant enzymes was insufficient to minimize the increase in cholinergic neurotransmitters caused by peroxidative damage and stress response in the brain.\u003c/p\u003e \u003cp\u003eIn our study, it was found that under the warming conditions (higher water temperature), the stress response and the activity of antioxidant enzymes were high, and the appearance of apoptosis was also high. This findings are consistent with that of a similar study by Madeira et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) who reported that excessive oxidative stress induced in the muscles, liver, and brain of Gilt-head bream S\u003cem\u003eparus aurata\u003c/em\u003e at high water temperature (30\u0026deg;C) increased the activity of antioxidant enzymes (SOD, CAT, GST) to counteract the effect of increasing ROS. Additionally, it was similar to the results of Kim et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) who reported that a high water temperature environment increased the antioxidant enzyme activity of olive flounder and induced apoptosis in liver tissue by increasing the activity of CASP3. Additionally, in our study, the stress response, the antioxidant enzyme activity, and the appearance of apoptosis were found to be high in the combined warming (high water temperature) and acidification (pH decrease) conditions, similar to the overall warming conditions. This result was confirmed in a study by Rosa et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Ara\u0026uacute;jo et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) who state that the combined effect of the two environments appears to act negatively to induce cytotoxicity, which results in increased emergence of apoptosis. Specifically, this implies that the antioxidant enzyme to remove ROS acted not just in a high temperature environment but also in a complex environment with warming (high water temperature) and acidification (pH decrease), although the appearance of apoptosis was not inhibited because the action capacity of the antioxidant enzyme exceeded due to a large amount of ROS.\u003c/p\u003e \u003cp\u003eThe activity of CAT in our study was consistently high under the conditions in which warming and acidification were combined. This is described in Carney Almroth et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) who state that Atlantic halibut \u003cem\u003eHippoglossus\u003c/em\u003e maintained a significant correlation of high SOD and CAT activity in a warming (high water temperature) environment but not in a high CO\u003csub\u003e2\u003c/sub\u003e concentration (low pH level) environment. Fish may have increased amounts of hydrogen ions (H\u003csup\u003e+\u003c/sup\u003e) in their body to normalize the pH levels around blood cells by increasing HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in an environment exposed to CO\u003csub\u003e2\u003c/sub\u003e (Heuer and Grosell, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This excess presence of H\u003csup\u003e+\u003c/sup\u003e in the blood can combine with H\u003csub\u003e2\u003c/sub\u003eO to form H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Sampaio et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which is believed to induce a sustained increase in CAT, the key enzyme that generally scavenges H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe results of this study under the combined environmental conditions of warming and acidification, stress and antioxidant enzyme activity, and apoptosis all tend to be high at 20\u0026deg;C. Grans et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) found that warming and acidification conditions increased oxygen demand and cardiac performance in Atlantic halibut \u003cem\u003eHippoglossus hippoglossus\u003c/em\u003e, although these increases were not interpreted as growth improvements. Instead, it was reported that Atlantic halibut was more affected by acidification under lower water temperature conditions than the optimum growth water temperature, and hence the growth decreased. When cells are exposed to oxidative stress, ROS is also generated in the electron transport chain of mitochondria, but considering the generation of ROS due to increased respiration in a high-temperature environment, in our study, the effects of acidification under the combined warming and acidification conditions do not exceed the effects of warming. Rather, the effect of acidification appears to be greater in low-temperature than in high-temperature conditions.\u003c/p\u003e \u003cp\u003eSummarizing the results of our study on juvenile olive flounder, 1) Oxidative stress and apoptosis tend to increase under both the warming (high water temperature), combined warming (high water temperature), and acidification (pH decrease) conditions. 2) Considering the increase in ROS due to oxidative stress as well as the increase in ROS generation due to increased respiration in the high water temperature condition, the effect on acidification in the combined warming and acidification condition does not seem to exceed the effect of warming. 3) It is implied that the effect on acidification is greater at a relatively low water temperature (20\u0026deg;C) than at a high temperature condition.\u003c/p\u003e \u003cp\u003eExcessive induction of oxidative stress due to warming and acidification may lead to a decrease in the growth of fish, induce disease due to reduced immunity, and shorten their lifespan. Therefore, understanding the physiological responses of marine organisms in combination with the prediction of future changes in the marine environment is important for predicting the direct impact of climate change on marine organisms and their vulnerability. The data thus obtained can also be very important for predicting the sustainability of the fishery and aquaculture industries. Additionally, in the mid-latitude regions where Korea is located, seawater temperature fluctuates greatly in many cases due to seasonal changes. Therefore, we suggest that additional research on oxidative stress and antioxidant responses of marine organisms is needed in an environment in which low water temperature and acidification conditions coexist in winter, which cannot guarantee sufficient nutritional status due to poor food intake.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA,analysis of variance;CASP3, caspase 3; CAT, catalase; CO\u003csub\u003e2\u003c/sub\u003e, carbon dioxide; CWA, combined warming and acidification conditions; H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hydrogen peroxide; HSP70, heat shock protein 70; IHC, immunohistochemistry; NBS, National Bureau of Standards; O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e,superoxide radical; ROS, reactive oxygen species; SOD,superoxide dismutase; TUNEL,Terminal transferase dUTP nick end labelling.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e- Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the \u003cstrong\u003eMinistry of Oceans and Fisheries, Korea\u003c/strong\u003e [PE99922].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- \u003c/strong\u003e\u003cstrong\u003eConflicts of interest/Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Availability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets and materials used during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Code availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Authors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDae-Won Lee: Data curation, Writing - original draft.\u003c/p\u003e\n\u003cp\u003eJin Ah Song: Data curation, Writing - editing\u003cbr /\u003e Heung-Sik Park: Funding acquisition,Data curation, Formal analysis, Review \u0026amp; editing.\u003cbr /\u003e Cheol Young Choi: Conceptualization, Project administration, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Ethics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted in accordance with the guidelines and ethical principles of the Institutional Animal Care and Use Committee operation established by ministry of agriculture, food and rural affairs of Korea.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAra\u0026uacute;jo, J.E., Madeira, D., Vitorino, R., Repolho, T., Rosa, R., Diniz, M., 2018. Negative synergistic impacts of ocean warming and acidification on the survival and proteome of the commercial sea bream, \u003cem\u003eSparus aurata\u003c/em\u003e. J. Sea. Res. 139, 50-61.\u003c/li\u003e\n\u003cli\u003eCaldeira, K., Wickett, M.E., 2003. Oceanography: anthropogenic carbon and ocean pH. Nat. 425, 365.\u003c/li\u003e\n\u003cli\u003eCarney Almroth, B., Bresolin De Souza, K., J\u0026ouml;nsson, E., Sturve, J., 2019. Oxidative stress and biomarker responses in the Atlantic halibut after long term exposure to elevated CO\u003csub\u003e2\u003c/sub\u003e and a range of temperatures. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 238, 110321.\u003c/li\u003e\n\u003cli\u003eGrans, A., Jutfelt, F., Sandblom, E., Jonsson, E., Wiklander, K., Seth, H., Olsson, C., Dupont, S., Ortega-Martinez, O., Einarsdottir, I., Bjornsson, B.T., Sundell, K., Axelsson, M., 2014. Aerobic scope fails to explain the detrimental effects on growth resulting from warming and elevated CO\u003csub\u003e2\u003c/sub\u003e in Atlantic halibut. J. Exp. Biol. 217, 711-717.\u003c/li\u003e\n\u003cli\u003eH\u0026auml;nsel, M.C., Schmidt, J.O., Stiasny, M.H., St\u0026ouml;ven, M.T., Voss, R., Quaas, M.F., 2020. Ocean warming and acidification may drag down the commercial Arctic cod fishery by 2100. PLOS ONE 15, e0231589.\u003c/li\u003e\n\u003cli\u003eHeuer, R.M., Grosell, M., 2014. Physiological impacts of elevated carbon dioxide and ocean acidification on fish. Am. J. Physiol. Regul. Integr. Comp. Physiol. 307, R1061-1084.\u003c/li\u003e\n\u003cli\u003eIPCC, 2014. Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, R.K. Pachauri and L.A. Meyer (eds.)]. IPCC, Geneva, Switzerland, 151 pp.\u003c/li\u003e\n\u003cli\u003eKim, B.-S., Jung, S.J., Choi, Y.J., Kim, N.N., Choi, C.Y., Kim, J.-W., 2016. Effects of different light wavelengths from LEDs on oxidative stress and apoptosis in olive flounder (\u003cem\u003eParalichthys olivaceus\u003c/em\u003e) at high water temperatures. Fish Shellfish Immunol. 55, 460-468.\u003c/li\u003e\n\u003cli\u003eKregel, K.C., 2002. Invited Review: Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J. Appl. Physiol. 92, 2177-2186.\u003c/li\u003e\n\u003cli\u003eKroeker, K.J., Kordas, R.L., Crim, R., Hendriks, I.E., Ramajo, L., Singh, G.S., Duarte, C.M., Gattuso, J.P., 2013. Impacts of ocean acidification on marine organisms: quantifying sensitivities and interaction with warming. Glob. Chang. Biol. 19, 1884-1896.\u003c/li\u003e\n\u003cli\u003eLesser, M.P., 2006. Oxidative stress in marine environments: Biochemistry and Physiological Ecology. Ann. Rev. 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Total Environ, 618, 388-398.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"abiotic stress, cell damage, high water temperature, low pH, olive flounder","lastPublishedDoi":"10.21203/rs.3.rs-902059/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-902059/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe use of fossil fuels by anthropogenic activities causes ocean acidification and warming, and the combined conditions of these two environments can negatively affect fish metabolism, growth, and survival. Additionally, it can affect the antioxidant enzyme activity and cell damage caused by lipid peroxidation. In this study, we explores the adaptive potential for future marine conditions by investigating the stress and antioxidant enzyme activity, and apoptosis of juvenile olive flounder in an environment where ocean acidification and warming coexist. We found that juvenile olive flounder had increased oxidative stress and apoptosis under both warming and combined warming, and acidification conditions. Additionally, the effect on acidification under warming conditions does not seem to exceed the effect of high temperature conditions, and it is considered that the effect of acidification at low water temperatures is greater.\u003c/p\u003e","manuscriptTitle":"Combined Effects of Low pH and High Water Temperature Conditions on Oxidative Stress and Cell Damage in Juvenile Olive Flounder Paralichthys Olivaceus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-10-01 21:37:39","doi":"10.21203/rs.3.rs-902059/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-09-27T08:06:06+00:00","index":0,"fulltext":""},{"type":"editorInvited","content":"Fish Physiology and Biochemistry","date":"2021-09-19T12:45:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-09-14T04:27:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fish Physiology and Biochemistry","date":"2021-09-13T09:11:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f1ca0b3c-2e36-4cd9-b704-d61320cfd065","owner":[],"postedDate":"October 1st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":7595814,"name":"General Biochemistry"},{"id":7595815,"name":"Molecular Genetics"},{"id":7595816,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2022-08-08T13:13:28+00:00","versionOfRecord":[],"versionCreatedAt":"2021-10-01 21:37:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-902059","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-902059","identity":"rs-902059","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}