The combined effects of temperature and salinity on the digestion and respiration metabolism of Pinctada fucata

The combined effects of temperature and salinity on the digestion and respiration metabolism of Pinctada fucata were evaluated via response surface methodology and box-benhnken design under laboratory condition. Results indicated that the primary and secondary effects of salinity and temperature had significant effects on amylase (AMS) of P. fucata (P < 0.05)., The digestive enzyme reached the maximum activity when temperature was 26 °C. The AMS and trypsin (TRYP) increased at first, and then decreased with increasing temperature. The Lipase (LPS) was positively correlated with either salinity or temperature. Salinity had no significant effect on TRYP as a primary effect (P > 0.05), but had a significant effect on TRYP as a secondary effect (P < 0.01). These effects were completely opposite to the effect of temperature on pepsin (PEP) as primary and secondary effects. The combined effects of salinity and temperature on AMS, TRYP and PEP were significant (P < 0.01), but had no significant effect on LPS (P > 0.05). The primary, secondary and interaction effects of salinity had significant effects on NKA (Na+-K+-ATPase) of P. fucata (P < 0.05), and NKA presented a U-shaped distribution with increasing salinity. The quadratic and interactive effects of temperature had a significant effect on AKP (P < 0.05), and AKP showed a U-shaped distribution with increasing temperature. Lactate dehydrogenase (LDH) activity decreased at first, and then increased when temperature and salinity changed from 20 to 30 °C and 23–33 ‰, respectively. The expression of GPX gene affected by temperature in gills may be delayed compared with that in hepatopancreas, and its expression is tissue-specific. The appropriate digestion and respiratory metabolism index models were established under the combined temperature and salinity conditions. The optimization results showed that the optimal combination of temperature and salinity was 26.288 °C/28.272‰. The desirability was 0.832. Results from the present study will provide a theoretical reference for shellfish culture affected by environmental interactions and the establishment of related index models.


Results
Model significance analysis. The  Variance analysis of digestive indexes and immune genes in hepatopancreas were presented in Tables 1. The results showed that the established models of salinity and temperature on digestive physiology of P. fucata were significant (P < 0.05), and the lack of fit was nonsignificant (P > 0.05), indicating that the regression model was significant. The fitting between experimental data and the model was good, and the model was suitable. Variance analysis was performed on respiratory metabolism indexes and immune genes in gills ( Table 2). The results showed that the established models of the effects of salinity and temperature on respiratory metabolism physiology of P. fucata were significant (P < 0.05), and the lack of fit was nonsignificant (P > 0.05), indicating that the regression model was significant, and the fitting between the experimental data and the model was good, the model was suitable, and the experimental results caused by unknown factors had little interference. The Model coefficient estimation. The established regression model of digestive and respiratory metabolism indexes were estimated by coefficients, and the results were shown in Table 3 and Table 4. The coefficients in Table 3 and Table 4 were coded values (elimination of units among coefficients), and their effects were directly reflected by numerical values. 95% confidence interval (C.I.) explained the change of coefficient coding value in 95% interval. The test and analysis results of the model coefficients showed that the primary and secondary effects of salinity and temperature and their interaction effects had significant indigenous effects on AMS (P < 0.05). The primary and secondary effects of salinity and temperature had significant effects on LPS (P < 0.05), while the secondary effects of salinity and their interaction with temperature had no significant effects on LPS (P > 0.05). The primary effect of temperature and the interaction effect of temperature and salinity had significant influence on PEP (P < 0.05), while the primary effect of salinity and the secondary effect of temperature and salinity had no significant influence on PEP (P > 0.05). The primary, secondary and interactive effects of salinity and temperature had significant effects on TRYP (P < 0.05). The primary and secondary effects of temperature, the secondary effects of salinity and the interaction between temperature and salinity had significant effects on the relative gene expression of GPX in hepatopancreas (P < 0.05), while the primary effect of salinity had no significant effect on the relative gene expression of GPX in hepatopancreas (P > 0.05). The primary, secondary www.nature.com/scientificreports/ and interaction effects of temperature and salinity had significant effects on the relative gene expression of SOD in hepatopancreas (P < 0.05). Table 4 showed that the minimum values predicted by the 95% C.I. of the intercept of respiratory metabolism indexes were 288.61, 1.82, 207.92, 0.43 and 0.40, respectively, and the maximum values were 324.47, 2.17, 248.15, 0.56 and 0.65, respectively. The test results of the model coefficients showed that the primary, secondary and interaction effects of salinity and temperature had significant effects on LDH (P < 0.05). Primary effect of salinity and temperature and their interaction effect, secondary effect of salinity had significant indigenous effect on NKA (P < 0.05), secondary effect of temperature had no significant indigenous effect on NKA (P > 0.05). The primary effect of temperature and the secondary effect of salinity had no significant effect on AKP (P > 0.05), while the primary effect of salinity, the secondary effect of temperature and the interaction between temperature and salinity had significant indigenous effect on AKP (P < 0.05). The primary and secondary effects of salinity and temperature had significant effects on the relative expression of GPX gene in gills (P < 0.05), and the interaction of salinity and temperature had no significant effect on the relative expression of GPX gene in gills (P > 0.05). The primary and secondary effects of temperature and the interaction of salinity and temperature had significant effects on the relative expression of SOD gene in gills (P < 0.05), while the primary and secondary effects of salinity had no significant effects on the relative expression of SOD gene in gills (P > 0.05).

Response surface analysis of digestive and respiratory metabolism.
Under all different combinations of salinity and temperature, the digestive enzyme activity and the relative expression of immune genes in hepatopancreas tissue estimated according to the fitted response surface were shown in Fig. 1. By examining the response surface or contour, it was found that the relative expression levels of AMS, TRYP and hepatopancreas SOD genes were unimodal. AMS and TRYP increased at first and then decreased with increasing temperature or salinity, while the relative expression of SOD gene in hepatopancreas decreased at first and then increased with increasing temperature or salinity. LPS increased with increasing two factors. PEP increased with increasing temperature and decreased with increasing salinity. The relative expression level of hepatopancreas GPX gene showed an upward trend with increasing temperature, and decreased at first and then increased with increasing salinity.
Under all different combinations of salinity and temperature, the activities of respiratory metabolic enzymes and the relative expression of immune genes in gills estimated by the fitted response surface were shown in Fig. 2. By examining the response surface or contour, it was found that LDH was a single peak, and its activity showed a U-shaped trend with the increase in salinity or temperature. NKA showed a decreasing trend with the Table 2. Model variance analysis of respiratory metabolic indicators. LDH Lactate dehydrogenase activity in gills, NKA Na + -K + -ATPase activity in gills, AKP Alkaline phosphatase activity in gills, GPX Relative expression levels of GPX antioxidant genes in gills, SOD Relative expression levels of SOD antioxidant genes in gills. www.nature.com/scientificreports/ increase in temperature, and a U-shaped trend with the increase in salinity. AKP showed a U-shaped trend with the increase in temperature, and decreased with the increase in salinity. The relative expression of GPX gene in gills showed a decreasing trend with the increase in temperature, and a U-shaped trend with the increase in salinity. The relative expression of SOD gene in gills showed a U-shaped trend with the increase in temperature, and decreased with the increase in salinity.

Optimization.
The results showed that the optimal combination of temperature and salinity was 26.288 °C/28.272 ‰, and the maximum value of AMS, LPS, PEP and TRYP was 1.107, 2.136, 2.660 and 860.627, respectively. The minimum relative expression levels of GPX and SOD genes in hepatopancreas were 0.740 and 0.070, respectively. The lowest relative expression levels of GPX and SOD genes in gills were 0.439 and 0.463, respectively, and the lowest values of LDH, NKA and AKP were 319.477, 1.677 and 232.716, respectively. The desirability was 0.832.

Discussion
Temperature and salinity are the key factors affecting the digestive enzyme activity of shellfish 41 . As an osmotic animal, shellfish will actively discharge salt or water to adapt to environmental changes when the environmental salinity is too low or too high 42 . In the process of osmotic pressure regulation, the digestive ability of shellfish decreases with the consumption of large amounts of energy 43 . The results of the present study showed that the www.nature.com/scientificreports/ digestive enzyme activity of P. fucata was relatively high at the salinity of 28 ‰, and the activities of AMS and TRYP decreased when the salinity was low or high, which was consistent with the above laws and consistent with the results of juvenile scallop Chlamys nobilis 44 and Solen grandis 43 . There are few reports on the effect of salinity on digestive enzyme activity in shellfish, except for those consistent with the above view 45,46 , Chiu and Benitez 47 also pointed out that inorganic ions in seawater can be used as activators of digestive enzymes at appropriate concentrations, and inhibitors below the appropriate range.
According to the kinetics of enzymatic reaction and the protein properties of digestive enzymes, the speed of enzymatic reaction is accelerated with the increase in temperature in a certain temperature range, and begins to decrease beyond a certain temperature range. As a thermophilic animal, the digestive enzyme activity of shellfish is directly affected by environmental temperature changes. Digestive enzymes need optimal temperature to better participate in the biological reaction process 44 . The present study showed that when temperature was about 26 °C, digestive enzymes reached the maximum activity value, and AMS and TRYP showed an inverted U-shaped trend with the increase in temperature, which was in line with the above laws and was consistent with Haliotis diversicolor 48 , Chlamys nobilis 44 , Chlamys farreri 49 , Lutraria sieboldii Reeve 50 , Solen grandis 43 , and Pinctada martensii 51 had the same results. The increase of LPS with temperature or salinity may be due to the low-fat intake in shellfish diet and the fluctuation of LPS in the lower activity range. Different digestive enzyme activities in vivo are related to different feeding habits of different shellfish. Bivalves such as Mytilus edulis have higher protease and AMS activities due to their preference for unicellular algae, while gastropods such as Littorina sp. have higher cellulase activities due to their preference for macroalgae 52 . The inconsistency may be related to the difference in enzyme activity determination methods, species, age, feeding habits and breeding conditions.
Results from the present study showed that the primary and secondary effects of temperature and salinity had significant indigenous effects on the AMS of P. fucata, indicating that the AMS activity was susceptible to temperature and salinity, and it was in line with the above law of digestive enzyme activity changing with temperature and salinity. This may be because the salinity changes affected the osmotic pressure of the body, and AMS was a biological macromolecule. The enzyme activity was affected by the increasing of temperature, and high temperature may even caused denaturation (inactivation). Therefore, the maximum value of AMS will only  www.nature.com/scientificreports/ reach at the optimum temperature and salinity 46,53 . Incidentally, the peak value here was the optimum temperature of digestive enzyme activity in P. fucata. In the study of Chlamys farreri 49 , Haliotis discus hannai Ino 41 and Sinonovacula constricta 54 , it was found that the suitable temperature for the growth of shellfish was lower than the temperature of its main digestive enzyme activity. The optimal temperature obtained in the present study was 26 °C, which was close to the result of 27 °C of juvenile P. martensii by Zhu 55 . This may be because the present study and Zhu 55 experiment were the activity of digestive enzymes secreted by shellfish stimulated by ambient temperature, and other studies were environmental stimulation enzyme solution. The primary effect of salinity on TRYP was not significant, but the secondary effect was significant, and the primary effect of temperature on PEP was significant, but the secondary effect was not significant, indicating that TRYP activity was nonlinear with salinity, and PEP was linear with temperature. This may be because interaction between factors shields one or two effects 44,55 .
Previous studies mostly focused on the effects of single factor (factor) on digestive enzyme activity in aquatic organisms, but few on the interaction between factors. In the case of interaction between factors, the investigation of interaction between factors is much more important than the investigation of the main effect (primary or secondary effect) of a single factor 15 . In the present study, Box-Benhnken design (BBD) was used to conduct the significant analysis and test of the interaction between salinity and temperature. The results showed that the combined effect of salinity and temperature had significant influence on the activities of AMS, PEP and TRYP. It may be because the superposition of two main effects (salinity and temperature) has an impact on digestive enzyme activity, which was similar to the results reported by Zhu 55 and Qian, et al. 44 . However, in this study, the interaction between salinity and temperature on LPS in P. fucata was not significant, indicating that the effects of salinity and temperature on LPS were independent rather than antagonistic or synergistic, or the two effects (salinity and temperature) were mutually shielded, resulting in the weakening of the interaction between the two 44 , or the influence mechanisms of salinity and temperature on the enzyme activity of P. fucata were different. There were also no significant results of interaction between reproduction and embryonic development of P. fucata 1 , R. cuneata 21 , P. margaritifera 56 and noble scallop Chlamys nobilis 26 . Such variations of salinity and temperature may due to the species difference as different species have different osmoregulation modes and effects.
As an important part of energy metabolism research, respiratory metabolism is an important presentation and expression of metabolic activities in aquatic animals. Oxygen, metabolic level and physiological status required for maintaining the lowest metabolic level of aquatic animals can be directly or indirectly reflected by it 57 . During the life activities of shellfish, respiratory metabolism reflects the physiological status, metabolic characteristics and adaptability to external environmental stress 28 . Salinity and temperature are important environmental factors affecting the respiratory metabolism of aquatic organisms. Salinity can affect the regulation of osmotic pressure to affect the metabolism of the body, while temperature can affect biological oxygen consumption and osmotic pressure and ion regulation to affect the metabolism level 57 .
NKA is a kind of P-type ATPase, which can actively transport Na + out of cells and K + into cells. It is essential to maintain cell osmotic pressure 28 . Its activity provides a major driving force for activating other ion transport systems involved in osmotic regulation 58 . In this study, NKA showed a U-shaped distribution with the increase in salinity, which was consistent with the results of black and red shell P. fucata after 1.5 h salinity stress 28 and red shell P. fucata after 12 h salinity stress 29 . It indicated that P. fucata had strong adaptability to low or high salt. Different from the results of Meretrix lusoria 58 and Pomacea canaliculata 59 , salinity has different effects on respiration metabolism in different species. In the primary and secondary effects, the effect of salinity on NKA of P. fucata was significant, indicating that NKA was easily affected by salinity changes, and showed a U-shaped trend with the increase in salinity, which might be due to the change of osmotic pressure caused by salinity changes, and the enhancement of NKA activity to maintain the osmotic pressure balance in the body 44 . In this study, the interaction between temperature and salinity had a significant indigenous effect on NKA in P. fucata, while in the study of Chlamys nobilis 44 , the effect on NKA activity was not significant, which was different from the results of this study, one may be superimposed by the main effect, while the other was shielded from each other. This may be due to different species and different osmotic pressure regulation modes, so that the degree of NKA on the membrane was different under the interaction (synergistic effect) of salinity and temperature. Osmotic regulation of P. fucata is not entirely achieved by NKA enzyme in gills 28 .
AKP can catalyze the transfer reaction of phosphate groups and the hydrolysis of phosphate monolipids. As an important enzyme for the survival and growth of aquatic organisms, AKP also helps the body to form and secrete chitin, absorb calcium in water and form calcium phosphate 60 . The present study showed that AKP showed a U-shaped distribution with the increase in temperature, indicating that the increase of temperature accelerated the respiratory metabolism level of P. fucata to a certain extent and increased the ATP production to support the continuous movement of P. fucata. Contrary to the findings of Clinocardium californiense 60 . Evidence indicates that Clinocardium californiense is not tolerant to high temperature, while P. fucata has strong adaptability to high temperature and low temperature. Results from the present study showed that the secondary effect of temperature had a significant indigenous effect on AKP, indicating that there was a peak value of AKP within the set temperature range. The response surface diagram also clearly showed that there was a minimum value of AKP with the increase in temperature. This may be because P. fucata begins to secrete large amounts of AKP at lower or higher temperatures to resist adverse environments in order to regulate immunity 61 . The interaction between salinity and temperature had significant effects on AKP in P. fucata, indicating that osmotic pressure regulation and temperature were closely related to respiratory metabolism in P. fucata. This may be due to the fact that salinity provides metal ions to activate enzymes, and temperature changes the conformation of AKP, so that the two can jointly promote the regulation of enzyme activity in the process of substrate-phosphatase binding and activation 62 .
LDH is an important glycolytic enzyme required for cell energy metabolism, which can convert lactic acid, the main by-product of anaerobic glycolysis, into pyruvic acid and release energy. LDH plays a key role in www.nature.com/scientificreports/ maintaining aerobic metabolism, and its activity is closely related to cell metabolism, which can reflect the level of anaerobic respiration to a certain extent 28 . In the present study, when the temperature and salinity changed from 20 to 30 °C and from 23 to 33 ‰, respectively, the LDH activity decreased at first and then increased, indicating that the anaerobic respiration level of P. fucata presented a U-shaped distribution with the increase in temperature or salinity. In the study of C. californiense 60 , it was found that LDH activity increased with the increasing in temperature in the range of 16-28 °C. In the present study, the LDH activity of P. fucata was consistent with its law at 26-30 °C. When temperature was about 26 °C, and the salinity was about 28‰, the LDH activity was the lowest, indicating that the salinity and temperature were appropriate at this time, and the anaerobic metabolism level was the lowest in the body. It was speculated that aerobic metabolism was the main part at this time, and a large amount of energy generated could be used for the body to cope with environmental changes. The interaction between marine invertebrate environmental factors and antioxidant enzymes has been intensively studied. Mostly reflected in the stress of various factors, enzyme activity determination and expression level of space-time, tissue differences. There are still many studies on the genes of antioxidant enzymes related to stress resistance in marine organisms, such as GPX, SOD and GST 63 . However, there are few studies on the effects of temperature or salinity on the relative expression of SOD and GPX genes in shellfish. In this study, the primary and secondary effects of temperature had significant effects on GPX and SOD gene expression in hepatopancreas and gill, indicating that they were nonlinear with temperature, and temperature had significant effects. Among them, the relative expression of hepatopancreas SOD and gill SOD had the minimum value. The expression of GPX in hepatopancreas was up-regulated with the increase in temperature, while the expression of GPX in gills was just the opposite. This may be because the gene expression of GPX in gills affected by temperature is lagging behind that in hepatopancreas, and also showed the tissue specificity of gene expression.
In this study, the primary and secondary effects of salinity have significant effects on hepatopancreas SOD gene expression and gill GPX gene expression, indicating that salinity has a significant effect on it and a nonlinear relationship. The secondary effect of salinity on the relative expression of SOD in gills was shielded by the interaction of temperature and salinity. The secondary effect of salinity had a significant impact on the relative expression of GPX and SOD genes in hepatopancreas and GPX genes in gills of P. fucata. It can be seen that they have the optimal value within the specified range. At this time, the environment was suitable for P. fucata, and the relative expression of antioxidant genes was down-regulated, which indirectly supported the above results on digestive enzymes and respiratory metabolic enzymes.

Conclusion
In summary, environmental salinity and temperature changes may regulate the growth, energy utilization and metabolism of P. fucata by affecting digestive and respiratory metabolism and antioxidant capacity of hepatopancreas and gill. Our study showed that the combined effect of salinity and temperature had a significant indigenous effect on the digestion and respiratory metabolism of P. fucata. Therefore, the effects of single factor and synergistic effect should be considered in the artificial breeding and breeding of P. fucata. Results from the present study provide a theoretical basis for future research and digestive enzyme model establishment.

Materials and methods
Experimental animals. The pearl oysters (total weight: 34.19 ± 1.39 g, shell length: 50.75 ± 1.43 mm) were collected from South China Sea, and then transferred to the laboratory of Lingshui Station (Hainan, China) for acclimation. They were conditioned in some 5000 L cement tanks in the laboratory at ambient temperature (25 ± 1 °C) and salinity (33 ± 1 ‰) prior to experiment. During one week of acclimation, ambient parameter remained DO > 6.5 mg / L, pH 8.0 ± 0.1, light intensity < 500 Lx with natural photoperiod and Platymonas subcordiformis (200 × 10 3 cells/mL) was fed once a day at regular intervals (9: 00-9: 30 a.m.). Half of the seawater was replaced and feces, residues were siphoned off daily, and dead pearl oysters were removed from the tank immediately.
Experimental design. The respiratory metabolism and digestion were assessed utilizing the Box-Benhnken design (BBD) with either 2-factors or explanatory variables, salinity (S, ‰) and temperature (T, °C). Each factor contained 3-levels, which were coded as − 1, 0 and 1, respectively (Table 5). There were nine salinity-temperature combined treatments in the experiment, each treatment had three replicates, and each replicate contained 15 P. fucata ( Table 6, Table 7). The experiment was carried out in twenty-seven 800-L cement tanks.
Throughout the experiment, salinity ranged between 23 and 33 ‰, and the minimum temperature was set at 20 °C, the maximum at 30 °C. Preliminary trials, based upon the seasonal changes in salinity and temperature of the seawater in the South China Sea, were conducted to define the settings of salinity and temperature chosen for the combination experiment. Temperatures for the respiratory metabolism and digestion experiments were manipulated by heating rod or ice bottles. All salinities were prepared through addition of sea salts or tap water Collection and determination of tissue samples. Three shellfish were randomly collected from each parallel, and the hepatopancreas and gill tissues were cut off on an ice tray with scissors. Rinsed with pre-cooling 0.9% normal saline, blotted with clean filter paper, the tissue samples were quickly placed in 2 mL centrifuge tubes and stored at − 80 °C. The biochemical parameters of gills and hepatopancreas tissue were determined according to the instructions of the manufacturer (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) i.e.,    According to the kit manufacturer's instructions, regarding the use of arbitrary unit (AU) and King unit as units of enzyme activity in this study, the specific meaning of these units in each enzyme is as follows: For AMS, one unit of amylase activity (AU/mgprot) was defined as the hydrolysis of 10 mg of starch per mg of protein in tissue treated with substrate at 37 °C for 30 min. For LPS, each gram of tissue protein reacted with the substrate in this reaction system for 1 min at 37 °C, and each 1 μmol of substrate consumed was one unit of enzyme activity (AU/gprot). For PEP, 1 μg of tyrosine per milligram of tissue protein at 37 °C per minute was equivalent to one unit of enzyme activity (AU/mgprot). For TRYP, at pH 8.0, 37 °C, each milligram of trypsin contained in the protein changes the absorbance by 0.003 per minute, which was one unit of enzyme activity (AU/mgprot). For LDH, each gram of tissue protein was treated with substrate at 37 °C for 15 min, and 1 µmol pyruvate was produced in the reaction system as one unit (AU/gprot). For NKA, the amount of ATP breakdown to produce 1 µmol of inorganic phosphorus per milligram of tissue protein per hour was specified as one unit of atpase activity (AU/mgprot). For AKP, 1 mg of phenol per gram of tissue protein produced by interaction with substrate at 37 °C for 15 min was defined as 1 King unit/gprot. (Table 8) for qPCR analysis were conducted based on Adzigbli, et al. 64 and Gu, et al 65 . RNA was extracted according to the method of Fu, et al. 66 . The ND 5000 spectrophotometer (BioTeke Corporation, China) and 1% agarose gel electrophoresis were used to evaluate the quantity and integrity of isolated RNA, respectively. Finally, the reverse transcription and relative gene expression were determined according to the method of Yang, et al. 67 .

Primer design and gene expression. Genes selected and primers designed
Statistical analysis. Box-Benhnken design (BBD) was used in the experiment, namely the specific combination of salinity and temperature at different levels. According to Ryan and Morgan 68 and Montgomery 15 , this experimental design could more accurately estimate the influence of different factors (salinity and temperature in the present study), and expand the range of validity of the conclusion when additional factors are inserted. The relationship between quantitative correlation factors and response largely depended on the response surface of fitting experimental data. Therefore, the three-dimensional response surface map could be obtained by drawing and processing the response of different factors (here is salinity and temperature). Image rendering and data analysis were performed using Design Expert 10 (32-bit). Assuming that the response surface properties are described by the following models: In the formula, Y was the response (digestive and respiratory metabolism related indicators), β 0 was a constant, β 1 represented the linear effect of salinity, β 2 represented the linear effect of temperature, β 12 represented the interaction effect of salinity and temperature; β 11 was the secondary effect of salinity, β 22 was the secondary effect of temperature, ε was the random error, the mean was zero (in line with normal distribution). The regression coefficients of these experimental points were calculated by the least square method. P < 0.05 of the items included in the above model was considered statistically significant or significant. The adequacy and importance of establishing the model could be proved by the generated variance analysis table. The fitting of the above model was expressed by the determination coefficient R 2 , and its statistical significance was determined by F test. After the model equation was established, the optimization program of Design Expert 10 software was used to optimize and analyze several responses. By maximizing the conversion of the expected function, the optimal condition set was finally obtained. According to Wang, et al. 1 , it was necessary to optimize the response surface analysis. The model equations of digestive and respiratory metabolism were optimized according to the method of Montgomery 15 .

Data availability
The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.