Tolerance, Growth, and Physiological Responses of the Juvenile Razor Clam (Sinonovacula constricta) to Environmental Ca2+ and Mg2+ Concentrations

To facilitate transplanting razor clam (Sinonovacula constricta) populations to inland saline-alkaline waters (ISWs), we evaluated the tolerance of juvenile S. constricta (JSC) to Ca²⁺ and Mg²⁺ concentrations, and determined the effects of these ions on JSC growth and physiological parameters. After 30 days stress, the tolerable ranges of JSC to Ca²⁺ and Mg²⁺ were determined to be 0.19 mmol⋅L^-1–19.46 mmol⋅L^-1 and 0 mmol⋅L^-1–29.54 mmol⋅L^-1, respectively. The concentrations of Ca²⁺ (less than 0.65 mmol⋅L^-1 or more than 3.24 mmol⋅L^-1) and Mg²⁺ (less than 0.37 mmol⋅L^-1 or more than 14.17 mmol⋅L^-1) significantly inhibit JSC growth. Physiological enzyme activity no significant response when the concentrations range of Ca²⁺ and Mg²⁺ are 0.93 mmol⋅L^-1–6.49 mmol⋅L^-1 and 0.37 mmol⋅L^-1–14.77 mmol⋅L^-1, respectively. For transplantation practice, these data indicate that only high concentrations of Ca²⁺ (3.24–6.825 mmol⋅L^-1) and Mg²⁺ (14.77–33.69 mmol⋅L^-1) in target inland saline-alkaline water had significantly impact on growth and physiological response. In addition, present study suggests that the increase in Ca²⁺ and Mg²⁺ ion concentrations caused by ocean acidification will not affect the survival, growth and physiology of S. constricta. Current research suggests that S. constricta can adapt to extreme changes in the marine environment (Ca²⁺ and Mg²⁺) and may be an excellent candidate for inland saline-alkaline water transplantation practice.

Introduction

Saline-alkali soil and inland saline-alkaline waters (ISWs) exist in more than 100 countries (Shi, 2009). In China, there are approximately 1 million km² of saline-alkaline soil and approximately 0.3 million km² of ISW (He et al., 2010). Saline water resources have long been used for agriculture, but traditional terrestrial crops are not salt-tolerant and cannot be irrigated with salt water. Therefore, ISWs are often low-yielding land (Qadir et al., 2001; Wang et al., 2011). The use of ISWs to culture economically important marine organisms represents an effective means to use inland saline-alkali resources, such as the most successful species Litopenaeus vannamei (Roy et al., 2007, 2010). Inland aquaculture of economically important marine organisms worldwide is mainly concentrated in ISW areas, and is practiced in several countries. However, there are few reports on marine mollusc transplanting in ISWs; these reports only describe aquaculture of Mytilus edulis (Dinh and Fotedar, 2016), Ruditapes philippinarum (Hiele et al., 2014), Haliotis laevigata (Doupé et al., 2003), Trochus niloticus (Lee, 1997), and Crassostrea gigas and Saccostrea glomerata (Ingram et al., 2015).

In China, some ISWs have been used for aquaculture, but most of them have historically been in a state of disuse (Lv et al., 2012). According to incomplete statistics, more than 33 economically important organisms are cultured in ISWs in China (mainly freshwater fish) (Zhang and Zhang, 1999). However, the practice of aquaculture of economically important marine mollusc in ISW has only been attempted with Cyclaina sinensis (Lin et al., 2012) in China; this venture ultimately did not succeed.

The ion composition of ISW is quite different from that of seawater. Monovalent and divalent cations and their interactions have an important influence on the physiology of organisms (Duchen, 2000). All of the main ionic components in ISW (Ca²⁺, Mg²⁺, Na⁺, K⁺, SO₄^2-, HCO₃^-, OH^-, Cl^-, Ca²⁺: Mg²⁺, Na⁺: K⁺, SO₄^2-: Cl^-) are factors that affect the survival and growth of aquatic animals (Hadfield et al., 2012; Hiele et al., 2014; Raizada et al., 2015). Ca²⁺ is a crucial element of the outer shell of marine crustaceans and mollusc, is involved in muscle activity, shell formation, neurotransmission, and osmotic regulation, and plays an important role in the growth of marine invertebrates such as crustaceans and mollusc (Wilbur and Yonge, 2013; Yang, 2011). Mg²⁺ is found in the active region of many enzymes, it is a metabolic cofactor, and is an important participant in the active transport of substances involved in maintenance of the cell membrane. Studies have shown that Mg²⁺ has an important effect on the survival, growth, and osmotic pressure of marine crustaceans (Mantel and Farmer, 1983). The imbalance of ionic components in ISW tends to affect aquaculture practices more than salinity, as demonstrated in L. vannamei by Saoud et al. (2003). In addition, it has been reported that the high content of Mg²⁺, Ca²⁺, and K⁺, and low Ca²⁺/Mg²⁺ and Na⁺/K⁺ ratios in ISW affect the survival of cultured L. vannamei.

Many marine invertebrates are osmotic conformers: the osmotic concentration (and usually the ionic composition) of the extracellular fluid is similar to that of the ambient seawater (Larsen et al., 2014). Thus, in aquatic mollusc can be found the stenohaline, euryhaline or oligohaline osmotic conformers, and, also the freshwater bivalves (Deaton, 2008). Most of the intertidal zone bivalve belongs to the euryhaline osmotic conformers, and usually their conformational floor are reported to be between 0.7 ppt and 3.4 ppt (Otto and Pierce, 1981; Deaton, 1992, 2008). There is also volume regulation in marine mollusc, the process that returns the cytoplasmic and extracellular fluid compartments to osmotic equilibrium with the ambient medium (Smith and Pierce, 1987; Deaton, 1992; Larsen et al., 2014). The capacity for volume regulation in marine osmotic conformers varies from limited to high (some molluscs) (Virkar, 1966; Pierce, 1971; Gainey, 1978). Although the study of osmotic and ionic regulation in bivalve is not sufficient, Ca²⁺ and Mg²⁺ play an important role in this system.

The Ca²⁺ and Mg²⁺ ion contents vary widely in ISWs in different regions of China. For example, the Ca²⁺ content of ISWs in Haolebaojinao (Wu Shen banner, Ordos City, the Inner Mongolia Autonomous Region, China) is only 0.199 mmol⋅L^-1 of seawater (Tian, 2000). In contrast, the Ca²⁺ content of Bosten Lake is 5.61 mmol⋅L^-1 that of seawater with the same salinity (Shi, 1989). Shi (2009) found that the Mg²⁺ content in ISW in the middle reaches of the Yellow River in China was 2.33–8.15 mmol⋅L^-1. Therefore, studying the effects of Ca²⁺ and Mg²⁺ on marine invertebrates can help facilitate transplanting marine invertebrates in China ISW.

Sinonovacula constricta is a eurythermal and euryhaline marine mollusc that is distributed in estuary and intertidal zones (Lin and Wu, 1990). S. constricta feeds on microalgae, and fixes calcium ions in its aquatic environment during the formation of its shell, which can contribute to the improvement of an ISW environment. Peng et al. (2018) reported that S. constricta tolerate high pH and carbonate alkalinity under long-term stress, and suggested that S. constricta is a potentially good species for culture in ISW. However, the effects of different concentrations of Ca²⁺ and Mg²⁺ on the survival, growth, and physiological responses of S. constricta have not been reported. In addition, ocean acidification and climate warming also directly affect the concentration of Ca²⁺ and Mg²⁺ ions in the ocean (Orr et al., 2005). Therefore, understanding the critical tolerances and physiological responses of S. constricta to Ca²⁺ and Mg²⁺ stress is crucial for the development of optimal ISW culture conditions and ocean environments changing. Thus, this study investigated the effects of Ca²⁺ and Mg²⁺ on the survival, growth, and physiological responses of juvenile S. constricta (JSC), and the possibility of this species as a potential candidate for transplantation to ISWs, providing a theoretical basis for this process.

Materials and Methods

Experimental animals were treated according to the guidelines of the Institutional Animal Care and Use Committee of Shanghai Ocean University (IACUC-SHOU), Shanghai, China, under examination and approval number SHOU-DW-2018-014. In this tests the animals were artificially propagated as juveniles.

Healthy JSCs (20 days old) were obtained commercially (Zhejiang Sanmen Donghang). Before experimentation, all JSCs were kept in at 8 ppt seawater for 3 days, then were kept at 6 ppt artificial seawater (Ca²⁺: 1.71 mmol⋅L^-1, Mg²⁺: 9.23 mmol⋅L^-1) for 4 days. In these conditions the total salinity of water is set at 6 ppt, since the salinity of ISW (carbonate type) in China is around 6 ppt (Yao et al., 2010). Artificial seawater was formulated using chemical reagents (AR degree, Sangon Biotech) and distilled water according to the Artificial Seawater Recipe (Chu) in Table 1A (Harvey, 1957; Yao et al., 2010). Approximately 10,000 specimens of JSCs were obtained before experiments, and then 270 JSCs were randomly selected to measure shell length (0.2102 ± 0.058 cm) and body weight (0.000892 ± 0.00015 g). Water temperature in experiments was maintained between 20 to 22°C.

TABLE 1A

Table 1A. Artificial seawater recipe (Chu).

Long-Term Stress Study

Juvenile S. constrictas were divided into 29 groups for long-term stress testing (Table 1B), consisting of a control group (CK), 14 groups with different concentrations of Mg²⁺ (DCMg: G1-G14), and 14 groups with different concentrations of Ca²⁺ (DCCa: G1–G14). Each of the 29 groups had 3 parallel trials. For each parallel stress trial, 90 JSCs were cultured in an 11 cm diameter round plate for a month. 150 mL test water (with the appropriate salinity and Ca²⁺ or Mg²⁺ concentration) was added to the round plate, and the test water was changed once every single day. Feed-water was prepared as follows: test water was used to replace the culture water of Chaetoceros calcitrans by centrifugation, and the concentration of C. calcitrans was adjusted to 400–480 cells μL^-1 in test water. For every single feeding, 15 mL feed-water was used to replace 15 mL of test water. JSCs were fed thrice a day.

TABLE 1B

Table 1B. Design of different Ca²⁺ and Mg²⁺ concentrations for the long-term stress test.

Live JSCs were counted at day 0 (0-day) and at day 30 (30-day). At 0-day, 36 JSCs (6 repeats: 6 JSCs per repeat) were selected to determine the activities of the following enzymes: Na⁺/K⁺-ATPase (NKA), aspartate aminotransferase (AST), superoxide dismutase (SOD), acetylcholinesterase (AChE), and lysozyme (LZM). At 30-day, live JSCs were used to measure shell length, body weight, and enzymes activity (NKA, SOD, AST, AChE, and LZM). Nine JSCs (representing three separate tests with three JSCs in each group) were used to measure enzyme activities. Briefly, after shell removal, JSC tissue was homogenized in normal saline water in a ratio of 1 mg: 9 μl. After homogenization, the total protein content of the sample was measured by Coomassie Brilliant Blue Total Protein Assay kit (Nanjing Jiancheng Bioengineering Institute, China), according to the manufacturer’s instructions. NKA, AST, SOD, AChE, and LZM enzyme activities were measured by corresponding kits from Nanjing Jiancheng Bioengineering Institute Ltd., as previously described (Peng et al., 2018).

Na⁺/K⁺-ATPase activity was measured by Pi released from ATP, in the incubation solution containing: 100 mM NaCl, 20 mM KCl, 4 mM MgCl₂, 1 mM EGTA, 0.2 mM 2-methyl-8-(phenylmethoxy)imidazol(1,2-α) pyridine-3-acetonitrile 40 mM Tris–HCl (pH 7.4) and 3 mM Na₂ATP. The incubation time with substrate was 10 min. Pi concentration was calculated from the regression line based on standard Pi solutions. The units of NKA activity are expressed as U (μmol Pi mg prot^-1 h^-1). AST can transfer amino and keto groups between alpha ketoglutarate and aspartic acid, then generate glutamic acid, and oxaloacetate. Oxaloacetate can be decarboxylated to pyruvic acid by itself during the reaction. Pyruvic acid reacts with 2,4-Dinitrophenylhydrazine to form 2,4-Dinitrophenylhydrazone, which is reddish brown in an alkaline solution. After measuring the OD, the standard curve was checked to obtain AST activity, which is expressed as U (μmol Pyruvic acid mg prot^-1 min^-1). SOD activity was tested using SOD assay kit by WST-1 method. The WST-1 method depends on produces a water-soluble formazan dye upon reduction with the superoxide anion. The rate of the reduction of WST-1 with O2^- are linearly related to the xanthine oxidase activity, and this reduction is inhibited by SOD. Therefore, the 50% inhibition activity of SOD can be determined by the colorimetric method. The SOD activity is expressed as units per mg of protein (U mg prot^-1), where the one U represents the amount of enzyme required to achieve a SOD inhibition rate of 50%. AChE hydrolyzes acetylcholine to produce choline and acetic acid. Choline can react with sulfhydryl chromogenic reagent to form Sym-Trinitrobenzene (yellow compound), after measuring the OD, the standard curve was checked to obtain AChE activity, which is expressed as units per mg of protein (U mg prot^-1), where the one U represents at 37°C for 6 min of reaction conditions, the amount of enzyme required to hydrolyze 1 mmol of acetylcholine. The activity of LZM was detected using a turbidimetric method, which light transmittance of the bacterial turbid solution. Lysozyme can lysis bacteria by hydrolyze peptidoglycan on the bacterial wall to increase the light transmission of the bacterial turbid solution. After measuring the light transmission, the standard curve was checked to obtain LZM activity, which is expressed as units per mg of protein (U mg prot^-1), where the one U equivalent to the activity capacity of standard LZM. All data of enzyme activities are ultimately represented as a ratio of 30-day to 0-day.

Acute Stress Study

Within 3 days of the start of the long-term stress study, we found that all JSCs died in the following groups: DCMg-G10, DCMg-G11, DCMg-G12, DCMg-G13, DCMg-G14, DCCa-G13, and DCCa-G14. To more fully explain the physiological effects of Ca²⁺ and Mg²⁺ on JSCs, we designed and conducted an acute stress study. The CK, DCMg-G1, DCMg-G10, DCCa-G1, and DCCa-G13 were used in the acute stress experiment. In this experiment, the animals were exposed to the same conditions used in the long-term stress tests, except that the clams were not fed algae. At 0-h, 12-h, 24-h, and 48-h, ten JSCs from each group were used to measure oxygen consumption, then the hemolymph of JSCs was collected, as previously reported (Peng et al., 2017), to separate haemocytes and to measure metabolic and phagocytic activities.

The method used for measuring oxygen consumption rate was previously reported (Peng et al., 2019). After hemolymph collection, 20 μL of JSC haemocyte suspension solution was diluted with TBS to 100 μL. Then 10 μL of 1/10 diluted carboxylate-modified microspheres (diameter 1 μm, yellow-green fluorescent, FluoSpheres^®, Invitrogen) was added, and samples were incubated in the dark at room temperature for 1 h (Wang et al., 2014). A BD C6Plus (BD Biosciences, United States) flow cytometer was used to measure the phagocytosis of haemocytes. The same haemocyte concentration without fluorescent microspheres in each group was analyzed by flow cytometry to obtain the cell concentration, and we set the threshold to be Gate A (cell only). The relative size of haemocytes (FSC value) and FITC fluorescence intensity (FITC value) were selected to limit the data. Finally, the data was extracted for analysis and figure mapping.

The metabolic activity of haemocytes was measured with a Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay. In every parallel group, 20 μL haemocyte suspension solution was diluted with TBS to 200 μL. Then, 20 μL of CCK-8 reagent was added and incubated at 28°C for 3 h. For the CK, 20 μL serum was obtained from the haemocyte suspension solution, and was diluted with TBS to 200 μL; then 0 μL of CCK-8 was added and incubated at 28°C for 3 h. After the OD value was measured, the relative metabolic activity was calculated using OD value and the total number of haemocytes (obtained from the phagocytosis test).

Calculation and Data Analytical Methods

Survival rate (SR):

$$\begin{array}{c}SR(\%) = 100 \times N_{30}/N_0\end{array}$$

N₃₀ and N₀ are the numbers of surviving individuals in the 30-day and 0-day groups, respectively.

Shell length growth rate (SGR):

$$\begin{array}{c}SGR(\%da{y}^{-1}) = 100 \times (L_{30} - L_0)/T,\end{array}$$

L₃₀ and L₀ are the average lengths of the individuals in the 30-day and 0-day groups, respectively. T is 30 days.

Weight gain rate (WG):

$$\begin{array}{c}WG(\%da{y}^{-1}) = 100 \times (W_{30} - W_0)/T\end{array}$$

W₃₀ and W₀ are the average wet body weights of the individuals in the 30-day and 0-day groups, respectively. T is 30 days.

Oxygen consumption rate:

$$\begin{array}{c}(mg{O}_2·g^{-1}·L^{-1}·h^{-1}) = [A_i(initialoxygenconcentrations) -A_f(finaloxygenconcentrations)] \times V \\ (volumeoftestwater) / [W(wetbodyweight) \times T(testtime)]\end{array}$$

Phagocytosis rate and relative metabolic activity:

$$\begin{array}{c}Phagocytosisrate\% = (H_{\text{t}} - H_{\text{u}})/H_{\text{t}} \times 100\end{array}$$

$$\begin{array}{c}Relativemetabolicactivity = [(O{D}_{\text{G}} - O{D}_{\text{GB}})/H_{\text{tG}} \times 1000]/[(O{D}_{\text{CK}} - O{D}_{\text{CKB}})/H_{\text{tCK}} \times 1000]\end{array}$$

H_t and H_u are the total haemocyte and un-phagocytic haemocyte numbers, respectively. OD_G, OD_GB, and H_tG are the OD of test sample, the blank OD of test sample, and the total haemocyte counts of the test sample, respectively. OD_CK, OD_CKB, and H_tCK are the OD of control sample, the blank OD of control sample and the total haemocyte count of the control sample, respectively.

Statistical Analysis

Sigmaplot 12.3 software was used to identify significant differences analysis and to plot results. Significant differences were analyzed by using single factor analysis of variance (one-way ANOVA analysis) and the LSD method.

Results

Survival and Growth of JSC Under Long-Term Ca²⁺ or Mg²⁺ Stress

During the long-term stress, we found that the concentrations of Mg²⁺ (higher than 73.86 mmol L^-1) and Ca²⁺ (higher than 207.55 mmol L^-1) cause JSCs to die rapidly (all JSC death groups are listed in Table 2). The concentrations of Ca²⁺ (lower than 0.093 mmol L^-1) causes JSCs to die over an extended period of time. Meanwhile, we observed that the bodies of JSCs were lacked calcium carbonate shells in groups stressed with Ca²⁺ concentrations lower than 0.093 mmol L^-1. However, the JSCs stressed with low concentrations of Mg²⁺ remained viable (Figure 1). In the range of Mg²⁺ concentration from 0 mmol L^-1 to 29.54 mmol L^-1, there is a trend of JSC survival ratio show as first rise and then fall. Similarly, in the range of Ca²⁺ concentration from 0.19 mmol L^-1 to19.46 mmol L^-1, there is a trend for JSC survival to first rise and then fall. The Mg²⁺ concentration from 0.18 mmol L^-1 to 3.69 mmol L^-1 and Ca²⁺ concentration from 0.93 mmol L^-1 to 2.32 mmol L^-1 showed no significant differences in survival compared to the CK.

TABLE 2

Table 2. Detailed juvenile Sinonovacula constricta mortality information in the test groups which cannot survive under long-term stress with different Ca²⁺ and Mg²⁺ concentrations.

FIGURE 1

Figure 1. Survival rates of JSCs under long-term stress with different Ca²⁺ and Mg²⁺ concentrations. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups (G1–G14) and control group (CK).

The shell growth rate was significantly inhibited in the Mg²⁺ concentration from 0 mmol L^-1 to 0.37 mmol L^-1 (P < 0.05), and from 14.77 mmol L^-1 to 29.54 mmol L^-1 (P < 0.001), and was significantly inhibited in the Ca²⁺ concentration 0.19–0.65 mmol⋅L^-1 (P < 0.05) and 6.49–19.46 mmol⋅L^-1 (P < 0.05) (Figure 2). The body weight gain rate was significantly inhibited in the Mg²⁺ concentration 0–0.37 mmol⋅L^-1 (P < 0.05) and 14.77–29.54 mmol⋅L^-1 (P < 0.001), and was significantly inhibited in the Ca²⁺ concentration 0.19–0.65 mmol⋅L^-1 (P < 0.05) and 3.24–19.46 mmol⋅L^-1 (P < 0.05) (Figure 3).

FIGURE 2

Figure 2. Daily shell length growth rate of JSCs under long-term stress with different Ca²⁺ and Mg²⁺ concentrations. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups (G1–G10) and CK.

FIGURE 3

Figure 3. Daily weight gain rate of JSCs under long-term stress with different Ca²⁺ and Mg²⁺ concentrations. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups (G1–G10) and CK.

Enzyme Activity of JSC Under Long-Term Ca²⁺ or Mg²⁺ Stress

Na⁺/K⁺-ATPase activity was significantly decreased in JSCs stressed with low concentrations range (0–0.046 mmol⋅L^-1) of Mg²⁺ (Figure 4). However, 14.77 mmol⋅L^-1 of Mg²⁺ significantly increased NKA activity in JSCs (P < 0.05). Low concentrations and high concentrations of Ca²⁺ affected JSC NKA activity. NKA activity was increased significantly in the Ca²⁺ concentration of 0.19 mmol⋅L^-1 (P < 0.001), 0.28 mmol⋅L^-1 (P < 0.001), and 19.46 mmol⋅L^-1 (P < 0.05). NKA and SOD activity patterns are totally consistent when Ca²⁺ varying. In contrast with NKA, SOD activity is not affected by low Mg²⁺ (Figure 5). AST activity (Figure 6) was significantly reduced in low concentrations (0–0.37 mmol⋅L^-1) of Mg²⁺ (P < 0.05), but was significantly increased in the high Mg²⁺ concentration, 14.77 mmol⋅L^-1 (P < 0.05) and 29.54 mmol⋅L^-1 (P < 0.001). However, both 0.19–0.65 mmol⋅L^-1 (P < 0.05) and 6.49–19.46 mmol⋅L^-1 (P < 0.05) concentrations of Ca²⁺ significantly reduced AST activity in JSCs. AChE activity increased as the concentration of Mg²⁺ increased (Figure 7). AChE activity was inhibited significantly in the Mg²⁺ concentration of 0 mmol⋅L^-1 and 0.046 mmol⋅L^-1 (P < 0.05), but was increased significantly in the of 14.77 mmol⋅L^-1 and 29.54 mmol⋅L^-1 (P < 0.001). The same trend was observed in the effects of Ca²⁺ concentration on AChE activity. AChE activity was inhibited significantly in the Ca²⁺ concentration of 0.19 mmol⋅L^-1 and 0.28 mmol⋅L^-1 (P < 0.001), but was increased significantly in the 6.49 mmol⋅L^-1 and 19.46 mmol⋅L^-1 (P < 0.001). Only the Mg²⁺ – 1.85 mmol⋅L^-1, Mg²⁺ – 3.69 mmol⋅L^-1, Ca²⁺ – 2.32 mmol⋅L^-1, and Ca²⁺ – 3.24 mmol⋅L^-1 showed no significant inhibition of LZM activity in JSCs (Figure 8).

FIGURE 4

Figure 4. Relative NKA activity of JSCs under long-term stress with different Ca²⁺ and Mg²⁺ concentrations. Results show NKA activity at 30-days normalized to the 0-day. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups (G1–G10) and CK.

FIGURE 5

Figure 5. Relative SOD activity of JSCs under long-term stress with different Ca²⁺ and Mg²⁺ concentrations. Results show SOD activity at 30-days normalized to the 0-day. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups (G1–G10) and CK.

FIGURE 6

Figure 6. Relative AST activity of JSCs under long-term stress with different Ca²⁺ and Mg²⁺ concentrations. Results show AST activity at 30-days normalized to the 0-day. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups (G1–G10) and CK.

FIGURE 7

Figure 7. Relative AChE activity of JSCs under long-term stress with different Ca²⁺ and Mg²⁺ concentrations. Results show AChE activity at 30-days normalized to the 0-day. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups (G1–G10) and CK.

FIGURE 8

Figure 8. Relative LZM activity of JSCs under long-term stress with different Ca²⁺ and Mg²⁺ concentrations. Results show LZM activity at 30-days normalized to the 0-day. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups (G1–G10) and CK.

Oxygen Consumption Rate, Phagocytic Rate, and Metabolism Activity of JSC Under Acute Ca²⁺ or Mg²⁺ Stress

The oxygen consumption rate of JSCs changed under acute stress due to different concentrations of Ca²⁺ and Mg²⁺ (Figure 9). Within 48 h, the oxygen consumption rate at 0 mmol⋅L^-1 of Mg²⁺ did not show any significant difference. However, the oxygen consumption rate in the Mg²⁺ concentration 73.86 mmol⋅L^-1 was significantly increased at 24 h, and then was significantly decreased at 48 h. The oxygen consumption rate of the Ca²⁺ concentration 0 mmol⋅L^-1 was significantly increased at 48 h. The oxygen consumption rate of the Ca²⁺ concentration 207.55 mmol⋅L^-1 was significantly increased at 12 and 24 h, and then was significantly decreased at 48 h. The phagocytosis rate of all groups decreased over time (Figure 10). Among them, the Mg²⁺ concentration 0 mmol⋅L^-1 showed a significant decreased at 48 h (P < 0.05), the Mg²⁺ concentration 73.86 mmol⋅L^-1 showed a decrease at 24 h (P < 0.05) and 48 h (P < 0.001), and the Ca²⁺ concentration 0 mmol⋅L^-1 and 207.55 mmol⋅L^-1 began to decrease significantly at 12 h. The Mg²⁺ concentration 0 mmol⋅L^-1 showed no significant change in metabolic activity (Figure 11). Metabolic activity was only significantly increased at 48 h in the Ca²⁺ concentration 0 mmol⋅L^-1. However, metabolic activity of the Mg²⁺ concentration 73.86 mmol⋅L^-1 and Ca²⁺ concentration 207.55 mmol⋅L^-1 began to increase significantly at 12 h.

FIGURE 9

Figure 9. Acute oxygen consumption rate of JSCs under extreme Ca²⁺ and Mg²⁺ concentrations. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups and CK.

FIGURE 10

Figure 10. Acute phagocytosis rate of JSCs under extreme Ca²⁺ and Mg²⁺ concentrations. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups and CK.

FIGURE 11

Figure 11. Acute metabolism of JSCs under extreme Ca²⁺ and Mg²⁺ concentrations. Results show metabolism activity of test groups normalized to the 0 h group. Bars (mean ± SE, n = 3) with an asterisk denote a significant difference (^∗P < 0.05, ^∗∗P < 0.001) between the test groups and CK.

Discussion

Changes of Ca²⁺ and Mg²⁺ content in water not only affect the survival and growth of bivalves, but also affect their physiological responses. ISW in northwest China, especially the Gansu area (a target area for the transplantation of S. constricta), is characterized by broad variation in pH, carbonate alkalinity, and Ca²⁺ (0.775 mmol⋅L^-1–6.825 mmol⋅L^-1) and Mg²⁺ (3.07 mmol⋅L^-1–33.69 mmol⋅L^-1) content. Studying the ion tolerance range of S. constricta is the first and crucial step toward transplanting S. constricta cultures to ISW.

There is a significant difference in the tolerance of Ca²⁺ and Mg²⁺ between different invertebrate species. Zhou et al. (2007) reported that the optimal concentrations of Ca²⁺ and Mg²⁺ for the survival of L. vannamei larvae were 0.76–8.78 mmol⋅L^-1 and 0.36–35.51 mmol⋅L^-1, respectively. Roy et al. (2007) found that Mg²⁺ concentrations are very important for shrimp. The concentration of Mg²⁺ in inland saline-alkali water is at least 25% higher than that of seawater at the same salinity. Sprung (1987) reported that if environmental Ca²⁺ concentrations are less than 0.3 mmol⋅L^-1, the veliger larvae of the zebra mussel (Dreissena polymorpha) cannot survive. Furthermore, the New Zealand mud snail (Potamopyrgus antipodarum) is reported to have a minimum Ca²⁺ concentration tolerance of 0.125 mmol⋅L^-1 (Herbst et al., 2008). Lin et al. (2012) studied the effects of acute Ca²⁺ and Mg²⁺ concentrations on the survival of C. sinensis to assess the possibility of cultivating C. sinensis as a cultured species in ISW. It was found that when Ca²⁺ concentrations were between 0.11 mmol⋅L^-1 and 115 mmol⋅L^-1, and Mg²⁺ concentrations were between 0 mmol⋅L^-1 and 126.67 mmol⋅L^-1, the C. sinensis survival rates were 66 and 91.5%, respectively. It was determined that Mg²⁺ concentration may not be a primary limiting factor for transplanting C. sinensis in ISW. Our findings are similar to those of Lin et al. (2012) namely that JSC can maintain a high survival rate for at least a month when under Mg²⁺ (0 mmol L^-1) stress. However, the tolerance of JSC to high concentrations of Mg²⁺ is far less than that of C. sinensis. It is worth noting that the maximum tolerated concentration of JSC for Mg²⁺ is also slightly lower than the maximum measured value of the target-ISW. This suggests that perhaps Mg²⁺ be one of the limiting factors to the S. constricta transplantation. Thus special attention have to be paid to whether the concentration of Mg²⁺ ions too high when practicing transplantation. Mg²⁺-free medium have little effect on the survival rates of JSC and C. sinensis; this may be caused by the presence of dietary Mg²⁺ ions, or may indicate that the effect of free Mg²⁺ on survival is a long-term process. Because Dietz et al. (1994) have reported that D. polymorpha depleted of Mg²⁺ did not survive beyond 51 days. JSC can survival in a wide range of concentrations of Ca²⁺ (0.19–19.46 mmol⋅L^-1), suggesting that transplantation of JSC to target-ISW may without effect of Ca²⁺, and could be performed in a broad range of Ca²⁺ concentrations.

The shell of bivalves serves as the first barrier to resist external threats, and the main component of the shell is calcium carbonate. The growth of shells is directly related to the growth of the bivalve. Since Ca²⁺ is the main cation involved in the shell formation of bivalves, it was chosen in previous growth models (Hincks and Mackie, 1997). Most of the environmental calcium (approximately 80%) that is actively taken up from the water is deposited in the shell (Van Der Borght and Van Puymbroeck, 1966). However, these reactions are reversible, and under condition of low Ca²⁺ concentrations, calcium is removed from the shell (Van Der Borght and Van Puymbroeck, 1966). Accordingly, in this test we observed that JSCs died from calcium carbonate shell loss when Ca²⁺ was less than 0.19 mmol⋅L^-1. Such a situation may stem from the results of Ca²⁺ regulation. On the one hand, it is the release of Ca²⁺ in the shell caused by the efflux of free Ca²⁺ from the hemolymph and shell-mantle fluid. On the other hand, the efflux of Ca²⁺ also causes an increase in free amino acids and acidic metabolites in the body. These acidic end products are buffered by the mobilization of Ca²⁺ from the shell (Akberali et al., 1977). In addition, Wu et al. (2003) studied the effect of Ca²⁺ on the activity of digestive enzymes in S. constricta. It was found that an imbalance of Ca²⁺ concentration significantly decreased the activities of amylase and protease, thereby inhibiting growth. The higher Ca²⁺ concentration range of target-ISW 6.49–6.825 mmol⋅L^-1 and 3.24–6.825 mmol⋅L^-1 may significantly inhibit the shell length and body weight growth of JSC, respectively. This suggest that transplanting S. constricta in the target-ISW may not have growth inhibition caused by Ca²⁺ deficiency, and there may be only a physiological response by excessive Ca²⁺. Other hand suggest that the effect of Ca²⁺ on the growth of JSC may be more than one aspect, which weight is more likely to be affected than shell.

Mg²⁺ is the most abundant divalent ion within living cells. It is a cofactor in many enzymes, and its free ionic concentration regulates metabolic and shell formation processes (Bentov and Erez, 2006; Roy et al., 2010; Marin et al., 2012; Suzuki and Nagasawa, 2013; Wilbur and Yonge, 2013; González-Vera and Brown, 2017). However, Wu et al. (2003) found that the concentration of Mg²⁺ in the water did not affect the digestive enzyme activity of S. constricta, indicating that the effects of Mg²⁺ on JSC growth likely involves the osmotic and ionic regulation caused by Mg²⁺ imbalance and its role as the activity center of metabolic and mineral enzymes. From the Mg²⁺ concentration distribution of the target-ISW, the higher Mg²⁺ concentration range 14.77–33.69 mmol⋅L^-1 will significantly inhibit the shell length and body weight growth of JSC. This suggest that transplanting S. constricta in the target-ISW will not have growth inhibition caused by Mg²⁺ deficiency, and there may be only a response caused by excessive Mg²⁺.

Most evidences in bivalves is based on observations that on simulative conditions, oxygen consumption rapidly rises to levels above normal (Moon and Pritchard, 1970; Akberali et al., 1977; Almada-Villela, 1984). Both cell volume regulation and taurine efflux are inhibited in Noetia ponderosa erythrocytes and Glycera dibranchiata coelomocytes exposed to Ca²⁺-free hypoosmotic artificial seawater, and both are potentiated in high Ca²⁺ hypoosmotic artificial seawater (Smith and Pierce, 1987). Divalent cations also stabilize the cell membrane. The increased membrane permeability to taurine caused by Ca²⁺-free isosmotic media may be due to a lack of divalent cations that bind to and stabilize membranes (Swinehart et al., 1980). Not difficult to imagine the volume regulation of JSC haemocytes is extremely active, it may be the main reason sustained increase in the metabolism. Meanwhile, we found that the oxygen consumption increases within 24 h but it decreases sharply in 48 h. This suggest that high Ca²⁺ and Mg²⁺ stresses affects ventilatory activity causing internal hypoxia at 48 h. This may be the main reason for the rapid death of JSCs. On the contrary, Lee et al. (2005) noted that in Mg²⁺-free artificial seawater, the respiration rate of hard clams is decreased. No significant changes in oxygen consumption were observed in the DCMg-G1 group (Mg²⁺-free group), and it is likely that this inhibitory effect will take longer in JSCs.

When osmotic concentration and the ionic composition of the extracellular fluid be changed, the cells release cytoplasmic ions and organic osmolytes into the extracellular fluid to eliminate the osmotic gradient across the plasma membrane (Deaton, 2008; Larsen et al., 2014). Changes in environmental Ca²⁺ and Mg²⁺ have important regulation for both inorganic ion and organic osmotic regulation. In research of G. dibranchiata, it was found that the lack of Ca²⁺ ions in the environment did not lead to the efflux of Na⁺ and Cl^-, but it caused the efflux of K⁺ (Costa and Pierce, 1983). Comparing our results, it suggesting that the effect of extracellular fluid Ca²⁺ concentration on NKA activity may be directly related to the flux of K⁺. We believe that the ionic regulation caused by Ca²⁺ may be much more than that. A decrease in the concentration of Mg²⁺ ions in water tends to inactivate NKA in shrimp (Roy et al., 2010). Similar results were also seen in our study, but Watts and Pierce (1978) reported that the ATPase in Modiolus demissus ventricles requires a minimum of 5 mM Mg²⁺ in body to maintain constant activity. These suggest that S. constricta may existence very high regulation ability. In addition, when cultured in Mg²⁺-free water, the NKA activity in JSC is not completely lost, as Mg²⁺ may be obtained through dietary Mg²⁺ ions in food (algae). Mollusc farming does not utilize compound feed directly, like shrimp farming, so the impact of dietary Mg²⁺ is difficult to eliminate. Of course, the target-ISW does not have the Mg²⁺-free type of water, and the target-ISW does not significantly inhibit the NKA activity when the Mg²⁺ content is the lowest. But the higher Mg²⁺ concentration range 14.77–29.54 mmol⋅L^-1 may significantly increase the NKA activity of JSC. In addition, the Ca²⁺ concentration range of target-ISW may not significant impact on NKA activity of JSC. This suggest that when transplanting S. constricta in the target-ISW, Mg²⁺ may be one of the main factors to induce individuals to produce intense osmotic and ionic regulation.

Mg²⁺ operates synergistically to facilitate glutamate dehydrogenase activity and interactions between this glutamate dehydrogenase and aminotransferase (Fahien et al., 1985). In bivalve, inorganic ions are the first efflux components that respond to ambient medium cation ions (Na⁺, K⁺, Ca²⁺, and Mg²⁺) balance changes (Akberali et al., 1977; Shumway, 1977; Smith and Pierce, 1987). The efflux of inorganic ions induces the osmotic regulation from the organic components (free amino acids). The regulation of divalent cations in JSC may affect the concentration of free amino acids and may be directly related to changes in AST activity. Decreases and increases in Mg²⁺ concentration directly affect AST activity, and these results were recapitulated in our study. However, decreases and increases of Ca²⁺ concentration inhibit AST activity in JSC. So from the Ca²⁺ and Mg²⁺ concentration distribution of the target-ISW, we can imagine that both the higher Ca²⁺ (6.49–6.825 mmol⋅L^-1) and Mg²⁺ (14.77–33.69 mmol⋅L^-1) concentrations range may significantly affect AST activity. This indicating that changes of Ca²⁺ concentration in water not only affect digestive enzyme activity but also affect enzyme activity related to protein metabolism, thereby inhibiting growth (Wu et al., 2003).

The imbalance between Ca²⁺ and Mg²⁺ in water leads to increased oxygen consumption in aquatic animals, resulting in the production of more oxygen free radicals (Lin and Kao, 2000; Culotta, 2001). The increase in oxygen free radicals induces an increase in SOD activity to scavenge these radicals. In the present study, SOD activity is only augmented at moderately low calcium concentrations, which are coincident with high NKA activity and partial restoring of shell growth. SOD is also increased at the highest concentrations of both ions, which are very stressful conditions. But in target-ISW may be only the higher Mg²⁺ concentration range (14.77–33.69 mmol⋅L^-1) may significantly increase the SOD activity of JSC. This suggest that Mg²⁺ may cause excessive metabolism and produce a large amount of oxygen free radicals when transplanting S. constricta in the target-ISW.

The AChE is a hydrolase for the signal termination at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine (Quinn, 1987). AChE paly as a important role in neurotransmission process also in fish and invertebrates (Fulton and Key, 2001). The inhibition of AChE activity has been used widely as a biomarker of exposure to organotoxic substance and heavy metal ions in mollusk (Rickwood and Galloway, 2004; Devin et al., 2017). In addition, Pfeifer et al. (2005) reported that the activity of AChE in Mytilus sp. is sensitive to environmental salinity. Although the effect of the concentration of Ca²⁺ and Mg²⁺ on AChE activity in mollusc has not been clearly reported, in fish, Tomlinson et al. (1981) reported that AChE activity reached maximum saturation when both Ca²⁺ and Mg²⁺ concentrations were on the order of magnitude of 10 mmol, and the activity level were maintained at more higher Ca²⁺ and Mg²⁺ concentrations. The same situation be found in our study, AChE activity was continuously increased with increasing concentrations of Ca²⁺ and Mg²⁺ in water. Although there are some differences in the physiological functions of fish and bivalve, the physiological roles and functional mechanisms of AChE (the perspective of heavy metal ion binding) are relatively conservative. This indicating that the molecular mechanism of bivalve and fish AChE are similar, and the difference between the them may occur in the magnitude of the cation strength affecting the enzyme activity. In our study, indicating that the neurotransmission process of JSC be significant inhibition when the concentration of Ca²⁺ higher than 6.49 mmol L^-1 and concentration of Mg²⁺ higher than 14.77 mmol L^-1. Then the corresponding physiological processes of neuromodulation are inhibited. Obviously, this effect may be existed in both the higher Ca²⁺ (6.49–6.825 mmol⋅L^-1) and Mg²⁺ concentration range (14.77–33.69 mmol⋅L^-1) of target-ISW.

Lysozyme is a typical immunoenzyme that specifically lyses bacteria (Bachali et al., 2002; Xue et al., 2010). Usually LZM is divided into two categories, based on whether it can be combined with Ca²⁺ (Jielian et al., 2017). In mollusc, there is a class of LZM that binds to Ca²⁺ and interacts with calcium carbonate to participate in the shell formation process (Jimenez-Lopez et al., 2003). However, in mollusc is reported that LZM activity has a low tolerance to Ca²⁺ and Mg²⁺ concentrations (Nilsen et al., 1999; Olsen et al., 2003). In addition, the results of SOD and phagocytosis in this study also reflect the immune response of JSCs. So, we propose that the effects of Ca²⁺ and Mg²⁺ on the immune capacity of JSC are comprehensive. We suggest that (1) Ca²⁺ concentration (lower than 0.28 mmol L^-1, higher than 19.46 mmol L^-1) and Mg²⁺ concentration (higher than 14.77 mmol L^-1) affect metabolic immune enzymes; (2) function of haemocyte phagocytosis will be inhibited when the concentration of Ca²⁺ and Mg²⁺ in hemolymph sharply rise or fall in short period of time. Because adhesion and aggregation function be limited (Chen and Bayne, 1995); (3) Ca²⁺ concentration (lower than 0.93 mmol L^-1, higher than 6.49 mmol L^-1) and Mg²⁺ concentration (lower than 0.37 mmol L^-1, higher than 14.77 mmol L^-1) affect antibacterial enzyme activity. Therefore, this means that when the S. constricta is transplanted to the target-ISW, both the higher Ca²⁺ (6.49–6.825 mmol⋅L^-1) and Mg²⁺ (14.77–33.69 mmol⋅L^-1) concentration range may significantly enhances the metabolic immunity, but the cellular immunity (haemocyte) and bacterial immunity are weakened.

The Ca²⁺/Mg²⁺ ratio is often reported in shrimp and has important effects on survival, growth and physiology (Pan et al., 2006; González-Vera and Brown, 2017). The Ca²⁺/Mg²⁺ ratio not only has strong species specificity, but also has been widely researched in shrimp species. Tavabe et al. (2013) found that a Ca²⁺/Mg²⁺ ratio of 0.8 was optimal for larval development, while Latif (1992) found that a 1:1 Ca²⁺/Mg²⁺ ratio was desirable for juveniles of Macrobrachium rosenbergii. This suggests that the Ca²⁺/Mg²⁺ ratio is an important indicator for shrimp, but further study is needed to elucidate its importance in other aquatic species. The Ca²⁺/Mg²⁺ ratio in mollusc has not been reported on. We do not have definitive evidence to obtain the significant correlation between Ca²⁺/Mg²⁺ ratio and the survival, growth, and physiology of JSC, but this does not mean that Ca²⁺/Mg²⁺ ratio has no effect, so this requires future validation with independent test.

Over the past 250 years, atmospheric carbon dioxide (CO₂) levels increased by nearly 40% (Solomon et al., 2007). Ocean CO₂ uptake, causes pH reductions and alterations in fundamental chemical balances that together are commonly referred to as ocean acidification (Millero et al., 2009). Orr et al. (2005) predicted that ocean acidification will cause a pH drop of 0.3–0.4 for the 21st century, is equivalent to approximately a 150% increase in H⁺ and 50% decrease in CO₃^2- concentrations. Marine carbonates are mainly in the form of CaCO₃ and MgCO₃, which means that the concentration of Ca²⁺ and Mg²⁺ in the 21st century will increase within 50% than it is now. Comparing our findings, we can see that such an increase may not have a negative impact on the survival, growth and physiology of JSC. However, the negative impact of ocean acidification on the survival and growth of corals and molluscs is a proven fact (Hoegh-Guldberg et al., 2007; Fabry et al., 2008). Ocean acidification is a complex environmental change process involving not only changes in the concentration of metal cations, but also pH, pCO₂, CO₃^2-, temperature and marine food chains (Fabry et al., 2008). Therefore, we believe that other factors or comprehensive factors other than Ca²⁺ and Mg²⁺ may eventually lead to the negative impact of ocean acidification on the survival and growth of molluscs.

Conclusion

Stress caused by changes in the concentration of Ca²⁺ and Mg²⁺ in the environment may cause efflux and reflux of Ca²⁺ and Mg²⁺ in JSC. Metabolism and oxygen consumption are rapidly increased but haemocyte phagocytosis are decreased in short time. At this time, a large number of JSCs died when Ca²⁺ and Mg²⁺ exceed physiological limits. In the long-term stress, the imbalance both Ca²⁺ and Mg²⁺ may cause a comprehensive physiological response including ionic regulation, metabolic levels, neurotransmission process, and immunity. Then ultimately affect growth and even survival. When using such a “standard ruler” to measure the effect of Ca²⁺ and Mg²⁺ concentrations in the target-ISW on S. constricta, we found that only high concentrations of Ca²⁺ (3.24–6.825 mmol⋅L^-1) and Mg²⁺ (14.77–33.69 mmol⋅L^-1) had significantly impact on transplantation practice. In physiology, such effects include accelerated ionic regulation, accelerated metabolism, increased oxygen free radical production, increased metabolic immunity, decreased bacterial and cellular immunity, and inhibit neurotransmission process. The concentrations of Ca²⁺ and Mg²⁺ in the target-ISW does not cause all S. constricta to die in the short term, but it inhibits growth and causes a part of individual death during long-term aquaculture. Those implies that we have to choose the right concentration of Ca²⁺ and Mg²⁺ or to improve the water quality in target-ISW. Fortunately, the Ca²⁺ and Mg²⁺ content of most area target-ISWs is suitable for long-term transplantation of S. constricta. Our study also suggests that the increase in Ca²⁺ and Mg²⁺ ion concentrations caused by ocean acidification will not affect the survival, growth and physiology of S. constricta.

Data Availability

All datasets generated for this study are included in the manuscript and/or the supplementary files.

Ethics Statement

In this study, the animals were artificially propagated as juvenile clams.

Author Contributions

MP was responsible for experimental design, test operations, data processing, and manuscript writing. ZL was responsible for data processing and manuscript modification. XL was responsible for experimental design. BY was responsible for test operations. TL was responsible for experimental data collection. JL, DN, and ZD were responsible for experimental program guidance.

Funding

This study was supported by grants from the National Natural Science Foundation of China (Grant No. 31101897), the “863” Hi-tech Research and Development Program of China (Grant No. 2012AA10A400-3), and the Shanghai Universities Knowledge Service Platform (Grant No. ZF1206).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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