Osmotic regulation of free amino acid for adaptation to high salt in razor clam

doi:10.21203/rs.3.rs-2610164/v1

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Osmotic regulation of free amino acid for adaptation to high salt in razor clam

https://doi.org/10.21203/rs.3.rs-2610164/v1

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Free amino acids play an important role in osmotic regulation induced by high salt. In order to study the effect of free amino acids on the osmotic regulation of Sinonvacula constricta cells, the salinity was set at 20 and 32 in control group and experimental group. In the experimental group, proline, taurine and alanine amino acid solid powder with concentration of 5ug/ml was added. Serum and cell samples were taken from each group at different time points for detection by osmotic pressure and detection by flow cytometry. In summary, we found that serum osmolality, total free amino acid and single free amino acid content increased with the increase of salt stress, and remained at a high level. Under high salt conditions, the osmotic pressure in S. constricta could not equilibrate with the seawater environment. In the later stages of stress, the gap between the two gradually widened. Under the condition of amino acid addition, the osmotic pressure is gradually the same as that of foreign seawater. Furthermore, the mean number of fluorescent channels produced by high salt downflow cytometry was significantly lower than that of the other amino acid groups. Among the three amino acids, proline has the largest accumulation in the S. constrictaand the shortest regulation time for osmotic balance. It has been shown that free amino acids can help better osmotic regulation of hypersaline S. constricta. The results of this study provide a reference for exploring the physiological mechanism of the amino acid on salt tolerance in bivalves.

Sinonvacula constricta

High salt

Osmotic adjustment

Free amino acid

Proline

Salinity is one of the most significant environmental factors affecting marine organisms, as it can influence metabolism, growth, and osmoregulation (Ruan et al., 2022). Extreme events (e.g., tides, rainfall, high temperature, and drought) can increase the frequency and severity of seawater salinity fluctuations in coastal areas and estuaries. Leading to changes in salinity, resulting in varying degrees of shellfish damage (Carregosa et al., 2014). Most of the marine shellfish in the world's economy are distributed or farmed in intertidal areas, tidal flats, or estuaries. So did S. constricta. Therefore, changes in salinity are crucial to the growth and life of mollusks (Gagnaire et al., 2006). In order to adapt to changes in salinity, marine species living in the coastal intertidal zone must be able to change their physiological processes. Most aquatic invertebrates conform to osmosis. This demonstrates that their osmotic pressure is close to that of seawater, and that they do not have the capacity to adjust the osmotic pressure of the inner media (Willme, 2002). Although the patterns manifested and the challenges posed by the variations in each environment are distinct, in general they tend to converge on one common feature, osmotic variation. The balance of water gain and loss in aquatic organisms depends primarily on the movement of water across cell membranes, and this is known as water osmosis (Medeiros et al., 2020). Osmotic pressure is another example of a biophysical cue that modulates cell function. Osmotic pressure changes have been widely associated with growth arrest and proliferation in plants and seeds (Vicente et al., 2020), and more recently in bacteria (Lv et al., 2020) and yeast (Abuhattum et al., 2018). In different osmotic environments, animals use complex regulatory mechanisms to maintain the stability of water and solutes in their bodies. It is to make the osmotic pressure difference of body fluids stable within a certain range. This is the osmotic pressure regulation of body fluids, also known as osmotic regulation (Ortiz, 2001).

The hemolymph concentration of ions and free amino acids, accompanied by metabolites from the blood, accounts for most of the hemolymph osmotic pressure (Chen et al., 2020; Dalla et al., 1986). Free amino acids (FAA) play important roles in determining the cell volume and osmoregulatory processes of many organisms, particularly mollusca and crustacea, fish, amphibians, and reptiles (McNamara et al., 2004). Intracellular accumulation of free amino acids is a common response of many organisms to changes in ambient salinity (Yancey et al., 1982; Hare et al., 1998; Kempf et al., 1998; Gilles et al., 1997). Free amino acids that accumulate in response to hyperosmotic stress are called compatible osmoles because they can regulate cell volume and stabilize cellular macromolecules (Wang et al., 2012). Numerous studies on marine and brackish water bivalves have reported changes in the FAA content when the animals are subjected to osmotic stresses (Deaton et al., 1984; Heavers et al., 1985; Hosoi et al., 2003; Kube et al., 2005). It was found that the cells of Crassoas gigas were significantly increased in glycine, alanine, p-alanine, proline, arginine and taurine during hyperosmotic adaptation. Among them, alanine increases immediately, which is important for short-term adaptation to hyperpermeability. Taurine, on the other hand, increases slowly and in large quantities, helping to adapt to high permeability for a long time (Hosoi et al., 2003). It was found that the total FAA content of Atlantic salmon in fresh water was significantly lower than that in seawater (Duan et al., 2021). As environmental salinity increases, sodium and chloride are absorbed to up-regulate intracellular osmolarity, and the intracellular free amino acid level increases through synthesis and protein break-down. We found that L-aspartic acid in the 35‰ group was decreased. It may be used as an energy source for osmoregulation (Jiang et al., 2019). On the other hand, osmole systems in freshwater bivalves have rarely been reported, probably because the necessity of osmole systems is apparently less in freshwater bivalves due to their lower and more stable internal osmolality (Hosoi et al., 2008). However, a few studies have indicated that, despite the lower necessity, freshwater bivalves possess the ability to increase some FAAs under hyperosmotic conditions. In the freshwater clam Lampsilis teres, the FAA content in the gills increased with acclimation to high salinity (Jordan et al., 1999). In this study, the FAA pool increased from 20 to 115 mmol/g dry weight after a 70h exposure to water of salinity 5.0 (0.5% saline water) in Corbicula manilensis (Gainey, 1978). Previous studies indicate that proline, alanine, glutamic acid, glycine, and taurine play important roles in the osmoregulation of crustaceans but also differ among species (Willett et al., 2003). That the accumulation of alanine is the primary mechanism for initial increases in cytoplasmic osmotic concentration during hyperosmotic volume regulation in bivalves has long been accepted as dogma. Betaine is probably a major component of the osmole pool in most euryhaline bivalves (Deaton et al., 2001). Under salinity stress, an osmole such as praline maintains cellular homeostasis through osmotic regulation and induces physiological processes favorably. The role of phytohormones under salinity stress is critical in modulating physiological responses that eventually lead to adaptation of plants to an unfavorable environment (Iqbal et al., 2014). Similarly, increase in the accumulation of glycine and taurine levels in FAA in Crassostrea gigas was reported when oysters exposed to increased salinity from 30 to 39 (Lee et al., 2004). Furthermore, the FAA concentration enlarged by anaerobic mechanism during hyperosmotic adaptation which caused a decrease in oxygen uptake of ventricles (Henry et al., 1980). Constant salinity fluctuation resulted in the net protein loss which contributed to cell volume regulation (Haider et al., 2018).

S. constricta lives in estuarine and intertidal zones and is highly susceptible to salinity changes. However, there has not been much research on how amino acids assist in the osmotic regulation of S. constricta under high salt levels. In this study, the osmotic pressure of S. constricta was compared with that of S. constricta under different amino acid treatments and high salt stress. And the effects of different amino acids on cell relative volume under high salt stress were compared. The osmotic reaction mechanism of amino acid helping S. constricta adapt to high salt environment was discussed.

2.1 Experimental samples

Razor clams were obtained from Donghang Farm, Zhejiang Province, China. We chose healthy clams with an average length of 4.0-0.5cm. The razor clams were kept temporarily for a week in the laboratory at room temperature and a salinity of 20. In the laboratory, razor clams were cultured in a tank (40 cm length × 40 cm width × 65 cm height) with artificial seawater (70 L). The artificial sea salt was used to adjust salinity.

2.2 Sample collection and design of experiment

Razor clams were randomly selected for the salinity challenge experiment. The salinity values of the two treatments were set at 20 and 32, which represented the control and experimental groups, respectively. In order to study the role of amino acids in the osmotic regulation mechanism of the clam, an exogenous amino acid supplement experiment was designed. That is, there was another high-salt group, but the seawater was fortified with the three amino acids most involved in osmoregulation in living organisms. Thy are proline (Pro), taurine (Tau) and alanine (Ala).

According to literature review, seawater and proline, taurine and alanine solids (C2H7NO3S, Shanghai Macklin Biochemical Co., LTD., Shanghai, China) were prepared with a concentration of 5mM. Aerated fresh water and artificial sea salt were used to prepare the experimental seawater (32‰). Razor clams were selected randomly and divided into five groups in the experiment, which were control group, high salt group, proline group, taurine group and alanine group, respectively. They were 40 razor clams in each group, which were placed in a tank (0.60 m×0.40m×0.20 m) filled with 6 L seawater with animo added. The razor clams were transferred from holding conditions (20‰) to salinity treatments directly. Then, razor clams were collected to extract hemolymph and gill tissue in the test tube at 0, 3, 6, 12, 24, 48, and 72 h. The hemolymph should be placed in an ice box after extraction Three samples of gills were taken at each time point and stored at − 80℃ after liquid nitrogen flash freezing for tissue enzyme analysis. The biology of each group was repeated three times.

2.3 Measurement of serum osmotic pressure

The obtained hemolymph samples were immediately centrifuged at 4℃ at 3000 rpm for 10 min, and the supernatant was collected for detection. The osmotic pressure was measured by a Freezing point osmometer (Osmomat 030, Gonotec GmbH, Berlin, Germany) (Jiale et al., 2016).

2.4 Detection of amino acid content under high salt stress

After the gill tissue samples taken in 2.2 were thawed in the icebox, 9 volumes of PBS buffer at a weight-to-volume ratio of 1,9 were added. The sample was then mechanically centrifuged at a low temperature (4°C), and the supernatant was collected. The concentration of proline, taurine, alaine, and isoleucine in gills of the 3 groups was assayed by the corresponding ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) with double antibody one-step sandwich method (Ma et al., 2021).

2.5 Effect of proline, taurine and alanine on cell relative volume

The fresh gill tissue was immersed in PBS, and the cell suspensions were obtained by cell cleaning and gradient sterilization according to the cell extraction steps using PBS, 75% alcohol, and PBS containing triple antibodies. Finally, the samples were analyzed by flow cytometry (Li et al., 2019).

2.6 Measurement of cell volume

When the cells are ejected from the nozzle of the flow cytometer in a single row, the light will be scattered by laser irradiation. The FSC is mainly proportional to the cell volume, that is, the larger the cell size, the stronger the scattering of light.

The above cell suspension was mixed with 0∙5ml and 10,000 cells were counted by flow cytometry for detection. Meanwhile, the average strength of FSC was calculated (Jordan et al., 1996).

2.7 Data analysis

All of the data was presented as the mean ± standard deviation (S.D.). Draw graphics with Origin 2018 (Learning edition, OriginLab, Northampton, Massachusetts, USA) software. Flow cytometry results were analyzed using FlowJo 10.6.2 (BD, USA) software. One-way analysis of variance (ANOVA) was performed using SPSS software (SPSS 23, IBM SPSS software, Armonk, NY) to identify significant differences between the experimental and control groups. Differences were considered significant at P < 0.05.

3.1 Effects of high salt stress on total free amino acid (FAA) and serum osmotic pressure

Figure 1 shows that salinity stress had significant effects on total free amino acid (FAA) content and osmotic pressure changes in S. constricta (p<0.05). The results showed that the FAA content in S. constricta was always higher than the salinity of 20 at a salinity of 32. FAA levels at high salt (32‰) have been maintained at high levels. The serum osmolality of S. constricta increased significantly after high salt stress. The osmolality detected at high salt (32‰) remained at a high level with prolonged stress time. After 12h, the serum osmal pressure of S. constricta gradually stabilized at high salt (32‰). But there is still significant difference with the osmotic pressure of seawater (p<0.05).

3.2 Changes in free amino acid content under high salt stress

Figure 2 shows the increase of amino acid content in S. constricta under normal salinity and high salt. The results showed that the content of isoleucine (IIE), proline (Pro), taurine (Tau) and alanine (Ala) was significantly higher than the control group (P<0.05) in S. constricta after high salt stress. The accumulation of proline in S. constricta was the highest in both normal salinity and high salinity. Next comes alanine, then isoleucine, with taurine the lowest. As can be seen from Figure 2-c, the increase level of proline is also significantly higher than that of other amino acids after high salt stress (P<0.05). It increases the most after 3 hours. The rest of the time has also been at high levels.

3.3 Effects of exogenous amino acids on the serum osmotic pressure of S. constricta

Figure 3 (a, b and c) shows the changes of serum osmal pressure and external seawater osmal pressure in S. constricta after adding three different exogenous amino acids under the premise of high salt treatment. The results showed that the serum osmotic pressure in S. constricta could not be balanced with the external environmental osmotic pressure due to high salt. Under the condition of the addition of exogenous amino acids, the serum osmotic pressure and the external water gradually tended to be balanced.

The results show that after adding proline, taurine and alanine, the osmotic pressure of seawater increased. The serum osmotic pressure in S.constricta also increased sharply and it's always at a high level. With the extension of stress time, the osmotic pressure of the serum after the addition of exogenous amino acids was gradually the same as that of the external water. However, at later stages, the difference between serum osmolality and seawater osmolality at high salt (32‰) became larger. There were significant differences between the exogenous amino acid control group and the high salt group in each group (P<0.05).

The comparison of three different treatment groups is shown in Figure 3-d. The results showed that the adjustment time between serum osmolality of S. constricta and external seawater was the shortest after adding proline. After about 6 hours the osmotic pressure is almost the same. Next came the alanine addition group. Finally, it took about 12h for S. constricta to adjust osmotic pressure balance after taurine was added. There were significant differences between the exogenous amino acid control group and the high salt group in each group (P<0.05).

S_20/32-Serum represents the serum osmolality of S. constricta at a salinity of S_20/32. S₃₂T₁-Serumindicates the change in serum osmolality of S. constricta after the addition of proline. S₃₂T₂-Serum indicates the change in serum osmolality of S. constricta after the addition of taurine. S₃₂T₃-Serum indicates the change in serum osmolality of S. constricta after the addition of alanine.

S_20/32-Seawater represents the osmotic pressure of seawater at a salinity of S_20/32-Seawater. S₃₂T₁-Seawater indicates the change in seawater osmolality after the addition of proline. S₃₂T₂-Seawater indicates the change in seawater osmolality after the addition of taurine. S₃₂T₃-Seawater indicates the change in seawater osmolality after the addition of alanine.

a, b and c represent significant differences between different groups at the same time with different

letters (P<0.05).

3.4 Effects of proline, taurine and alanine on the relative volume of cells

Gill cells were taken at 3h, 6h, 12h, 24h, 48h and 72h. The mean FSC intensity of these cells was measured. The results are shown in the table 1. By comparing the results at the same time point, it was found that the average channel number of fluorescence intensity under high salt stress was significantly lower than that under control and other treatment conditions. It means that the cell volume under high salt is significantly smaller than that under control and other treatment groups. With the extension of stress time, the fluorescence intensity of three different amino acid treatments gradually approached.

Table 1 Test results of gill cell volume of S. constricta in different treatment groups

Cell Volume*（×10⁶）
Time (h)	20 ppt	32 ppt	Proline	Taurine	Alane
3	1.42	1.38	1.44	1.47	1.40
6	1.47	1.40	1.45	1.55	1.40
12	1.45	1.37	1.38	1.43	1.55
24	1.31	1.24	1.27	1.27	1.30
48	1.21	1.17	1.27	1.27	1.36
72	1.21	1.17	1.23	1.23	1.22

* Cell volume is expressed as the number of channels of average strength of its FSC. The values in the table are going to end up ×10⁶. The chart shows the number of channels of average fluorescence intensity of flow cytometry after passing through gill cells in different treatment groups at different time points.

The adaptability of marine shellfish to changes in environmental salinity is very important. For intertidal species, the salinity of the environment changes greatly every day, and shellfish need to adapt rapidly through self-regulation (Li et al., 2021). In natural environment, most of S. constricta live in brackish waters near estuaries. In the summer, rainfall or drought can lead to large variations in salinity in different coastal areas. The sudden change in salinity can cause an imbalance in the osmotic regulation system of S. constricta (Maoxiao et al., 2019; Wang et al., 2022). S. constricta, as a euronhalic bivalve, can also adapt to salinity mutations. But when salinity exceeds its ability to self-regulate, physiological metabolism can be disturbed. Long-term high salt inadaptation will lead to osmotic pressure imbalance (You et al., 2003).

4.1 Effects of high salt stress on total free amino acid (FAA) and serum osmotic pressure

The importance of total free amino acids (TFA) as osmotic effectors in vivo for osmotic regulation has been demonstrated in many organisms. These include bacteria (MEASURES, 1975), algae, plants (Gilles et al., 1981), mollusks and crustaceans (Schoffeniels et al.,1972), fish (Goldstein, 1981), amphibians and reptiles (Gilles-Baillien, 1973). In this diverse geobiota, the changes in the extracellular fluid osmotic pressure are compensated by the changes in the extracellular fluid osmotic pressure, constituting an isotonic, intracellular, volume-regulating reaction (McNamara, 2004). Most of the observed changes in amino acid levels in organisms appear to be directly or indirectly involved in osmoregulation (Aragão et al., 2010). In this study, S. constricta after the salinity stress showed that the free amino acids could increase significantly. And the total free amino acids produced by high salt stress at 6 and 72 hours were the most different from the control group. At the same time, the serum osmotic pressure of S. constricta under high salt stress also had the largest difference with that of outside water. This indicated that S. constricta did not adapt to a high salt environment under transient salinity stress. S. constricta adjusts osmotic pressure by accumulating osmoregulatory substances in the body, such as the accumulation of free amino acids. In addition, S. constricta may adjust its osmotic pressure to adapt to the high salt environment by regulating the production and accumulation of total free amino acids in the body. It was speculated that the changes in FAA content in S. constricta had an effect on the changes in osmotic pressure.

4.2 Effects of exogenous amino acids on the serum osmotic pressure

Under hypertonic stress, eurhaline mollusks accumulate amino acids in cells mainly in the form of organic osmotic pressure (Shumway et al., 1977; Pierce et al.,1981). They can regulate intracellular osmotic pressure by rapidly releasing or accumulating the organic osmotic pressure of cells or cells, respectively. The goal is to prevent extreme cell volume expansion (hypotonic stress) or contraction (hypertonic stress) (Pierce et al., 1982; Yancey et al., 2005). A general observation in euryhaline molluscs under hyperosmotic stress is that the patterns of the individual free amino acids recruited for intracellular volume adjustments are diverse (Mantel et al., 1983). Quantitatively relevant osmoles in different species are taurine, isoleucine, alanine, and proline (Hosoi et al., 2003; Pierce et al., 1982). Isoleucine, proline, taurine, and alanine, as free amino acids, are often involved in the osmotic regulation of organisms. In this study, it was found that the accumulation of isoleucine, proline, taurine and alanine in the gill tissue of S. constricta was higher than that of the control group after high salt stress. Taurine may be used more in the regulation of energy metabolism, and osmoregulation is a process that requires a lot of energy metabolism (Chen et al., 2021). Many studies have shown that taurine accumulation is more involved in energy metabolism than directly involved in osmoregulation (Lin et al., 2021). In this study, the content of proline in S. constricta was significantly higher than the other three amino acids, followed by alanine, before and after high salt stress. The results showed that the accumulation of proline may be more involved in the osmotic regulation of S. constricts under the conditions of high salt (Kuzmina et al., 2010). In hard clams, taurine is the most abundant penetrant in bivalves and is directly involved in their energy metabolism to relieve osmotic pressure (Gilles et al., 1972; Kube et al., 2007; Livingstone., 1979). In this experiment, the original content and increment of proline were higher than other amino acids in S. constricta. Therefore, we speculated that proline may play an important role in the osmotic regulation of S. constricta.

Moreover, it has been established that the predominant types of FAAs differ among different species of osmoconformer bivalves. For example, in the soft-shell clam Mya arenaria (Haider et al., 2019), glycine has been shown to be the dominant FAA, whereas alanine was the most abundant FAA in the Japanese basket clam Corbicula japonica (Matsushima et al., 1984) and baltic clam Macoma balthica (Sokolowski et al., 2003). Taurine was found to be the most dominant FAA in hard clams (Lin et al., 2016). The occurrence of proline and alanine as the major contributors to the intracellular FAA pool of Ampelisca brevicornis (Costa, 1853) concurs with the work of Schoeniels & Gilles (1970) on other marine crustaceans (Schoffeniels et al., 1970). After 10 days of exposure, the total FAA in adult pagei's hemolymph and muscles increased by 25%. Mainly due to the elevation of alanine and proline. This may result from increased FAA synthesis or protein catabolism in the muscle (Augusto et al., 2007). In this study, the addition of exogenous amino acid nutrients such as proline, taurine, and alanine could help S. constricta perform osmotic regulation. With the extension of stress time, the serum osmolality of S. constricta was gradually close to that of seawater in different treatment groups. But the serum osmolality of S. constricta was not close to that of seawater under high salt conditions. High salt stress led to the inability of S. constricta to adjust the balance of osmotic pressure between themselves and the outside world. It further indicated that amino acids could effectively help S. constricta adapt to the environment under conditions of high salt.

4.3 Effects of proline, taurine, and alanine on the number, structure, and relative volume of cells

The cells of bivalve tissues exposed to hyperosmotic media accumulate organic molecules to reduce the osmotic gradient across the plasma membrane and restore cellular volume. At the beginning, alanine, proline and glycine accumulated continuously during the hypertonic volume regulation of pine algae. However, the accumulation of taurine in cells decreased slowly over time (Baginski et al., 1977). Consistent with previous studies on molluscs, we found that the increase in the basic amino acids in the cytoplasm in response to hyperosmotic stress is relatively small (Augusto et al., 2007). In order to adapt to changes or fluctuations in the salinity of surrounding seawater, osmotic-compliant marine animals, including S. constricta, have mechanisms to regulate osmotic pressure concentration and thus cell volume. Many studies have studied the osmotic regulation mechanism of bivalves, in which intracellular FAA plays a dominant role in intracellular osmotic pressure and cell volume (Hosoi et al., 2003). In this experiment, proline, taurine and alanine were added for follow-up experiments. They found that the number of channels of fluorescence intensity in cells subjected to high salt stress was significantly lower than in the control group and the other three different treatment groups. It indicated that the relative volume of cells under high salt condition was lower than that in control group and amino acid addition group. Therefore, we think that cells may regulate the relative size of cells through the accumulation or metabolism of amino acids. And studies have shown that large accumulations of organic permeable substances do not cause harm.

The purpose of this study was to investigate how to adapt to a high-salt environment through osmoregulation in S. constricta. Rapid changes in salinity can affect the osmotic pressure of S. constricta. The razor clams can regulate osmotic balance through amino acid accumulation and intracellular amino acid regulation. Compared with other free amino acids, proline was the most affected amino acid by osmotic regulation in S. constricta under high salt condition. The regulation time of serum osmotic pressure by proline was faster than that of other free amino acids. The results provided a reference for the study of the osmotic regulation mechanism of S. constricta in a high salt environment.

Acknowledgements

This work was supported by the National Key R & D Plan “Blue Granary Science and Technology Innovation” Special Project (2019YFD0900700) and the National Natural Science Foundation of China (31472278).

Declaration of competing interest

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript Author contributions.

Author's statement

Min Deng, Writing – original draft, Conceptualization, Methodology, Formal analysis, Sample collection, Conduct the experiment, Data collection and analysis. Honglei Wang and xinxin Du, Check of article. Yifeng Li and Yuan Li, Conceptualization, Formal analysis. Donghong Niu, Experimental program guidance, Writing - review & editing, Supervision. The manuscript is approved by all authors for publication.

Data availability

Data will be made available on request.

Abuhattum S, Kim K, Franzmann T M, et al (2018) Intracellular mass density increase is accompanying but not sufficient for stiffening and growth arrest of yeast cells, Front. Phys, 6, 131.
Aragão C, Costas B, Vargas-Chacoff L, et al (2010) Changes in plasma amino acid levels in a euryhaline fish exposed to different environmental salinities, Amino Acids, 38, 311-317.
Augusto A, Greene L J, Laure H J, et al (2007) Adaptive shifts in osmoregulatory strategy and the invasion of freshwater by brachyuran crabs, evidence from Dilocarcinus pagei (Trichodactylidae), J Exp Zool A Ecol Genet Physiol, 307, 688-698.
Baginski R M, Pierce S K (1977) The time course of intracellular free amino acid accumulation in tissues of Modiolus demissus during high salinity adaptations, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 57, 407-412.
Carregosa V, Figueira E, Gil A M, et al (2014) Tolerance of Venerupis philippinarum to salinity, osmotic and metabolic aspects, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 171, 36-43.
Chen J-C, Chia P-G (1997) Osmotic and ionic concentrations of Scylla serrata (Forskål) subjected to different salinity levels, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 117, 239-244.
Chen Y, Ye B, Niu D, et al (2021) Changes in metabolism and immunity in response to acute salinity stress in Chinese razor clams from different regions, Aquac., 19, 100624.
Dalla Via G J (1986) Salinity responses of the juvenile penaeid shrimp Penaeus japonicus, I. Oxygen consumption and estimations of productivity, Aquaculture, 55, 297-306.
Deaton L E, Hilbish T J, Koehn R K (1984) Protein as a source of amino nitrogen during hyperosmotic volume regulation in the mussel Mytilus edulis, Physiol. Biochem. Zool, 57, 609-619.
Deaton L E (2001) Hyperosmotic volume regulation in the gills of the ribbed mussel, Geukensia demissa, rapid accumulation of betaine and alanine, J. Exp. Mar. Biol. Ecol, 260, 185-197.
Duan Z, Dong S, Sun Y, et al (2021) Response of Atlantic salmon (Salmo salar) flavor to environmental salinity while culturing between freshwater and seawater, Aquaculture, 530, 735953.
Gagnaire B, Frouin H, Moreau K, et al (2006) Effects of temperature and salinity on haemocyte activities of the Pacific oyster, Crassostrea gigas (Thunberg), Fish Shellfish Immun, 20, 536-547.
Gainey Jr L F (1978) The response of the Corbiculidae (Mollusca, Bivalvia) to osmotic stress, the cellular response, Physiological Zoology, 51, 79-91.
Gilles R, Péqueux A (1981) Cell volume regulation in crustaceans, Relationship between mechanisms for controlling the osmolality of extracellular and intracellular fluids, J. Exp. Zool., 215, 351-362.
Gilles R (1972) Osmoregulation in three molluscs, Acanthochitona discrepans (Brown), Glycymeris glycymeris (L.) and Mytilus edulis (L.), Biol. Bull 142, 25-35.
Gilles R (1997) “Compensatory” organic osmolytes in high osmolarity and dehydration stresses, history and perspectives, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 117, 279-290.
Gilles-Baillien M (1973) Seasonal variations and osmoregulation in the red blood cells of the diamondback terrapin Malaclemys centrata centrata (Latreille), Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 46, 505-512.
Goldstein L (1981) Free amino acids and cell volume regulation in the elasmobranch, Raja erinacea, J. Exp. Zool., 215, 371-377.
Haider F, Sokolov E P, Sokolova I M (2018) Effects of mechanical disturbance and salinity stress on bioenergetics and burrowing behavior of the soft-shell clam Mya arenaria, J. Exp. Biol., 221.
Haider F, Sokolov E P, Timm S, et al (2019) Interactive effects of osmotic stress and burrowing activity on protein metabolism and muscle capacity in the softshell clam Mya arenaria, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 228, 81-93.
Hare P D, Cress W A, Van Staden J (1998) Dissecting the roles of osmolyte accumulation during stress, Plant Cell Environ, 21, 535-553.
Heavers B W, Hammen C S (1985) Fate of endogenous free amino acids in osmotic adjustment of Crassostrea virginica (Gmelin), Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 82, 571-576.
Henry R P, Mangum C P, Webb K L (1980) Salt and water balance in the oligohaline clam, Rangia cuneata II. Accumulation of intracellular free amino acids during high salinity adaptation, J Exp Zool, 211, 11-24.
Hosoi M, Kubota S, Toyohara M, et al (2003) Effect of salinity change on free amino acid content in Pacific oyster, Fish Sci 69, 395-400.
Hosoi M, Kubota S, Toyohara M, et al (2003) Effect of salinity change on free amino acid content in Pacific oyster, Fisheries Sci, 69, 395-400.
Hosoi M, Yoshinaga Y, Toyohara M, et al (2008) Freshwater bivalve Corbicula sandai uses free amino acids as osmolytes under hyperosmotic condition, Fisheries Sci, 74, 1339-1341.
Iqbal N, Umar S, Khan N A, et al (2014) A new perspective of phytohormones in salinity tolerance, regulation of proline metabolism, Environ. Exp. Bot., 100, 34-42.
Jiale L, Maoxiao P, Donghong N, et al (2016) Draw operating means of two shell shellfish hemolymphs, CN205322337U.
Jiang W, Tian X, Fang Z, et al (2019) Metabolic responses in the gills of tongue sole (Cynoglossus semilaevis) exposed to salinity stress using NMR-based metabolomics, Sci. Total Environ, 653, 465-474.
Jordan C T, Yamasaki G, Minamoto D (1996) High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations, Exp. Hematol, 24, 1347-1355.
Jordan P J, Deaton L E (1999) Osmotic regulation and salinity tolerance in the freshwater snail Pomacea bridgesi and the freshwater clam Lampsilis teres, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 122, 199-205.
Kempf B, Bremer E (1998) Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments, Arch. Microbiol, 170, 319-330.
Kube S, Gerber A, Schiedek D (2005) The key role of taurine for osmotic adaptation in two marine bivalve taxa (Mytilus spp. and Macoma balthica) along their European distribution. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USA: ELSEVIER SCIENCE INC, 2005, 141, S214-S214.
Kube S, Sokolowski A, Jansen J M, et al (2007) Seasonal variability of free amino acids in two marine bivalves, Macoma balthica and Mytilus spp., in relation to environmental and physiological factors, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 147, 1015-1027.
Kuzmina V V, Gavrovskaya L K, Ryzhova O V (2010) Taurine. Effect on exotrophia and metabolism in mammals and fish, J. Evol.Biochem. Phys+, 46, 19-27.
Lee N-H, Han K-N, Choi K-S (2004) Effects of salinity and turbidity on the free amino acid composition in gill tissue of the pacific oyster, Crassostrea gigas, Fish Shellfish Immunol., 23, 129-134.
Li Y, Niu D, Bai Y, et al (2019) Identification of a novel C1q complement component in razor clam Sinonovacula constricta and its role in antibacterial activity, Fish & shellfish Immunology, 87, 193-201.
Li Y, Niu D, Wu Y, et al (2021) Integrated analysis of transcriptomic and metabolomic data to evaluate responses to hypersalinity stress in the gill of the razor clam (Sinonovacula constricta), Comp. Biochem. Physiol. Part D Genomics Proteomics, 38, 100793.
Lin C-H, Yeh P-L, Lee T-H (2016) Ionic and amino acid regulation in hard clam (Meretrix lusoria) in response to salinity challenges, Frontiers in Physiology, 7, 368.
Lin C-H, Yeh P-L, Lee T-H (2021) Time-course changes in the regulation of ions and amino acids in the hard clam Meretrix lusoria upon lower salinity challenge, Journal of Experimental Zoology Part A, Ecological and Integrative Physiology, 335, 602-613.
Livingstone D R, Widdows J, Fieth P (1979) Aspects of nitrogen metabolism of the common mussel Mytilus edulis, adaptation to abrupt and fluctuating changes in salinity, Mar. Biol, 53(1), 41-55.
Lv R, Wang K, Feng J, et al (2020) Detection and Quantification of Viable but Non-culturable Campylobacter jejuni, Front. Microbiol, 10, 2920.
Ma Q-W, Guo H-Y, Zhu K-C, et al (2021) Dietary taurine intake affects growth and taurine synthesis regulation in golden pompano, Trachinotus ovatus (Linnaeus 1758), Aquaculture, 530, 735918.
Mantel L H, Farmer L L (1983) Osmotic and ionic regulation, Internal anatomy and physiological Regulation, 5, 53-161.
Maoxiao P, Xiaojun L, Donghong N, et al (2019) Survival, growth and physiology of marine bivalve (Sinonovacula constricta) in long-term low-salt culture, Scientific Reports, 9, 1-9.
Matsushima O, Katayama H, Yamada K, et al (1984) Effect of external salinity change on the adenylate energy charge in the brackish bivalve Corbicula japonica, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 77, 57-61.
McNamara J C, Rosa J C, Greene L J, et al (2004) Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp Macrobrachium olfersii (Crustacea, Decapoda) during long-term salinity acclimation, Mar Freshw Behav Physiol, 37, 193-208.
MEASURES J C (1975) Role of amino acids in osmoregulation of non-halophilic bacteria, Nature, 257, 398-400.
Medeiros I P M, Faria S C, Souza M M (2020) Osmoionic homeostasis in bivalve mollusks from different osmotic niches, Physiological patterns and evolutionary perspectives, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 240.
Ortiz R M., 2001, Osmoregulation in marine mammals, J. Exp. Biol., 204, 1831-1844.
Pierce S K, Amende L M (1981) Control mechanisms of amino acid-mediated cell volume regulation in salinity-stressed molluscs, J. Exp. Zool., 215, 247-257.
Pierce S K (1982) Invertebrate cell volume control mechanisms, a coordinated use of intracellular amino acids and inorganic ions as osmotic solute, Biol. Bull, 163, 405-419.
Ruan W, Dong Y, Lin Z, et al (2022) Molecular Characterization of Aquaporins Genes from the Razor Clam Sinonovacula constricta and Their Potential Role in Salinity Tolerance, Fishes, 7, 69.
Schoffeniels E, Gilles R (1972) Ionoregulation and osmoregulation in Mollusca, Chemical Zoology, 7, 393-420.
Schoffeniels E (1970) Osmoregulation in aquatic arthropods, Chemical Zoology, 5, 255-286.
Shumway S E, Gabbott P A, Youngson A (1977) The effect of fluctuating salinity on the concentrations of free amino acids and ninhydrin-positive substances in the adductor muscles of eight species of bivalve molluscs, J. Exp. Mar. Biol. Ecol, 29, 131-150.
Sokolowski A, Wolowicz M, Hummel H (2003) Free amino acids in the clam Macoma balthica L. (Bivalvia, Mollusca) from brackish waters of the southern Baltic Sea, Comp. Biochem. Physiol. Part A Mol. Integr. Physiol, 134, 579-592.
Vicente M J, Martínez-Díaz E, Martínez-Sánchez J, et al (2020) Effect of light, temperature, and salinity and drought stresses on seed germination of Hypericum ericoides, a wild plant with ornamental potential, Sci. Hortic, 270, 109433.
Wang X, Guo Z, Lei X, et al (2022) Osmoregulation, physiological metabolism, and oxidative stress responses to water salinity in adult males of Chinese mitten crabs (Eriocheir sinensis), Quat. Int., 1-19.
Wang Y, Li E, Yu N, et al (2012) Characterization and expression of glutamate dehydrogenase in response to acute salinity stress in the Chinese mitten crab, Eriocheir sinensis, PLoS One, 7, e37316.
Willett C S, Burton R S (2003) Characterization of the glutamate dehydrogenase gene and its regulation in a euryhaline copepod, Comp. Biochem. Physiol. B, Biochem. Mol. Biol., 135, 639-646.
Willmer P (2002) Biochemical adaptation - Mechanism and process in physiological evolution, Science, 296, 473-473.
Yancey P H, Clark M E, Hand S C, et al (1982) Living with water stress, evolution of osmolyte systems, Science, 217, 1214-1222.
Yancey P H (2005) Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses, J. Exp. Biol, 208, 2819-2830.
You Z, Lu T, Ma B, et al (2003) Effect of Seawater Salinity on the Growth and Survival of Argopecten irradians concentricus Larvae and Juvenile, Chinese J. Ecol., 38, 58-60.

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Osmotic regulation of free amino acid for adaptation to high salt in razor clam

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Experimental samples

2.2 Sample collection and design of experiment

2.3 Measurement of serum osmotic pressure

2.4 Detection of amino acid content under high salt stress

2.5 Effect of proline, taurine and alanine on cell relative volume

2.6 Measurement of cell volume

2.7 Data analysis

3. Results

3.1 Effects of high salt stress on total free amino acid (FAA) and serum osmotic pressure

3.2 Changes in free amino acid content under high salt stress

3.3 Effects of exogenous amino acids on the serum osmotic pressure of S. constricta

3.4 Effects of proline, taurine and alanine on the relative volume of cells

4. Discussion

4.1 Effects of high salt stress on total free amino acid (FAA) and serum osmotic pressure

4.2 Effects of exogenous amino acids on the serum osmotic pressure

4.3 Effects of proline, taurine, and alanine on the number, structure, and relative volume of cells

5. Conclusion

Declarations

References

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