The one-year old experimental scallops were obtained from a scallop raft-culture farm at Chengmai, north of Hainan Province. They were transported to the laboratory within 2 hours in cool and aerated seawater. Healthy scallops were cleaned and reared for acclimation in three 900 L circulating water culture system for a week (30-40 scallops in each system) under the conditions of natural light, water temperature of 25 ± 1°C and salinity of 31. Microalgae Isochrysis galbana (2×10⁵ cell/L) was fed twice a day, and one-third of the seawater was replaced every two days to ensure water quality.

The Doppler ultrasonography system can measure a series of hemodynamic parameters (listed in Table 1 ). These indices can indicate several physiological situations of the circulatory system in different tissues, including the blood flow capacity, pumping capacity of the heart and whether blood vessels work well. The monitored ED values of the mantle and adductor muscle were discontinuous, so only the gill’s ED values were measured. Based on this, only the RI and S/D values of the gill were calculated.

Doppler ultrasonography system (FL-60181, Xuzhou Pyle Co., Ltd; Xuzhou, China) was equipped with an 8-12 MHz high frequency waterproof linear emission probe. The anterior and posterior auricle of the scallops were secured with plastic clips so that the opening was upward for easy monitoring. When measuring, the scanning mode was set to B mode (black & white), then the probe was put into the water with the position parallel to the two shells, carefully adjusted until clear image of the blood vessel was shown on the screen. Secondly, the scanning mode was set to C mode (color) to show the blood flow dynamic, then D mode (Doppler pulse) was switched on, and a 0.5mm sampling site was located at the middle of the vessel so as to acquire the most active blood flow dynamic image ( Figure 1 ). One minute duration video was recorded for further detailed measurement.

Adenosine triphosphate (ATP), cytochrome-C oxidase (COX) and pyruvate kinase (PK) were selected as metabolic indexes of scallop tissue under hypoxia and high temperature stress. The content of ATP in tissue reflects the state of energy supply. The activities of COX and PK reflect the aerobic and anaerobic metabolic capacity of tissue, respectively.

Three metabolic indexes were measured by commercial analyzing kits (Beijing Solarbio^® biological company). After stress treatment, the scallops were dissected on ice, and the tissue samples (gill, mantle, adductor muscle) were quickly frozen in liquid nitrogen and thereafter stored in - 80°C refrigerator.

In order to better reflect the changes of physiological cycle indexes and metabolic indexes of scallops under stress, we classified the experimental results. In hypoxia stress experiment, HR and metabolic indexes (ATP, COX and PK) were expressed as relative rate of change, whereas the blood velocity (PS and ED), FV, RI and S/D were expressed as measured values. In high temperature stress experiments, blood velocity (PS and ED), FV, and metabolic indexes (ATP, COX and PK) were expressed as relative change rates, whereas the HR, RI, and S/D were expressed as measured values. The formula for calculating the relative rate of change in hypoxia and high temperature stress is shown in formula (1).

The experimental data were analyzed by SPSS 24.0 statistical software. The significant differences among each group were analyzed by one-way analysis of variance (ANOVA), combined with LSD test and independent T test, respectively (p<0.05). All variables were tested for normality of variance and homogeneity before analysis. Values are expressed as mean ± standard error (SE).

According to the change process of circulating physiological indexes, the whole hypoxia stress process can be divided into three stages (0-2h) early stage, (4-6h) middle stage and late stage (8-10h). The scallop was under the greatest stress at 6 h, with the highest HR, the lowest total FV and the RI of gill vessels ( Figure 2 , Figure 4C and Figure 5C ). Correspondingly, we found that the metabolic indexes of the three tissues were also remarkably different in the three periods, reflecting their response to hypoxic stress.

The study found that COX in the gill was always suppressed under hypoxic conditions (except for 6 h) ( Figure 6B ), and the PK value was significantly increased after 2 h, indicating that the anaerobic metabolic pathway was activated ( Figure 6C ). ATP was positively accumulated after 6h, indicating that the energy supply of gills was restored ( Figure 6A ). In the late state, the relative change rate of PK in gills decreased rapidly, from 314.06% (4h) to 31.25% (10h). COX activity also decreased by -56.34% ( Figures 6B, C ). It means that the aerobic and anaerobic metabolic capacity of gill tissue decreased at the same time.

The COX in the mantle kept at a negative state throughout the stress process, indicating a loss of aerobic capacity under hypoxic conditions ( Figure 6B ). The relative change rate of PK value increased by about 240% between 4 and 8 h, and the ATP content at this time also accumulated in the mantle, indicating that the energy supply recovered by the improvement of anaerobic metabolic capacity. However, at 10h, the relative change rate of PK value decreased to 30.73%, and the ATP was largely consumed in this period ( Figures 6A, C ). It indicated that the anaerobic metabolism of the mantle was inhibited and the energy supply tended to be unbalanced.

The relative change rates of COX and PK in the adductor muscle were significantly increased in the middle stage and significantly decreased in the late stage (P<0.05) ( Figure 6 ). The ATP in adductor muscle was consistently consumed throughout the whole severe hypoxia stress process, and was largely consumed at 4-6h ( Figure 6A ). The simultaneous enhancement of COX and PK activities in the middle stage may mean that scallops are doing their best to improve their resistance to hypoxia stress ( Figures 6B, C ). However, although COX and PK activities increased simultaneously, the ATP in adductor muscle were still in depletion state, indicating that the metabolism in the muscle was completely disordered.

The ATP content of the mantle was continues accumulated during high temperature stress, which was significantly higher than the normal level, and the activity of COX continued to increase, indicating that the aerobic metabolism of the mantle was enhanced ( Figures 10A, B ). When the temperature increased from 26 to 29°C (at 7:30 on the first day), the change rate of ATP content was the largest (550.00%), and the mantle was very sensitive to temperature ( Figure 10A ). The change rate of ATP content in the mantle was significantly greater than that in the gill and adductor muscle (P<0.05) ( Figure 10A ).

The content of ATP in the adductor muscle also increased significantly by 126.67% at 7:30 on the first day (29°C), indicating that the adductor muscle can quickly generate ATP to cope with high temperature stress ( Figure 10A ). Subsequently, the increase rate of ATP gradually decreased, and dropped to -40.00% and -11.11 at 15:00 on the second day and 7:30 on the third day, respectively. ATP was consumed in large quantities, and the activity of COX was also remarkably reduced at this time. Subsequently, the activity of COX was significantly enhanced (P<0.05), and the supply of ATP was gradually restored ( Figures 10A, B ). The changes of ATP content in the gill under high temperature stress were small, and the significant decrease in ATP content on the second day indicated that the gill might be under high temperature stress at this time ( Figure 10A ).

Cardiac function is the most important in the circulatory system and is widely used as a representative of animal metabolism to reveal its immediate response to environmental changes (Trueman et al., 1973; Burnett et al., 2013; Chapperon et al., 2016). In our experiments, the relative change rates of the scallop’s HR under different dissolved oxygen levels were all positive, indicating that the HR would increase under hypoxic conditions to accelerate circulatory efficiency and obtain more oxygen. Under the condition of severe hypoxia (2 mg/L DO), the HR reached the highest, indicating that scallop was rapidly adjusting the circulatory rhythm to adapt to the stress. Extensive studies have been carried out on the cardiac response of bivalves under hypoxic stress (Bayne, 1971; Grieshaber et al., 1993). Gurr et al., 2018 found that heart rate and respiratory rates in bay scallop Argopecten irradians increased when dissolved oxygen was below 5 mg/L. This is consistent with our result.

The circulatory system transports blood to various organs to exchange nutrients and oxygen. Since the convective supply of oxygen is directly dependent on blood flow, the regulation of tissue oxygenation depends crucially on blood flow regulation (Pittman, 2011). In our study, moderate hypoxia (3mg/L DO) will stimulate the scallop to increase blood flow, especially to the gill, to acquire more oxygen from the water and transport to other tissues. Nicholson (2002) found that Perna viridis under hypoxia stress will increase blood output to maintain hemolymph circulation. But it is worth noting that the FV in each organ began to decrease after 6 h at 3mg/L DO, which indicates that prolonged moderate hypoxic stress brought a great load to the scallop’s circulatory system. FV in adductor muscle was remarkably higher than that in gills and mantle under three dissolved oxygen condition, which may be related to the resistance to the blood flow of different organs (Jorgensen et al., 1984).

At 2mg/L DO level, the overall blood flow of each organ decreased, the blood flow in the gill changed smoothly, whereas the blood flow of the mantle and adductor muscle decreased remarkably. This may be related to the change of blood vessel diameter. The blood vessel diameter of the gill increased by an average of 23.25%, whereas that of mantle and adductor muscle decreased by an average of 11.17% and 13.64%, respectively (unpublished data). Gill is the main respiratory organ of bivalves. Different from mantle and other organs, gill can maintain the respiratory stability of the whole body. Bivalves can obtain more oxygen by increasing heartbeat (perfusion) and expanding blood vessels in case of hypoxia (Bayne, 1971). The diameter of gill blood vessels of Mytilus edulis increases by 1.6-1.9 times in the face of hypoxia (González et al., 2019). The relationship between HR and FV was complex. Generally, the FV (ml/min) is decided by the HR, blood flow velocity and the diameter of the blood vessel. Under certain HR and blood flow velocity, if the vessel diameter increases, the FV will also elevate.

The blood flow velocity index of PS and ED can indicate the cardiac capacity and blood pumping ability and efficiency. ED is more sensitive to hemodynamic changes, while PS is less affected by Doppler artifacts, which is more suitable for dynamic blood flow regulation control system analysis (Rosengarten et al., 2001). PS is used as an index to judge whether the renal artery is damaged in medicine (Chen et al., 2014). Our study found that PS exhibited greater fluctuations under hypoxic stress, whereas ED was less affected. The PS of the gill elevated remarkably under mild hypoxia (4 mg/L DO), which shows that together with HR increment, the scallop was trying to increase its blood flow efficiency in the most important breathing organ to cope with hypoxia stress. Meanwhile, the PS fluctuation detected in the adductor muscle indicates that it is more vulnerable to the stress. Under moderate hypoxia conditions, the HR and PS in the three tissues didn’t elevate so much than mild condition ( Figures 2 , 3 ), yet FV in the gill and adductor tissue increased remarkably ( Figure 4 ). It may be caused by the blood vessel expanding. More blood was redistributed to the gill for oxygen acquirement and to the adductor muscle to avoid tissue damage. When facing with severe hypoxia, although HR, PS of all tissues increased largely, the FV in the tissues were still inevitably reduced which caused by the shrinking of blood vessel ( Figure 4 ). It means that the circulatory regulation failed and functional damage is inevitable under this stress degree.

The RI threshold and S/D threshold of the gill vessels of noble scallop C. nobilis under hypoxic conditions were slightly higher than that of C. farreri under normal conditions (RI: 0.50-0.63; S/D: 2.01-2.86) (Xu et al., 2019), but the resistance index (RI) and S/D ratio of gill increased over time, indicating potential tissue damage from prolonged exposure to hypoxic environments.

The results show that the metabolic regulation ability varied in different tissues under severe hypoxia (Venter et al., 2018). The gill has stronger self-regulation ability than others under hypoxia stress, and it can switch to anaerobic metabolic pathway timely, hence the energy supply ability is restored. This is also confirmed by the better RI and S/D indices of the gill under hypoxic stress. Moreover, the accumulation of ATP in the gill further alleviates the intensity of its anaerobic metabolism, further confirmed the better resistance of the gill to hypoxia stress among the three tissues.

The metabolic changes of the mantle are similar to that of the gill: the aerobic metabolic pathway is inhibited throughout the whole process, and the energy is completely supplied by anaerobic metabolic pathway. The self-regulation ability of the mantle is considered to be strong before 8h, yet the PK content in the mantle decreased rapidly at 10h, and the ATP accumulation also changed to consumption, which means that the long-term hypoxia brought great pressure on the anaerobic metabolism capacity of the mantle, and anaerobic metabolism could not provide enough energy. Continuous exposure to hypoxia may cause damage to the mantle, which corresponds to the greater potential damage of the mantle in cycling physiology experiments, but further research is needed.

When scallops avoid enemies and swim, the adductor muscle is responsible for strong and frequent opening and closing activities, and has a high demand for energy and oxygen (Chantler, 2006). Therefore, the adductor muscle may be more vulnerable to hypoxic stress. It is found that the tissue resistance of adductor muscle was the weakest and its self-regulation ability is very poor under hypoxia stress. It is proved by the results that the aerobic and anaerobic metabolic pathways didn’t effectively convert and the ATP content was always in depletion. Chantler (2006) found that adductor muscle of scallop mainly relies on anaerobic metabolism for energy supply in normal conditions, but our study didn’t find the effectively improved of anaerobic metabolism capacity. Under hypoxic conditions, the scallop showed an obvious escaping behavior with the shells clapping frequently, which probably responsible for the enormous energy in the adductor muscle. But the poor regulation ability of the tissue under severe hypoxia means that the scallop may lost its swimming ability to escape the “dead zone” to survive.

We found that the HR of scallops increased significantly when the water temperature increased from the optimum 26°C to 29°C. Moreover, the HR of the scallop fluctuated synchronously with temperature from 29°C to 31°C. This indicates that the noble scallop is also sensitive to temperature and can adjust the HR immediately, just like our previous study on the temperature scallop species C. farreri (Xu et al., 2019). Bivalves are very sensitive to temperature changes, and HR can be used as an indicator of their thermal tolerance (Xing et al., 2019). High HR relates to accelerated circulation rhythm and is essential to meet the need of faster body metabolism rate under thermal stress, yet if the duration of the stress exceed the limit, cardiac disorder caused by overburden may occur and would finally cause heart failure (Nicholson, 2002; Lannig et al., 2008; Xu et al., 2019). In present study, cardiac disorder or failure didn’t occur during our two and a half day’s experiment, and the reason may be that instead of a continuous extreme high temperature (31°C) stress, the water temperature fluctuated from 31°C to 29°C each day, just like the diurnal changing situation in the wild. It is vital for the scallop to have a breath and recover.

The blood velocity of each organ was also affected by the fluctuation of high temperature. Within the first two and a half days, the changes of the gill were consistent with temperature fluctuations, whereas the mantle blood velocity is negatively correlated with temperature fluctuation, which indicates that the regulation circulatory physiology is tissue-specific ( Figure 9 ). At 15:00 on the third day, the blood velocity indexes of the gill and mantle appeared disorder at the same time, indicating that the regulation has lost effect. The relative change rate of PS in the adductor muscle was relatively stable as a whole, and only decreased significantly at 7:30 on the second day, indicating that high temperature fluctuations had little effect on it.

The FV in the gill, mantle and adductor muscle of the scallop will increase significantly under high temperature stress. The blood flow in the gill was significantly reduced on the third day, which may be related to the increase of RI and S/D of blood vessels, and the increase of vascular resistance will lead to the decrease of blood flow. The relative change rate of the FV in the gill was consistent with temperature fluctuations in the first two and a half days. It means that as the most important respiration organ, the body will preferentially provide sufficient blood flow to it to maintain the oxygen acquisition efficiency during thermal stress. This also explains why the changes of FV and PS in the mantle were opposite to those in the gill. Williams and Ponganis, 2021 also found that blood flow of marine mammals preferentially flows to vital organs such as the brain and heart when facing environmental stress. On the third day, the increase rate of FV in each organ decreased, which means that the scallop gradually adapted to fluctuating high temperature stress and do not need more blood flow to obtain oxygen.

The RI variation of scallop’s gill at 26°C ranged from 0.51 to 0.66, which is close to that of C. farreri under optimal water temperature of 8°C-26°C (Xu et al., 2019). On the first day, RI and S/D values were the highest, indicating that scallops were sensitive to temperature changes and had a stress response, and the increased blood flow brought great pressure to the vessels. The decrease in RI and S/D values at 7:30 in the second day proved that the regulation ability at this time was normal. However, the subsequent continuous increase of RI and S/D indicated that the pressure on the branchial vessels gradually increased and the possibility of potential injury increased. At the same time, the rising trend of RI and S/D also indicated that prolonged time of high temperature stress may cause more serious damage to the scallop. It is worth noting that the RI and S/D of the gill under 3-day high temperature stress were lower than those under 10 h hypoxia stress, indicating that hypoxia caused more serious damage to scallop.

The results of the study showed that with the temperature increased from 26°C (normal state) to 29°C (at 7:30) and to 31°C (at 15:00) on the first day, the RI and S/D of the gill’s blood vessels increased rapidly, from 0.52 to 0.66 and 2.17 to 3.02, respectively ( Figures 9B, C ). With the temperature gradually dropped to 29°C at 7:30 in the second day, RI and S/D decreased significantly to 0.51 and 2.18, which were close to the normal state ( Figures 9B, C ). It shows that the gill has good physiological regulation function and is sensitive to temperature changes. However, in the following two days, RI and S/D continued to rise and did not change synchronously with temperature fluctuations ( Figures 9B, C ). The RI and S/D rose to 0.63 and 2.75 at the end of the experiment ( Figures 9B, C ). Although the RI and S/D values at this time were lower than those on the first day, their rising state means that prolonged high temperature may cause potential damage to the vascular function of the gill.

Considering all the above results, we can infer that the scallop could gradually adapt to high temperature stress through circulatory physiological regulation, and the gill, as the main respiratory organ, was not seriously damaged. The scallops were maintained in good condition, indicating that the stress of the scallop caused by the fluctuating high temperature of 29-31°C was limited.

Under high temperature stress, the contents and activities of metabolites in various tissues of scallops changed continuously. The activity of COX in the mantle increased continuously under high temperature stress, and ATP also accumulated, indicating that the improvement of aerobic metabolism of the mantle could provide sufficient energy for it. The accumulation of ATP in the mantle was significantly higher than that in the gill and adductor muscle, which may be because the mantle is more sensitive to the changes of high temperature and requires a large amount of energy supply to withstand high temperature stress when it was continuously exposed to high temperature. This corresponded to the increase of circulating physiological indexes in the mantle. The improvement of circulatory physiological ability in the mantle requires a lot of energy support, so the body may give priority to meet its energy needs. Abele et al., 2002 found that the heat stress of organisms will enhance the ATP demand, which is consistent with our findings.

Adductor muscle mainly relies on anaerobic metabolism to provide energy. Under high temperature stress, its PK and COX activities were enhanced as a whole, indicating that its aerobic and anaerobic metabolism have been greatly improved ( Figures 10B, C ). The increase of PK activity in extremely warm environment reflects the enhancement of ATP demand (Feidantsis et al., 2009). However, the ATP content in the adductor muscle did not accumulate significantly (except at 7:30 on the first day), and was consumed at 15:00 on the second day and 7:30 on the third day. It shows that the energy demand of the adductor muscle under high temperature stress is greater, and the overall improved metabolic capacity can only maintain its energy consumption. The results show that the three tissues of the scallop can restore energy supply by regulating the metabolic capacity under the fluctuating high temperature stress of 29-31°C.