Global climate change is responsible for increased frequency, intensity, and duration of extreme events, such as marine heatwaves (MHWs) are having far-reaching impacts on marine ecosystems (Gall et al., 2021; Mcpherson et al., 2021). MHWs are driving temperature increases at rates and levels well above projected warming scenarios (Babcock et al., 2019; Holbrook et al., 2019). The coupled impacts of acute temperature change and species thermal tolerance are causing havoc across the aquaculture industry as seen in the mass mortality of bivalves and seaweeds (Smale et al., 2019; He et al., 2021; Plecha et al., 2021; Xu et al., 2022). Increasing temperatures appear to be the more important contemporary stressor for many marine organisms (Hughes et al., 2017; Smale et al., 2019; Scanes et al., 2020a,b).

Increasing temperatures are not the only environmental source of stress to marine organisms. Increases in temperature are naturally accompanied by decreases in dissolved oxygen (DO) concentration. Globally, the prevalence of hypoxic areas caused by high temperatures in summer continues to increase, especially in offshore eutrophic areas (Diaz and Rosenberg, 2008; Suikkanen et al., 2013; Smith et al., 2020). Following the excessive reproduction of phytoplankton, massive amounts of organic matter sink to the seafloor where they are degraded via processes that consume oxygen and produce hypoxia or anoxia regions (Li et al., 2018). Especially in summer, haloclines and thermoclines weaken the vertical exchange of water, hindering the vertical mixing of DO, and aggravating the hypoxic or anoxic conditions of bottom waters (Wei et al., 2007). Additionally, biological sedimentation resulting from benthic shellfish culture has also led to increased sediment oxygen consumption, which exacerbated the risk of hypoxia or anoxia in the sediment environment of culture areas (Gilbert et al., 1997; Yang et al., 2007). In hypoxic or anoxic environments, the sedimentary environment shifts from an oxidative state to a reductive state (Nagasoe et al., 2011), which accelerates the process of acid volatile sulfur (AVS) reduction to produce hydrogen sulfide (H₂S) (Kodama et al., 2018). H₂S can be harmful, and even lethal, to benthic organisms (Smith et al., 1977; Joyner-Matos et al., 2010; Soldatov et al., 2018). To date, many studies regarding the environmental impact on shellfish have focused on the variation of DO concentration and the content of AVS in the water column, little attention has been paid to sedimental DO concentration and the H₂S content which is confirmed to be the only sulfide that causes harm to aquatic animals (Kodama et al., 2018; Liang, 2019). Therefore, the variation of DO and H₂S concentrations in the sedimental environment under high summertime water temperature are currently unknown.

Changes in environmental factors significantly affect the behavioral and physiological responses of marine organisms (Long et al., 2008; Li et al., 2019). When benthic shellfish experience hypoxia, they often seek access to additional oxygen by altering their digging behavior to reduce their burial depth (Lee et al., 2011). Bivalves mainly rely on glycolysis to meet their energy requirements when exposed to hypoxic conditions, and when the DO concentration continues to decrease, the dissimilation of glycolysis and the accumulation of succinate or propionate will be observed (Lee et al., 2011). Therefore, succinate concentrations can be used as an indicator of hypoxia stress in bivalve molluscs (Lee et al., 2011). Additionally, phenol oxidases (PO) and hydrolases, which are usually involved in detoxification, inflammation, and digestion, are considered to be the immune functions of bivalve molluscs that are most susceptible to environmental stressors (Hellio et al., 2006). Meanwhile, PO also plays an important role in the melanization of soft tissues and shells of bivalves (Cerenius et al., 2008). Even though the multiple environmental pressures often overlap in practical cases, most studies focus on individual pressures and few have considered the relationships between multiple pressure sources and how they affect marine organisms (Parisi et al., 2017).

The Manila clam Ruditapes philippinarum aquacultured in America and Europe was originated from Asia, and is now one of the most popular clams worldwide. However, Manila clam is threatened with mass death every summer (Jonsson and Andé, 1992; Wootton et al., 2003). Although there is evidence that environmental factors play a decisive role in the survival of Manila clam (Gestal et al., 2008; Matozzo and Marin, 2011), it does not seem possible to pin the cause of a large-scale Manila clam die-offs in summer on a single environmental pressure (Gajbhiye and Khandeparker, 2019). This is because any change in an environmental factor will trigger a series of environmental chain reactions. Therefore, a single change can drive changes in other environmental factors that may jointly affect the survival of marine organisms in an environment, resulting in large-scale mortality in conditions that would normally be considered tolerable (Parisi et al., 2017; Kim et al., 2018). In this study, temperature-controlled experiments were conducted to simulate sudden temperature changes in summer, to identify the characteristics of variation of DO and H₂S in different substrate types of sediments under different temperature conditions, and to clarify the environmental chain reactions brought about by temperature, as well as the effects on the behavioral and physiological responses of Manila clam. This study will serve as a reference for future research on artificial breeding, resource conservation, behavior, and early warning mechanisms of the Manila clam and related molluscs.

Typically, global pressure sources (e.g., global warming and ocean acidification) tend to vary at large time scales. Indeed, while climatic events are playing an important in marine organisms and the ecosystems they support, extreme environmental changes (e.g., MHWs) on a short time scale, in some cases, may generate even more profound impacts. This impact is more apparent for mudflat bivalves living in estuarine which are more likely to be exposed to drastic temperature change. These rapid temperature change (e.g., within a single tidal cycle) over small spatial scales may trigger unexpected environmental chain reactions (Helmuth et al., 2014). In this study, increased temperature and more prolonged duration resulted in a significant decrease in the DO of the sediment. The higher the temperature, the more dramatic the effect. Therefore, hypoxia or anoxic conditions are more likely to occur in summer with higher temperatures (Diaz, 2001). Furthermore, the effect of temperature on sedimental DO is much higher than that on the overlying water. Likely, the DO concentration of overlying water has not been reduced to hypoxic conditions (DO < 4 mg/L) when the sediment is already in anoxic condition (DO < 2 mg/L). So it may not be accurate to focus only on the DO concentration in the bottom water in benthic shellfish culture area.

When high temperatures and low DO conditions co-occur, the increase in H₂S release also becomes obvious. At the end of this study, the H₂S concentrations in the sediments under normal conditions (24°C) were about 3∼5 μmol/L, while at higher temperatures, the maximum concentration of H₂S in sediment samples was 18.10 μmol/L. However, even this high concentration was still far lower than the concentration of AVS in sampling area sediments (45∼180 mg/kg) (Liu et al., 2009; Yang et al., 2018), but it still approached or exceeded the half lethal concentration for many polychaetes with strong tolerances to H₂S (Wang and Chapman, 1999). So, it is important to accurately measure H₂S concentrations (Liang, 2019). In addition, H₂S is very easy to oxidize and hardly exists in oxygen enriched water, so increases of H₂S concentration have usually been associated with hypoxic or anoxic conditions (Nagasoe et al., 2011). H₂S concentration would increase under the condition of decreasing DO concentration in the sediment. The increased H₂S will also be oxidized by DO, which will accelerate the consumption of the remaining DO and lead to more serious hypoxia phenomenon. In summary, temperature changes may bring about a correlated environmental change of DO and H₂S anomalies in the sediment, resulting in benthic organisms that can be exposed to three stresses simultaneously.

In addition, we note that the effects of DO depletion and H₂S release vary significantly among substrate types in this study, and the environmental indicators of sediments with larger grain diameters are relatively better. Because the water is more easily exchanged, which prevents the accumulation of pollutants (Ichimi et al., 2019) and can effectively increase the oxidation intensity in sandy sediments (Yang et al., 1999). The finer the sediment particles are, the more quickly they accumulate organic matter (Hatcher et al., 1994; Mayer, 1994), which drives sediments to shift to a reductive state. Indeed, the phenomenon where DO concentrations decrease and H₂S concentrations increase does not occur every summer (Kodama et al., 2018). Since the increase of H₂S was also related to the consumption of DO in the bottom waters. Therefore, in a natural environment, under the influence of tidal and other hydrodynamic effects, the exchange of water might disassociate the effect of temperature from the increase in H₂S concentration. Because of this, it’s likely that sediment DO and H₂S concentrations in summer were related substrate in addition to magnitude and duration of elevated temperatures. It also reminds us that in summer, when there is a continuous high temperature, it is necessary to monitor H₂S concentrations in addition to changes in the sediment DO.

In this study, increasing temperature and decreasing DO levels did not affect the survival on Manila clams. While there is evidence that increasing temperature and decreasing DO did appear to influence Manila clam mortality additively because temperature increases narrow the tolerable DO range (Kim et al., 2018). However, this conclusion was based on prolonged stress conditions in the laboratory. In a natural environment, under the influence of tides and other hydrodynamic effects, benthic organisms rarely have the opportunity to be exposed to prolonged environmental stress. Manila clam is highly tolerant of temperature or DO variations. The results of succinate proved that Manila clam did not utilize anaerobic metabolism during the experiment. Therefore, the increasing temperature and decreasing DO within a specific range is not the direct cause of the massive mortality of Manila clam. Nevertheless, even increasing temperature and decreasing DO level within know tolerable ranges affected on the behavior of Manila clam. When faced with environmental pressure, marine organisms have various coping strategies, including behavioral and physiological responses (Wu, 2002). This study found that when the sedimental DO decreased, Manila clam will reduce their burial depth to be closer to the sediment-water interface. It also seemed that the Manila clam’s locomotory response to environmental pressure was relatively rapid. Firstly, Manila clam may emerge from sediment to breathe more efficiently to cope with stress and then through regulating physiological metabolism, energy supply strategy, and the other physiological processes (Kang et al., 2016). However, the shallower burial depth may increase the risk of predation, indirectly increasing the likelihood of high mortality (Lee et al., 2011; Howard et al., 2017).

Dissolved oxygen, rather than other environmental factors, is a significant stressor in the digging behavior of Manila clam (Kim et al., 2018). Due to the release of H₂S along with increased temperature and decreased DO, Manila clam may be harmed by higher H₂S concentrations in the sediment surface while seeking more oxygen. Indeed, changes in behavioral characteristics of Manila clam also increase the concentration of H₂S. Because the lack of movement (excavation) decreased the irrigation and penetration of DO into the sediments and, therefore, increased H₂S production. In this study, PO activity did change significantly. Our results showed that the release of H₂S might activate PO activity and damage the immune system of the Manila clam. In mollusks, PO is an important substance involved in immune defense that can be activated by external factors and is a key enzyme for melanin formation in the soft tissues and shells of bivalves (Asokan et al., 1997). Changes in the activity of this key component of the immune system may directly affect the survival of mollusks under environmental stress (Cajaraville et al., 1996; Xing et al., 2008). Nagasoe et al. (2011) reported that that exposure to high-H₂S concentrations can cause tissue melanization and damage of the Manila clam, including damage to the siphon, gills, adductor muscle, etc., and further affect physiological processes involved in breathing and feeding. Therefore, even though several environmental factors had no significant effect on the survival of the Manila clam during the initial exposure, as the environmental stress frequency or duration increased, damage to immune system could occur, potentially leading to significantly reduced performance in the future (Kozuki et al., 2013). MHWs induced elevated water temperatures are not the unique source of stress for marine organisms, and the environmental effects on the behavior and physiology of organisms should be considered in an integrated manner.

At higher temperatures, DO was consumed more rapidly and H₂S release increased in sediments. Sediment type also affected oxygen consumption and H₂S release rates. When Manila clams face environmental pressure, they respond quickly with locomotory behaviors (e.g., digging). Manila clams shows a strong tolerance to both high temperature and low DO and their physiological metabolism may not be affected by temperature increases or DO reduction within a certain range. However, when toxic substances such as H₂S start to accumulate, they may damage the immune system and tissues of the Manila clam, thereby affecting its future survival. Therefore, when temperatures are high for long periods in summer, and especially when the bottom water DO begins to decrease, it is necessary to take timely action to prevent and guard against harm caused by H₂S to aquaculture organisms. Therefore, areas that experience weak hydrodynamic exchange or that have small sediment particle sizes should be avoided in when selecting benthic bivalves culture locations.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

YL and JZ conceptualized the study. WW and QK were responsible for the experimental operation. XW was in charge of the data curation. YL prepared and wrote the original draft. CL reviewed and edited the manuscript. All authors read and approved the final manuscript.

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.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.