In recent decades, the seawater temperature keeps rising with the acceleration of global warming (Hoegh-Guldberg et al., 2007), and in the worst cases, seawater temperature could increase by 4.5°C by the end of this century (IPCC, 2013). As a consequent, many intertidal marine mollusks are living at temperatures close to or just below their upper thermal limits (Stillman, 2003; Tomanek and Zuzow, 2010; Clark et al., 2021; Ning et al., 2021). Unlike terrestrial animals, marine organisms inhabiting nearshore are usually unable to shelter from high temperature environment (Burrows et al., 2019). Therefore, investigation of their biological responses to thermal stresses may provide insights into underlying the adaptive mechanisms of these intertidal mollusk to elevated temperatures.

Habitat temperature is a prominent stressor that can affect the survival, growth, development, reproduction, metabolism, immunity and geographical distribution of marine organisms (Chen et al., 2007; Anestis et al., 2010; Jiang et al., 2016; Yang et al., 2017; Monaco and McQuaid, 2019). For instance, thermal stress can induce overproduction of reactive oxygen species (ROS), which damages biological macromolecules (proteins, lipids, and DNA) and initiates a cascade of cellular defense events (Meng et al., 2014; Luo et al., 2014). Moreover, the physiological performance and gene expression of marine organisms can be disorganized by thermal stress (Clark et al., 2018, 2021; Li et al., 2020; Shi et al., 2020). It has been reported that enzymatic defense system, antioxidant system and apoptosis pathway of marine mollusk can be activated to counteract the adverse effect of oxidative stress induced by temperature changes (Zhang et al., 2012; Zhou et al., 2019; Ning et al., 2021). However, intertidal organisms have poor capacities in acclimation to rapid climate warming (Stillman, 2003). For instance, the growth and gonad development of Crassostrea gigas can be inhibited by thermal discharge from a nuclear power plant (Dong et al., 2018). It is thus necessary to investigate the thermo-resistant mechanisms of intertidal organisms in response to elevating temperatures.

The ark shells, Scapharca subcrenata, are economically important species and widely distributed in the coastal waters of China, Japan, and Korea (Chen et al., 2009). In the past decades, the wild resources of ark shells in China have suffered serious damage owing to overfishing and ecological changes, resulting in the decline of genetic diversity and stress resistance. Moreover, cultured ark shells, especially those cultured in outdoor ponds in China, are vulnerable to high temperature and their growth and survival are often threatened severely by continuous or extreme high temperature in summers (Lin J. L. et al., 2020; Ning et al., 2021). The seawater temperature of outdoor shallow ponds in Northern China can reach as high as 33.5°C in summer (Ning et al., 2015), thus pond cultured ark shells are often exposed to acute or chronic high temperature stress during this period. Therefore, in this study, we investigated the effects of acute or chronic thermal stress on apoptosis, physiological performance, and gene expression of ark shells, which would help to understand the thermal tolerance and contribute to selection of heat-resistant strains in ark shells.

Temperature, as an important abiotic factor, can substantially affect the physiological responses in marine organisms (Windisch et al., 2011; Dong et al., 2019). In this study, the 96-h upper LT₅₀ of ark shells was determined to be 31.6°C, based upon acute temperature tolerance experiments. However, the highest temperature in outdoor ponds or tidal flats in summer is far beyond this temperature, indicating that the cultured ark shells might be subjected to severe thermal stress. Moreover, the high temperature stress could last for up to over 1 week, suggesting that the ark shells could suffer from a long-term thermal stress. Therefore, it is important to examine the responses of the ark shells to both acute and chronic thermal stress.

Apoptosis, also called programmed cell death, is a vital physiological protective mechanism of organisms in adaptive responses to environmental stress, which can be induced significantly by high temperature stress (Zhang et al., 2012; Cheng et al., 2018; Zhou et al., 2019). However, severe stress may also cause necrosis when the stress is beyond the tolerance of the animal. In consistent with previously published results in oyster (Yang et al., 2017), we also observed that the apoptosis and necrosis rates of hemocytes were increased significantly in a time-dependent manner under both acute and chronic thermal stress, suggesting the apoptosis process of hemocytes has been activated and the stress injury gradually became irreparable with the prolonging of stress time and eventually caused necrosis in the ark shells. This is supported by the significant upregulation of Casp-3, a member of cysteine-dependent protease families that plays important roles in the activation of apoptotic pathway (Qin et al., 2020) in the two thermal stress treatments. It is notable that higher apoptosis level was observed under the acute thermal stress than under the chronic stress, suggesting the ark shells suffered more damage when exposed to acute temperature changes. The higher cumulative mortality rates of ark shells in the AHT also support this point.

In order to counteract the irreparable injuries caused by apoptosis, anti-apoptotic pathways could be activated immediately when organisms suffer from abiotic stresses. The expansion of inhibitors of apoptosis proteins in the oyster genome and their response to various stresses are crucial for oysters in adaption to sessile life in the highly stressful intertidal zone (Zhang et al., 2012). Glucose regulated protein 78 kD (GRP78) is an essential molecular chaperone located in the endoplasmic reticulum (Melnick et al., 1994; Ni et al., 2011). It has been reported that GRP78 could reduce apoptosis induced injuries by suppression of Caspase 7 activation (Reddy et al., 2003). Tumor necrosis factor receptor-associated factors (TRAFs) TRAFs plays crucial roles in stress resistance. Among which, TRAF6 is an important signaling molecule involving in cell proliferation and metabolism through a series of signal transduction (Rothe et al., 1995). As reported in recent study, TRAF6 could suppress the apoptosis of oyster hemocytes through activation of Pellino (Lin Y. et al., 2020). In the present study, the expression of anti-apoptosis related genes (GRP78 and TRAF6) was up-regulated significantly, indicating anti-apoptotic system also exists in ark shells in response to thermal stresses. This has been confirmed by our previous study according to the transcriptomic analyses (Ning et al., 2021).

Marine invertebrates are equipped with antioxidant defense system such as SOD and CAT to maintain the balance between normal physiological function and ROS accumulation (Lesser, 2006; Soldatov et al., 2007). SOD can catalyze the conversion of intracellular superoxide radicals into hydrogen peroxide (H₂O₂) and molecule oxygen (O₂), whereas CAT can convert the H₂O₂ into O₂ and H₂O. Therefore, the elevated activities of antioxidant enzymes are essential for animal to counteract oxidative damage induced by thermal stress (Cheng et al., 2018). In this study, the activities of SOD and CAT can be induced and increased significantly in response to both acute and chronic thermal stress, which was consistent with previous studies in Yesso scallop (Jiang et al., 2016) and Pacific oyster (Ding et al., 2020). However, the activities of SOD and CAT were decreased and lower than the initial level after 12 h under the acute thermal stress, suggesting the antioxidant ability could be repressed with the extension of acute thermal stress. This is consistent with the conclusion observed in giant clam, Tridacna crocea (Zhou et al., 2019), indicating a transition occurs from aerobic to anaerobic metabolism. Just as shown in our previous study, ark shells may suffer irreparable injuries due to oxygen limitation and metabolic dysregulation if the stress sustains (Ning et al., 2021).

Malondialdehyde content has been widely used as a marker for the extent of oxidative damage in marine organisms (Talmage and Gobler, 2011; Ferreira et al., 2019). In this study, the content of MDA was increased significantly firstly under the two thermal stress modes, suggesting the elevated seawater temperature induced oxidative damage in ark shells. However, the content of MDA was decreased and restored to the initial level subsequently, probably because the increased activity of antioxidant enzymes in a short time removed harmful substances induced by thermal stress (Wang et al., 2020). The enhanced activities of SOD and CAT were also observed under the two thermal stress modes in this study. The restored expression levels of most stress-related genes (GST, GRP78, TRAF-6, Casp-3, MRP, sHSP, HSP70, and HSP90) at day 4 also support the above viewpoint. Moreover, the higher MDA content under the acute thermal stress than under the chronic stress is consistent with the results found in Yesso scallop (Jiang et al., 2016), suggests that ark shells suffered more damage during the acute temperature changes.

Except for antioxidant enzymes, non-enzymatic antioxidant defense mechanisms also existed in marine invertebrates to prevent oxidative damage (Jiang et al., 2016). Peroxiredoxin is widely existed in prokaryotes and eukaryotes, which plays a vital role in the elimination of metabolically produced peroxides (Rothe et al., 1995; Fujii and Ikeda, 2002; Papadia et al., 2008). MT is responsible for removing metal ion toxicity through combination with them, which is involved in scavenging free radicals and the process of stress resistance (Masters et al., 1994; Carginale et al., 2000). RGN has the function of maintaining the dynamic balance of Ca²⁺ in cells and overexpression of RGN could inhibit cell death and apoptosis caused by a variety of signaling factors (Laurentino et al., 2011). In marine mollusks, the anti-oxidative responses to thermal stress are complicated and often affected by the differences in temperature gradient and exposure duration (Ning et al., 2021). Similar results were also observed in this study, with expression of GST and MRP up-regulated significantly and PRX, MT, and RGN down-regulated in both thermal stress modes. In consistence with previous results observed in oyster (Lim et al., 2016) and ark shells (Ning et al., 2021), our results suggest that marine mollusks may consume more energy for their redox responses.

Heat shock proteins (HSPs) are a group of highly conserved proteins which play important roles in cellular homeostasis after exposure to drastic environmental changes (Kayhan and Duman, 2010). HSP70s and HSP90s are well-known stress proteins in response to thermal stress in many marine mollusks (Farcy et al., 2007; Ivanina et al., 2009; Kim et al., 2009; Lim et al., 2016). HSP20s (stress induced protein 1) could promote the regeneration of denatured proteins and prevent stress induced protein aggregation when suffered elevated temperature stress (Nover and Scharf, 1997; Ning et al., 2021). In this study, the expression of HSPs (sHSP, HSP20, HSP70 and HSP90) were up-regulated significantly under both thermal stress modes. These results are consistent with our previous study (Ning et al., 2021), suggesting that the significant up-regulation of HSPs may help ark shells become more resistant to elevated seawater temperature. Moreover, the fact that the expression levels of HSPs (sHSP, HSP20, and HSP70) in the AHT were much higher than that in the CHT mode may imply that the ark shells were subjected to more severe cellular damage induced by acute thermal stress. Similar results were also observed in Yesso scallop (Jiang et al., 2016) and pufferfish, Takifugu obscurus (Cheng et al., 2018).

In summary, the physiological performance and gene expression were affected strongly by both acute and chronic thermal stress in ark shells. The apoptosis and necrosis rates of hemocytes were stimulated significantly in a time-dependent manner, and the content of MDA was increased in gills, indicating elevated seawater temperature has caused oxidative stress to ark shells. Thermal stress also induced actions in the anti-apoptotic system, antioxidant defense system and HSPs to protect ark shells from oxidative damage. The activities of antioxidant enzyme and expression of most genes in the CHT could be restored to initial level, which did not occur in the AHT, suggesting that the damage caused by rapid ambient temperature increase is irreversible, and thus more harmful to marine mollusks. Our results may provide more clues for management strategy of ark shell husbandry and further understanding of the mechanisms underlying the tolerance of ark shells to high temperature stress.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

DZ: investigation and roles/writing – original draft. JN: methodology, validation, software, and writing – review and editing. XL: supervision, formal analysis, and visualization. XW: validation and data curation. MC: software and data curation. BL: conceptualization and resources. JF: conceptualization and resources. CW: funding acquisition, writing – review and editing, and project administration. All authors contributed to manuscript revision, read, and approved the submitted version.

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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