Climate change, characterized by global warming (Hoegh-Guldberg et al., 2018) and more frequent extreme heat events (Frölicher et al., 2018), represents a pressing concern that amplifies the energy requirements of ectothermic organisms (Alton and Kellermann, 2023) and jeopardizes the long-term survival of these species (Dahlke et al., 2020). To better understand how organisms will respond to climate change, scientists have developed mechanistic modeling approaches to predict population extinction risks and future geographic distributions (Kearney and Porter, 2009). One mechanistic approach to assess an organism’s tolerance to future warming is by comparing its upper thermal limit to the highest temperature it encounters in the field (Sunday et al., 2014; Pinsky et al., 2019). Traditionally, quantification of the upper thermal limit involves controlled conditions utilizing the dynamic method (Lutterschmidt and Hutchison, 1997), wherein organisms are exposed to a continuous heating rate until they exhibit symptoms of thermal stress or reach the point of death (Fernández-Loras et al., 2019). However, a limitation of upper thermal limit studies arises from the influence of the heating rate during tolerance measurement on the obtained results (Dong et al., 2022). Understanding the physiological and molecular mechanisms governing organismal sensitivity, and assessing whether such sensitivity varies with heating rate, is crucial for enhancing predictions of organism vulnerability to future climate change.

In the range of examined heating rates (0.5°C/h - 60°C/h), faster heating resulted in an elevated upper thermal limit in diverse organisms, including ants (Atta sexdens rubropiosa, Ribeiro et al., 2012), tropical marine fishes (Acanthochromis polyacanthus, Amphiprion melanopus, and Lates calcarifer, Illing et al., 2020), and flies (Glossina pallidipes, Terblanche et al., 2007). Conversely, moth (Cydia pomonella, Chidawanyika and Terblanche, 2011) and crabs (Eurypanopeus abbreviatus and Menippe nodifrons, Vinagre et al., 2015) exhibited reverse correlations, as individuals subjected to faster heating displayed significantly reduced tolerance. Additionally, Antarctic fish Harpagifer antarcticus demonstrated no significant difference in upper thermal limits across various heating rates (Saravia et al., 2021). These findings highlight the species-specific nature of the relationship between heating rate and upper thermal limit, necessitating individualized analysis for each species when assessing realistic upper thermal limits in laboratory studies.

Furthermore, research has shown that the effect of heating rate on upper thermal limits depends on the characteristics of the habitat. Research on a variety of tropical marine ectotherms suggested that organisms inhabiting rapidly changing thermal environments, such as the upper shore, exhibit steeper relationships between thermal limits and temperature change rates compared to those living in less variable environments on the lower or subtidal shore (Nguyen et al., 2011). Moyen and colleagues (2019) demonstrated that the upper thermal limit of high-zone mussels was significantly higher at the faster heating rate compared to the slower heating rate. Consequently, the rate at which heating occurs is a critical factor in thermal acclimatization, emphasizing the need for a thorough understanding of heating rates in natural environments to accurately determine the maximum thermal limits of species (Han et al., 2023).

Previous studies have shown how heating rates affect upper thermal limits and emphasized the importance of considering these effects in models predicting population dynamics (Liao et al., 2021). However, little research has been done to investigate the physiological or molecular mechanisms used by organisms to deal with thermal stress at different heating rates (Sørensen et al., 2013; Illing et al., 2020; Saravia et al., 2021). The cellular stress response (CSR) is essential for organisms to manage thermal damage to proteins, nucleic acids, and membranes. When proteins unfold due to heat, they trigger the prompt activation of gene transcription for HSPs (Somero, 2020). In ectotherms, physiological responses are temperature-dependent, with metabolic rates generally increasing with body temperatures (Pörtner, 2001; Somero et al., 2017). Heat stress augments energy metabolism in ectotherms, and the increased tissue oxygen demand is satisfied by an elevation in heart rate (Christen et al., 2018). Consequently, heart rate serves as a potential indicator of metabolic rate (Hui et al., 2020). Thermal stress induces a range of transcriptomic responses, encompassing ATP-generating pathways, DNA and protein damage repair, and the elimination of cellular and molecular debris generated by stress (Sokolova et al., 2012). Transcriptional studies thus offer valuable insights into the molecular mechanisms employed by organisms under different heating rates.

In the present study, we tested dark unbanded shell type Batillaria attramentaria exposed to gradual temperature increase of 3°C/h (slow-heated) or 9°C/h (fast-heated) starting at 27°C. The snail is a dominant species within temperate tidal flats, playing a crucial role in the ecosystem due to its influence on carbon flows within the ecosystem (Kawasaki et al., 2019). The selected heating rates represented the typical slow and fast heating rates observed in the field (see Supplementary Figure S1 for details). Using median lethal temperature (LT₅₀) as an index, we compared the upper thermal limit of snails exposed to slow and fast heating rates. Additionally, we measured the heart rate (HR) under constant heating at both slow and fast heating rates. Studies on the response of gastropods to heat stress have primarily concentrated on cardiac physiology, assessing the impact of sudden temperature variations on HR. Acute thermal stress typically leads to heightened metabolic requirements, with increased tissue oxygen demand being met through enhanced cardiac function (Marshall et al., 2011). And we investigated the transcriptomic response in snails at the temperature where heart rate falls to zero (FLT). Our key objective was to contribute insights into the physiological and molecular underpinnings of the observed variations in upper thermal limit measurements resulting from different heating rates. The implications of our findings extend to facilitating a more accurate assessment of upper thermal limits in laboratory investigations.

The investigation of heating rate impacts on thermal limits has garnered significant attention in enhancing our mechanistic understanding of physiological responses to changing thermal conditions in the context of climate change. Nevertheless, there has been limited exploration of the physiological and molecular mechanisms employed by organisms to cope with thermal stress under varying heating rates. This study aims to fill this gap by demonstrating that the mudflat snail B. attramentaria reveals the physiological and molecular adaptations in response to distinct heating rates.

At a fast heating rate, heart rate of B. attramentaria exhibited a notable increase at 32°C, plateauing between 33 and 40°C. This observation implies that, during fast heating, B. attramentaria suppresses resting metabolism within the moderate thermal range, which is similar to other marine intertidal gastropods (Marshall et al., 2011; Chen et al., 2021). The metabolic rate depression serves as an adaptive strategy, enabling prolonged survival under thermal extremes by conserving energy stores while ensuring an adequate supply of ATP for essential physiological functions (Sokolova et al., 2012; Marshall and McQuaid, 2020). In contrast to the response in fast-heated snails, heart rate exhibited insensitivity to temperature variations during slow heating, with no significant increase between 27 and 40°C. The heart rate peaked at approximately 45°C. Thermal stress usually results in increased metabolic demands, and the increased requirement for tissue oxygen is met through improved cardiac performance (Marshall et al., 2011). On average, the heart rate of snails subjected to slow heating was 11 beats per minute lower than that of snails exposed to rapid heating when the temperature was below 45°C. A lower heart rate during slow heating signifies a decreased need for oxygen. Thus, our results indicate that the metabolic rate during slow heating is lower than that during rapid heating under comparable thermal stress conditions.

The MHR in ectotherms displays sensitivity to energy content, with individuals harboring greater energy stores exhibiting a heightened MHR under thermal stress (Hardison et al., 2023). When subjected to constant heating, snails experiencing slow heating manifested a lower MHR compared to fast-heated snails, indicative of diminished energy stores in slow-heated snails as the temperature reached 45°C. Despite the fact that snails exhibit a reduced metabolic rate at the equivalent temperature during slow heating, slow heating also prolongs exposure time to stressful conditions, resulting in higher energy consumption during slow heating as opposed to fast heating. In contrast to the ABT, slow-heated snails showed a significant decrease in their FLT compared to the fast-heated snails, similar to the difference observed in the LT₅₀. These results suggest that FLT can be used as an effective measure to determine the maximum temperature tolerance in B. attramentaria snails.

When temperatures approach or surpass the FLT in intertidal mollusks, cellular damage becomes irreversible, as evidenced by the upregulation of genes responsible for encoding proteins involved in eliminating irreversibly damaged proteins from the cell (Gracey et al., 2008). Functional enrichment analyses revealed that DEGs were notably enriched in “ATP-dependent protein folding chaperones” during both fast and slow heating when the temperature increased to FLT. The translocation-associated proteins (TRAP), recognized as membrane channels facilitating protein transport during translation into the endoplasmic reticulum (ER) (Jaskolowski et al., 2023), and protein disulfide isomerase (PDIA5), another ER-resident protein (Wilkinson et al., 2005), were identified in the analyses. This suggests an augmentation in protein translocation into the ER and unfolding of ER proteins during both fast and slow heating.

ATP-dependent chaperones necessitate ATP hydrolysis to assist in protein folding, preventing the accumulation of aberrant protein conformations that may lead to proteotoxicity (Mitra et al., 2022). Our findings indicate that snails activate an energy-costly heat shock response during both fast and slow heating rates. The expression levels of most chaperones were slightly higher during slow heating than fast heating, except for one HSP70B2 gene, which exhibited a dramatic up-regulation during slow heating ( Figure 6 ). These results suggest an increased production of chaperones aimed at removing irreversibly damaged proteins from the cell during slow heating when temperatures approach the FLT. Among these chaperones, we identified eight HSP70B2 genes with significantly increased expression under heat stress. It has been documented that HSP70B2 genes undergo expansion in intertidal invertebrates (Dong et al., 2023; Liang et al., 2023; Yuan et al., 2023). Furthermore, HSP70B2 expression is predominantly up-regulated during air exposure, indicating its pivotal role in intertidal adaptation (Yuan et al., 2023). Our results indicated the importance of HSP70B2 in response to heat stress in snail B. attramentaria.

The finding that a slower heating rate elicits a more pronounced CSR compared to a faster heating rate implies that the slower heating rate causes greater heat-induced damage at the cellular level. Specifically, two chaperones involved in binding unfolded proteins, GRP94 and Calnexin, were found to be up-regulated in slow-heated snails. GRP94 helps detect misfolded proteins in the endoplasmic reticulum and sends them for degradation (Buc Calderon et al., 2018). Calnexin is a molecular chaperone that assists in the folding and quality control of proteins associated with membranes and secretion (Paskevicius et al., 2023). These findings suggest that misfolded proteins accumulate at a slow heating rate, requiring additional energy for their degradation (Somero, 2002). Slow heating also leads to the enrichment of “oxidative phosphorylation” and “thermogenesis” pathways. Genes encoding proteins involved in redox regulation, such as cytochrome C oxidases (COX5A, COX5B), ATP synthases (ATP5MC2), and cytochrome reductase (Cytc), were up-regulated, indicating improved energy production when the temperature reaches the FLT ( Figure 7 ). The accumulation of misfolded proteins and the increased energy consumption may contribute to the lower FLT and LT₅₀ observed during slow heating.

The folding of newly synthesized proteins into their native conformation relies on the assistance of a network of heat shock protein chaperones. Initially, these proteins are produced as linear chains on ribosomes and must undergo folding to adopt their functional conformations (Hu et al., 2022). Our study found that the expression of several ribosomal protein genes was up-regulated in B. attramentaria when subjected to a slow heating rate. Ribosome activity has been identified as a potential biomarker for cellular stress. For instance, mudflat clams exposed to heat stress in mud-air conditions showed an increase in the expression of several ribosomal protein genes (Zhang et al., 2020). The up-regulation of ribosomal protein genes associated with both the large and small subunits can enhance the tolerance of fish to thermal stress (Quinn et al., 2011). Our findings suggest an increase in ribosome biogenesis in slowly heated snails, indicating that it plays a role in conferring tolerance to slow heating but not fast heating.

The GO terms “carbohydrate metabolic process,” “chitin binding,” and “chitin synthase activity,” as well as the KEGG pathway “amino sugar and nucleotide sugar metabolism,” were specifically enriched during fast heating. These categories are related to the metabolism of carbohydrates. Several DEGs were identified as being involved in chitin hydrolysis or synthesis, such as CHIT1, CDA8, and Hex. The expression of these genes decreased during fast heating, suggesting that chitin metabolism is temporally constrained when the temperature increases to FLT. Chitin is an insoluble linear polymer composed of β-(1,4) linked N-acetyl-D-glucosamine units and is commonly found in mollusk shells. For instance, the sea snail Conus inscriptus shell chitin has a yield of 21.65% on a dry weight basis (Mohan et al., 2019). The restricted chitin metabolism may conserve energy and allocate more resources to repair processes, which could contribute to the higher upper thermal limits of B. attramentaria during fast heating.

Four genes involved in carbohydrate metabolism, namely XYN1, BXL5, FUT1, and Fuc, were found to be upregulated in both treatment groups. XYN1 is a glycoside hydrolase that can cleave the β-1,4-glycoside linkages in the xylan backbone (Kim et al., 2023). BXL5 belongs to the exoglycosidase enzyme category and has a unique ability to break down xylo-oligosaccharides into xylose (Saleem et al., 2012). Fuc plays a role in catalyzing the hydrolysis of terminal fucose residues or trans-fucosylation (Wan et al., 2020). These findings suggest that xylan and fucose may serve as important energy sources for the snail B. attramentaria when exposed to high temperatures.

In conclusion, our study provides insights into the physiological and molecular foundations underlying the differences in upper thermal limit measurements obtained using different heating rates. We observed distinct patterns of cardiac performance in snails subjected to fast and slow heating rates. The heart rate during slow heating was lower compared to fast heating under equivalent thermal stress. However, slow heating also prolonged exposure to stressful temperatures, resulting in higher energy consumption. When the temperature reached the temperature of FLT, snails exhibited a common heat shock response characterized by the up-regulation of genes involved in protein folding, such as HSPs and PDIA5 ( Figure 8 ). Interestingly, slow-heated snails uniquely up-regulated genes involved in unfolded protein binding, namely GRP94 and Calnexin, indicating the accumulation of misfolded proteins. This accumulation, along with the additional energy consumption, may contribute to the lower upper thermal limit observed in slowly heated snails. Our findings have implications for determining realistic upper thermal limits and improving predictions of organism vulnerability to future climate change.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number can be found below: https://ngdc.cncb.ac.cn/gsa, CRA015574.

The animal study was approved by Ethics Committee for Experimental Animals of Yantai University. The study was conducted in accordance with the local legislation and institutional requirements.

L-ND: Formal analysis, Writing – original draft. Y-HD: Data curation, Formal analysis, Methodology, Writing – original draft. G-DH: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.