A total of 200 Yesso scallops were collected from Longwangtang Bay (Lvshunkou District, Dalian, China) in November 2022 and transported to the Key Laboratory of Aquaculture, Ministry of Agriculture and Rural Affairs (MARA), in insulated tanks. Scallops were removed from the substrate and rinsed. They were temporarily raised in a 1,000 L seawater tank at 15°C, with 50% of the water volume replaced daily. Scallops were fed once daily with powdered microalgae feed (Spirulina: Chlorella at 3:1) at a concentration of 10 mg/L. In addition, 30 northern Pacific sea stars were collected from the same location and transported to the laboratory under cool, moist conditions. After cleaning, 15 sea stars with arm lengths of 10–13 cm were selected as predators for the experimental group and temporarily cutured under the same conditions as the scallops, without feeding.

Only scallops that exhibited normal shell-closing behavior were selected for the experiment. Their shell length, height, and width were measured to 0.01 mm using vernier calipers (16EWR[M13] 0–150 mm, Marr Precision Measuring Instruments, Suzhou, China), and wet weight was recorded using an electronic balance (JJ500, Changshu Shuangjie Testing Instrument Factory, China) accurate to 0.01 g. Then they were grouped into three size groups based on shell length ( Table 1 ).

This behavior involves two phases of adductor muscle contraction (Tremblay et al., 2012). The first is a phasic contraction, involving rapid shell closures driven by striated muscle, resulting in a force peak referred to as the clapping force. A series of rapid claps over a short period constitute the phasic contraction force, which is the cumulative force of multiple claps (n). The second phase is a tonic contraction, involving slower, sustained shell closing (lasting >0.5 s), generated by smooth muscle. The corresponding force is termed the tonic contraction force. In this study, the following parameters were measured using a modified force gauge method (Zhang et al., 2021): the maximum shell closing force (F_max), the number of phasic contractions (T_phasic), the phasic contraction force (F_phasic), the times of tonic contractions (T_tonic), the tonic contraction force (F_tonic), the duration of tonic contractions (t_tonic), and the frequency of shell claps.

The scallop was placed in a glass tank (50 × 30 × 35 cm) with the lower valve fixed to a platform, while the upper valve was connected to a digital force gauge (Nscing SH - III - 100N, Suce, Nanjing, China). The gauge recorded the force, number and time data, which were transmitted to a computer ( Figure 1 ). The experiment was conducted in two groups: a control group (sea star absent) and an experimental group (sea star present). To minimize variability, sea stars of similar size were selected, and each experimental tank contained one sea star and one scallop, with the sea star’s arm in contact with the scallop shell. Each stimulation lasted for 3 minutes. Fifteen scallops from each size class were tested per group as biological replicates.

To assess physiological responses to predation stress, the activities of four enzymes, superoxide dismutase (SOD), catalase (CAT), arginine kinase (AK), and octopine dehydrogenase (ODH) were measured. These enzymes are involved in oxidative stress defense, energy metabolism, and muscle function. Three tissues (adductor muscle, gill, and mantle) were sampled from scallops in each size class following the 3 - minute predator exposure. Samples were immediately flash - frozen in liquid nitrogen and stored for analysis. Each group included three biological replicates. Enzyme activities were measured using commercial ELISA kits (Fancovi, Bio - Techne China Co. Ltd., Shanghai), following the manufacturer’s instructions.

Data were analyzed using SPSS 25.0 (IBM, New York, USA). Mean and standard deviation were calculated for behavioral parameters and enzyme activity. Differences between groups were assessed using independent-sample t-tests. Differences were considered statistically significant at P < 0.05 and highly significant at P < 0.01.

Force gauge recordings of M. yessoensis across different size classes revealed distinct phasic and tonic contraction patterns ( Figure 2 ). In control conditions, shell closure force increased with scallop size. Upon exposure to A. amurensis, scallops of all sizes consistently exhibited higher closure forces compared to their respective controls. Notably, small and medium scallops showed significantly higher phasic contraction counts following predator stimulation (P < 0.05), while large scallops demonstrated significantly longer tonic contraction durations ( Figure 2 ). Over time, all groups exhibited a decline in closure force and frequency, indicating fatigue or habituation.

Maximum closure force increased significantly with size across all groups (P < 0.05) and remained consistently higher in predator - exposed individuals ( Figure 3A ). Phasic contraction force and frequency were significantly elevated in small and medium scallops post - exposure, while large scallops did not show significant differences in these parameters. Tonic contraction force and duration increased significantly in all size groups following stimulation (P < 0.05), with large scallops exhibiting more than a threefold increase in tonic duration. Shell closure frequency was unaffected in small and medium scallops but significantly decreased in large scallops under predator stimulation. During the first 30 seconds of exposure, small scallops displayed the highest closure frequency, significantly greater than medium scallops (P < 0.05), with no significant difference observed between medium and large individuals ( Figure 3E ).

Enzymatic activity assays revealed significant alterations in SOD, CAT, AK, and ODH levels across tissues and size classes following predator exposure ( Figure 4 ). In gill tissue, SOD and AK activities were highly significantly altered (P < 0.01), while AK activity in the mantle also showed significant changes (P < 0.05). In adductor muscle tissue, SOD and ODH were significantly elevated (P < 0.05), while CAT and AK exhibited highly significant increases (P < 0.01) following stimulation.

A more detailed breakdown of enzyme activity in adductor muscles by size class is shown in Figure 5 . In large scallops, CAT and ODH levels dropped significantly (P < 0.01), from 7.74 ± 0.21 U/mL and 43.08 ± 1.42 U/mL in controls to 6.98 ± 0.20 U/mL and 42.60 ± 0.62 U/mL, respectively. Medium - sized scallops showed significant changes in all four enzymes: SOD declined from 40.47 ± 1.61 IU/mL to 37.20 ± 1.29 IU/mL (P < 0.05), and CAT, ODH, and AK were all highly significantly reduced (P < 0.01). In small scallops, CAT activity remained unchanged, while SOD, ODH, and AK were significantly altered (P < 0.01).

Based on these observations, medium - sized scallops exhibited the most pronounced enzymatic responses to predation stress and were therefore selected for transcriptomic analysis.

The shell closure behavior of scallops serves as a primary escape response against benthic predators. Our results demonstrated that the maximum shell closing force (F_max) increased significantly with scallop size, supporting previous findings that larger scallops possess stronger adductor muscles (Tremblay et al., 2012). When exposed to A. amurensis, all size classes showed elevated shell closure force compared to controls, indicating an acute defensive response. Notably, small and medium - sized scallops exhibited significantly higher phasic contraction frequency and force, while large scallops relied on prolonged tonic contractions with reduced closure frequency. This suggests a size - dependent behavioral adaptation: smaller scallops respond with rapid and repeated shell clapping to deter predators, whereas larger individuals depend on sustained force and shell thickness for protection. These findings are consistent with escape strategies observed in other scallop species, such as Aequipecten opercularis, which exhibit more frequent closures in juveniles compared to adults (Schmidt et al., 2008).

Predator - prey interactions elicit acute stress responses in prey species, often triggering physiological and biochemical changes. In shellfish, antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) are critical for mitigating oxidative stress by regulating reactive oxygen species (ROS) levels (Gao, 2016; Liu et al., 2003). Excessive ROS, generated during stress or high metabolic activity, can damage cellular structures unless effectively neutralized. Our results showed a size - dependent pattern of antioxidant response in Yesso scallops exposed to A. amurensis. Specifically, SOD activity increased significantly in medium and large scallops, while small individuals exhibited a marked decrease. Conversely, CAT activity declined across all size groups, with medium - sized scallops showing the most substantial drop. This pattern may reflect a rapid initial surge followed by depletion of enzymatic activity in small scallops, possibly due to their limited physiological reserves. The increase in SOD activity in larger scallops suggests a more robust antioxidant defense, aligning with prior studies that associate greater body size or age with enhanced stress resistance and immune capacity (Hao et al., 2014; Le et al., 1999).

Energy metabolism also plays a crucial role in stress response. Arginine kinase (AK), a key enzyme for ATP buffering in invertebrate muscle, supports rapid energy turnover during sudden escape movements (Morris et al., 2005; Yao et al., 2008). Our data revealed significant downregulation of AK activity in the adductor muscle of small and medium - sized scallops following predator exposure. This downregulation likely reflects acute energy demand during swimming behavior and subsequent depletion of ATP stores. Previous studies have demonstrated similar decreases in AK activity following intensive muscle activity in bivalves (Gäde et al., 1978; Smits et al., 2010), supporting the idea that predator stress imposes a high metabolic load on scallops. Octopine dehydrogenase (ODH), an enzyme involved in anaerobic glycolysis, was also significantly affected across all size groups. ODH catalyzes the formation of octopine as a terminal product of anaerobic metabolism, analogous to lactic acid formation in vertebrates (Livingstone et al., 1981; Bailey et al., 2003; Cavallini et al., 1966). Increased ODH activity likely supports short-term bursts of swimming and is consistent with earlier studies showing higher ODH levels in scallops following predator exposure (Guerra et al., 2013). The observed changes in AK and ODH activities are likely directly linked to the energetic demands of escape behavior, indicating that these enzymes may serve as reliable physiological markers of predator-induced locomotion in scallops.

Transcriptome analysis revealed that exposure to A. amurensis led to significant changes in gene expression within the adductor muscle of medium - sized Yesso scallops. A total of 514 differentially expressed genes (DEGs) were identified (raw P < 0.05), with 20 remaining significant after multiple testing correction. These DEGs included genes encoding muscle structure and function proteins such as paramyosin and myosin heavy chain, as well as KLF, which is known to regulate muscle development and stress response (Funabara et al., 2013; Prosdocimo et al., 2015). The upregulation of paramyosin and myosin heavy chain genes supports the observed enhancement of muscle contraction during predator exposure. These proteins play essential roles in maintaining the structural integrity and contraction dynamics of the adductor muscle, which are critical for the scallop’s escape response. The increased expression suggests a transcriptional reprogramming aimed at boosting locomotor capacity under acute stress conditions (Li et al., 2021). Krüppel - like factor (KLF), a transcription factor, has been implicated in muscle differentiation and cellular stress responses in various species. Its differential expression in scallops exposed to predators suggests that it may be involved in modulating muscle function or repair under stress. This aligns with physiological findings that indicate altered enzymatic activity supporting increased energy demand and oxidative stress management during predator evasion (Prosdocimo et al., 2015).

Additionally, several metabolic pathway-related genes, including those involved in amino acid metabolism (e.g., valine, leucine, isoleucine degradation), coenzyme biosynthesis, and oxidative metabolism (e.g., cysteine and methionine metabolism), were significantly enriched in KEGG pathway analysis. These findings reflect the underlying metabolic reorganization in response to predation stress, supporting rapid energy mobilization and reactive oxygen species (ROS) detoxification. Although GO enrichment did not identify significant terms related to muscle structure directly, several biological processes associated with organic acid metabolism and oxidoreductase activity were enriched, highlighting shifts in biochemical processes crucial for immediate stress adaptation. Overall, the transcriptomic responses corroborate behavioral and enzymatic data, confirming that predator - induced stress in Yesso scallops triggers a coordinated response involving enhanced muscle performance, metabolic adaptation, and stress signaling. These molecular signatures offer potential biomarkers for assessing scallop fitness and could inform selective breeding strategies aimed at enhancing resilience to environmental stressors, including predation.

Understanding predator - induced responses in scallops has direct or indirect applications for improving survival in bottom culture systems. Our findings show that scallops, particularly juveniles, undergo substantial physiological changes when confronted with predators. These responses, while adaptive, incur metabolic costs that may compromise long - term health and growth. Early detection of predation stress and the use of predator deterrents or protective seeding strategies (e.g., larger initial seed size, temporary netting) could help reduce mortality during critical early grow - out phases. The enzymatic assays revealed consistent patterns of stress-induced changes, especially in SOD, CAT, AK, and ODH levels. These enzymes reflect oxidative stress and energy demands in scallops under predation or other stresses. Such biomarkers may be used to monitor stress levels in real time and assess scallop health. Moreover, the identified genes could be used for development of molecular markers associated with stronger locomotor ability, supporting a selected breeding to develop scallop strains with enhanced predator resistance and stress tolerance.