Cold Stress and Molecular Adaptations in Aquatic Organisms: A Comparative Review of Fish, Crustaceans, and Mollusks

Abstract

Cold stress poses a significant challenge to aquatic organisms, affecting their survival, growth, and metabolic processes. This review explores the molecular mechanisms by which fish, crustaceans, and mollusks respond to cold stress, highlighting the shared and species-specific pathways that facilitate adaptation. Common responses to cold stress include modulation of energy metabolism, regulation of oxidative stress, immune responses, and maintenance of proteostasis. In particular, the activation of the adenosine 5′-monophosphate-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) pathways plays a critical role in regulating energy balance and autophagy in response to low temperatures. Furthermore, we examine the specific adaptive mechanisms employed by different groups of aquatic organisms. Fish utilize pathways such as peroxisome proliferator-activated receptor alpha/peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPAR/PGC-1α) and fatty acid oxidation to optimize energy utilization and improve cold tolerance. Crustaceans rely on crustacean hyperglycemic hormone (CHH) signaling and AMPK pathway activation, while mollusks employ metabolic suppression and glycogen storage to survive cold exposure. Moreover, the regulation of autophagy and apoptosis, mediated by p53 and cyclin-dependent kinase 1 (Cdk1), ensures the survival of healthy cells under prolonged cold stress, with autophagy maintaining energy homeostasis and apoptosis eliminating damaged cells. This review also discusses the role of molecular chaperones like heat shock protein 70 (HSP70) and the ubiquitin-proteasome system (UPS) in protein homeostasis, highlighting their importance to protect cells under cold stress. The combined action of these molecular pathways allows aquatic organisms to cope with and adapt to cold environments, ensuring cellular integrity and enhancing survival. Future research should focus on integrating molecular, physiological, and ecological approaches to better understand cold tolerance mechanisms and improve aquaculture practices under climate change scenarios.

1. Introduction

In the context of global climate change, in addition to persistent warming, cold waves, cold air invasions, seasonal sharp temperature drops, and localized upwelling events have also become frequent occurrences in multiple water bodies [1,2]. These events have become important environmental stressors impacting the stability of aquaculture and the health of aquatic ecosystems. For ectothermic aquatic species such as fish, crustaceans, and mollusks, environmental temperature directly governs their metabolic rate, feeding behavior, growth, immune defense, and reproductive performance [3]. Recent studies further confirm that temperature changes can alter neuroendocrine and molecular responses in metabolically active tissues such as the liver and skeletal muscle, while also affecting feed intake, appetite regulation, gastrointestinal transit, energy allocation, and growth performance in aquaculture species such as Atlantic salmon [4,5]. Thus, fluctuations in low temperature, especially sudden drops that exceed their heat tolerance range, often lead to physiological imbalances, tissue damage, reduced disease resistance, and even large-scale mortality, causing significant economic losses and risks to genetic resources. Current research indicates that cold stress not only affects individual survival and growth but also, by altering behavior, tissue function, and energy allocation, further impacts population structure and aquaculture management outcomes [6,7].

Conceptually, the response of aquatic animals to low temperatures should not be regarded as a single uniform process, but rather as a continuum that includes three distinct yet interconnected levels: acute cold shock, persistent cold stress, and cold acclimation/adaptation [8]. Acute cold shock refers to the immediate physiological response triggered by a rapid temperature decrease within a short period, and is typically characterized by rapid neuroendocrine activation, metabolic disturbance, oxidative stress, and behavioral inhibition. Persistent cold stress refers to the sustained physiological and cellular disturbance caused by prolonged exposure to suboptimal or critically low temperatures, often involving chronic tissue damage, energy metabolic reprogramming, immune dysregulation, and impaired organ function. In contrast, cold acclimation/adaptation refers to compensatory adjustments formed during gradual or long-term exposure to low temperatures, including metabolic remodeling, changes in membrane lipid composition, maintenance of protein homeostasis, and improvement of cold tolerance [7].

Although fish, crustaceans, and mollusks differ significantly in their phylogenetic positions and physiological structures, existing evidence suggests that they share several common adaptation themes under cold stress, including energy metabolic reprogramming, oxidative stress and antioxidant defenses, immune regulation, protein homeostasis, and cell fate regulation. For instance, in fish, cold temperatures can cause a decrease in blood cell count, liver damage, changes in antioxidant enzyme expression [9], and altered gene expression related to apoptosis [10], affecting cold tolerance through lipid metabolism [11], autophagy [12], MAPK pathways [13], and mitochondrial homeostasis [11]. In crustaceans, cold stress is often accompanied by metabolic inhibition in the hepatopancreas, mobilization of lipids and glycogen, changes in antioxidant and nonspecific immunity, as well as the involvement of CHH, biogenic amines, AMPK, and cell cycle/apoptosis factors [6,14]. In mollusks, low temperatures can reduce metabolic rates, alter glycogen and lipid reserves, induce transcription factors, and remodel metabolic pathways, along with behavioral strategies like burrowing and microhabitat selection to improve overwinter survival [15,16].

However, two major issues persist in current research. First, cold stress studies across different taxa have long remained fragmented within their respective disciplinary contexts. In fish, most studies have focused on neuroendocrine regulation, mitochondrial function, autophagy, and transcriptional regulatory networks. In crustaceans, research has mainly emphasized hepatopancreatic energy metabolism, antioxidant and immune responses, and neurohormonal regulation. In mollusks, greater attention has been paid to metabolic reserves, microhabitat buffering, and overwintering strategies. As a result, a truly comparative framework across major aquatic taxa is still lacking. Second, while recent advancements in transcriptomics, metabolomics, proteomics, and functional gene validation have significantly advanced cold stress research, much of the work remains limited to differential expression and physiological indicator descriptions. There is still a lack of systematic integration addressing the core question of which mechanisms are conserved, which are taxon-specific, and which can be translated into cold adaptation and molecular breeding targets. To avoid an overly broad and purely descriptive synthesis, the present review primarily focuses on aquaculture-relevant and temperate/subtropical representatives of fish, crustaceans, and mollusks, while also incorporating selected studies on cold-adapted or polar species when they provide mechanistic insight into long-term evolutionary adaptation. Cross-taxon comparisons are made by distinguishing conserved stress-response modules, taxon-specific strategies, and the strength of available evidence. Particular attention is paid to whether the cited studies are based on acute laboratory cold exposure, chronic low-temperature challenge, acclimation experiments, field observations, or evolutionary adaptation contexts.

This review aims to systematically summarize the physiological and molecular adaptive mechanisms of aquatic animals to cold stress, focusing on the neuroendocrine and behavioral responses, energy metabolic reprogramming, oxidative stress and antioxidant defense, immune regulation, protein homeostasis, autophagy and apoptosis, and core regulatory molecules and transcriptional networks in fish, crustaceans, and mollusks. Furthermore, it will discuss the potential applications of these mechanisms in cold acclimation, precision aquaculture management, and cold-resistant breeding strategies. By establishing an integrated framework of “common mechanisms—taxon-specific differences—application transformation”, this review seeks to provide a theoretical basis for the biological research of aquatic animals under cold stress, cold trait improvement, and optimization of aquaculture strategies in low-temperature environments.

2. Literature Selection and Comparative Framework

This review was designed as a narrative and comparative synthesis rather than a formal systematic review or meta-analysis. Literature was selected from peer-reviewed articles and reviews addressing cold shock, cold stress, low-temperature acclimation, thermal priming, overwintering responses, and cold adaptation in fish, crustaceans, and mollusks. Priority was given to studies that clearly reported the species examined, habitat or aquaculture background, life stage, exposure temperature, exposure duration, sampled tissue, and physiological, biochemical, molecular, or behavioral endpoints. Recent studies published from 2022 onward were prioritized where available, while earlier foundational studies were retained when they provided essential conceptual or mechanistic support.

Studies were interpreted cautiously or excluded from direct comparison when exposure conditions were insufficiently described, when the thermal treatment could not be related to the species-specific thermal window, or when conclusions were based only on broad differential expression without physiological, biochemical, or functional support. Because cold stress depends strongly on ecological background and thermal history, studies on polar or naturally cold-adapted species were not directly pooled with studies on warm-water or temperate aquaculture species. Instead, such studies were used to illustrate long-term evolutionary cold adaptation. Cross-taxon comparisons were made by separating conserved mechanisms, taxon-specific strategies, and evidence strength across comparable experimental or ecological contexts.

3. General Framework of Aquatic Animal Cold Stress Response

3.1. Layered Responses to Cold Stress: Primary, Secondary, and Tertiary Responses

Cold stress in aquatic animals operates through a hierarchical process that can be divided into primary (initial), secondary (core), and tertiary (ultimate) responses. This framework, first proposed in fish stress physiology, is applicable to crustaceans and mollusks as well.

3.1.1. Primary Response: Neuroendocrine and Rapid Signal Perception

The primary response is the earliest reaction of the body to temperature changes, mainly mediated through the nervous and endocrine systems. In fish, cold exposure quickly activates the hypothalamus–pituitary–interrenal (HPI) axis, promoting the release of stress hormones such as cortisol, thereby regulating metabolism, immunity, and ion balance [17]. In crustaceans, similar regulation is achieved through neurosecretory systems and hormones like crustacean hyperglycemic hormone (CHH) [18]. Mollusks, while lacking a typical endocrine axis, still utilize neuroregulation and humoral factors to sense and transmit cold stress signals [19]. Although direct evidence linking these neuroendocrine components to cold stress in bivalves remains limited, recent work on the warm-water bivalve Chlamys nobilis has shown that chronic cold exposure induces tissue damage, antioxidant remodeling, and altered expression of stress-related genes, suggesting that cold responses in mollusks involve coordinated neuro-humoral, redox, and cellular signaling processes [20]. At the cellular level, temperature perception also involves changes in membrane fluidity, ion channel regulation, and expression of cold-inducible proteins like cold-inducible RNA-binding protein (CIRBP), collectively forming the initial steps of cold stress signal transduction [21].

3.1.2. Secondary Response: Metabolism, Oxidative Stress, and Immune Regulation

The secondary response represents the core phase of cold stress and involves the reconfiguration of multiple physiological and biochemical processes, including:

(1) Energy Metabolic Reprogramming: Low temperatures typically cause a decrease in metabolic rates, but different taxa adopt different strategies to maintain energy supply. Fish generally achieve energy redistribution during cold exposure through the coordinated regulation of lipid catabolism, fatty acid β-oxidation, gluconeogenesis, amino acid metabolism, and mitochondrial energy production [22,23]. Recent transcriptomic and metabolomic studies further indicate that cold stress can activate PPAR-related metabolic pathways and reshape hepatic glucose and lipid metabolism in fish, thereby helping maintain energy homeostasis under low-temperature conditions [24]. In contrast, crustaceans often exhibit reduced respiration, glycogen and lipid mobilization, hepatopancreatic metabolic adjustment, and overall metabolic inhibition [14]. Mollusks rely more strongly on lowering metabolic rates and utilizing stored energy reserves, particularly glycogen and lipids, to survive prolonged cold periods [25].

(2) Oxidative Stress and Antioxidant Defense: Oxidative stress refers to a physiological or cellular state in which the production of reactive oxygen species (ROS) exceeds the capacity of antioxidant and repair systems, resulting in potential damage to lipids, proteins, nucleic acids, and cellular structures. Cold temperatures can disrupt mitochondrial function and electron transport chains, leading to increased ROS production. Organisms counteract excessive ROS accumulation by enhancing antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and by inducing molecular chaperones and repair pathways to mitigate oxidative damage [26].

(3) Immune System Modulation: Cold stress often leads to changes in immune function, including alterations in the complement system, antimicrobial peptides, and lysozyme. In fish, low temperatures suppress certain immune responses and increase disease susceptibility [27]. In crustaceans, nonspecific immune indicators like acid phosphatase (ACP), alkaline phosphatase (AKP), and antimicrobial peptides like Crustin exhibit phase-specific changes [28]. Mollusks modulate immune-related gene expression and energy distribution to balance defense and survival [29].

(4) Protein Homeostasis, Autophagy, and Apoptosis Regulation: Cold exposure induces protein misfolding and organelle damage, triggering molecular chaperones, the ubiquitin–proteasome system, and autophagy processes to clear damaged components. If damage exceeds repair capacity, apoptosis is activated via MAPK, p53, and caspase signaling pathways [30].

3.1.3. Tertiary Response: Behavioral Manifestations and Ecological Consequences

The tertiary response represents the ultimate manifestation of cold stress at the individual and population levels, including changes in behavior, growth, reproduction, and survival. For example, fish reduce feeding, activity, and growth at low temperatures [31]; crustaceans enter low metabolic or hibernation-like states to conserve energy [4]; mollusks adopt behavioral strategies such as burrowing and migration or selecting microhabitats to avoid extreme temperatures [32]. Over the long term, cold stress can also impact reproductive cycles and population structure, thereby affecting aquaculture yields and ecosystem stability.

3.2. Common Mechanisms and Differential Patterns in Aquatic Animals’ Cold Stress Response

From the comparison of different taxa, we can identify several conserved mechanisms, such as energy metabolism regulation, oxidative stress defense, immune modulation, and cell fate control. As shown in Figure 1, these mechanisms are ubiquitous across taxa but are regulated in different ways and hold different levels of importance.

From a commonality perspective, cold stress generally leads to metabolic reprogramming and ROS accumulation, activating antioxidant, defense, and repair mechanisms. From a differential perspective, fish typically exhibit more complex transcriptional regulation networks and metabolic control, employing pathways like PPAR/RXR, PGC-1α, and miRNA–TF–mRNA networks. Crustaceans rely more on neuroendocrine regulation and hepatopancreatic metabolic control, while mollusks primarily utilize metabolic depression, energy reserves, and behavioral strategies to adapt to low temperatures. Additionally, extreme environment species have formed long-term evolutionary adaptations. For example, Antarctic fish exhibit gene family expansions and reinforced protein homeostasis networks to sustain life in sub-zero conditions, showing that cold adaptation is not merely a short-term stress response but can be fixed as a stable physiological trait through evolutionary processes [33].

Overall, the response of aquatic animals to cold stress can be viewed as a multi-layered dynamic process consisting of “rapid signaling perception—metabolic and cellular regulation—individual performance”, where conserved mechanisms and taxon-specific strategies jointly determine cold tolerance across different species.

4. Fish Response Mechanisms to Cold Stress

4.1. Neuroendocrine Response and Behavioral Adaptations

As ectothermic vertebrates, fish rely on coordinated cellular, neural, and endocrine mechanisms to perceive and respond to changes in environmental temperature. At the sensory and cellular levels, cold exposure can be detected through changes in membrane fluidity and ion-channel activity, including transient receptor potential (TRP) channels, which function as important thermal sensors and contribute to the initiation of neural signaling under low-temperature conditions. These early events are further associated with the induction of cold-responsive molecules such as CIRBP, thereby linking cellular cold perception to systemic stress responses [34]. Upon exposure to cold stress, fish rapidly activate the HPI axis, triggering the release of cortisol and other stress-related hormones that regulate energy allocation, immune function, and behavioral adaptation [17,35].

Fish, being ectothermic, rely heavily on their neuroendocrine system to detect and respond to changes in environmental temperature. Upon exposure to cold stress, fish rapidly activate the HPI axis, triggering the release of cortisol and other stress-related hormones that regulate metabolic and immune responses [15]. These hormones play a crucial role in energy allocation, immune modulation, and thermoregulation during cold stress.

At the behavioral level, cold exposure typically leads to reduced feeding activity, swimming behavior, and overall movement [36]. Recent studies in aquaculture species further support this pattern. In juvenile hybrid sturgeon, low-temperature exposure significantly reduced growth performance and feed utilization [37], while a study on darkbarbel catfish reported pronounced cold-stress responses, including behavioral suppression, when water temperature dropped below the species-specific tolerance range [38]. Similar observations have also been reported in red tilapia, in which cold stress during overwintering is considered a major bottleneck for production and is associated with broad transcriptomic and proteomic changes in the brain, gill, liver, and skin [39]. These findings indicate that cold-induced reductions in activity and feeding are closely linked to metabolic reallocation, energy conservation, and stress adaptation in cultured fish. Therefore, rather than true torpor in a strict physiological sense, many fish exhibit metabolic suppression or overwintering dormancy-like states during prolonged cold exposure, thereby reducing energy expenditure and improving survival under low-temperature conditions [40,41].

Many fish species exhibit a decrease in metabolic rate and enter a period of decreased activity to conserve energy [40]. This is especially evident in species like salmonids, which exhibit a marked reduction in movement and feeding during cold acclimation. Some species can also enter a state of metabolic suppression or torpor, thereby reducing the energy demands of maintaining body temperature [41]. These behavioral adaptations are essential for surviving prolonged exposure to cold environments. However, when cold stress exceeds a certain threshold, the disruption to metabolic processes and immune functions may lead to increased susceptibility to pathogens and ultimately, mortality.

4.2. Metabolic Reprogramming: Lipid and Carbohydrate Metabolism

Cold exposure in fish causes a significant reprogramming of metabolic pathways. Metabolic depression is a common response, where energy expenditure is minimized, and cellular functions are altered to conserve energy. In particular, lipid metabolism plays a key role in maintaining energy balance under cold stress [42].

Cold exposure can reshape fish energy metabolism by coordinating mitochondrial activity, glucose mobilization, lipid catabolism, fatty acid β-oxidation, and redox homeostasis. In Nile tilapia (Oreochromis niloticus), acute cold stress causes marked physiological and biochemical changes, including altered ventilation rate, heart rate, oxygen saturation, mitochondrial activity, hydrogen peroxide production, malondialdehyde accumulation, nitric oxide levels, and total antioxidant capacity, indicating that abrupt cooling rapidly affects mitochondrial function and oxidative metabolic balance [43]. In salmonids, prolonged cold exposure can also impair liver metabolism and function; for example, Atlantic salmon (Salmo salar) exposed to prolonged low temperature showed disrupted hepatic lipid metabolism and liver dysfunction, suggesting that cold-induced metabolic adjustment may become maladaptive when exposure is prolonged or exceeds the physiological tolerance range [41]. At the molecular level, PPARα and carnitine palmitoyltransferase 1 (CPT1) remain important regulators of lipid utilization, because PPARα regulates genes involved in fatty acid oxidation, whereas CPT1 controls the mitochondrial entry of long-chain fatty acids for β-oxidation [44]. Therefore, cold-induced regulation of the PPARα–CPT1 axis may contribute to the use of lipid reserves as an energy source during low-temperature exposure, although the direction and magnitude of this response are species-, tissue-, and exposure-duration dependent.

Additionally, cold exposure can influence glycogen metabolism in the liver and muscle tissues of fish. Glycogen, being one of the primary energy reserves in fish, is mobilized during cold stress to support basic metabolic functions. This response, however, depends on the species and environmental conditions, as some fish may rely more heavily on lipid reserves, while others may utilize carbohydrate stores more efficiently [45]. This metabolic flexibility allows fish to adapt to cold environments by shifting energy usage according to the available resources.

4.3. Oxidative Stress and Antioxidant Defense

Exposure to cold temperatures leads to an imbalance between the production of ROS and the antioxidant capacity of cells, resulting in oxidative stress. Cold-induced oxidative stress is particularly harmful to cellular structures, including lipids, proteins, and nucleic acids, and can significantly impair cellular function [46].

Fish exhibit a marked increase in antioxidant enzyme activity under cold stress, with enzymes such as SOD, CAT, and GSH-Px being upregulated to counteract the damage caused by ROS [47]. These antioxidant enzymes play a crucial role in protecting cells from oxidative damage and maintaining redox homeostasis. However, when cold exposure is prolonged or exceeds the physiological tolerance range, antioxidant defenses may become insufficient, resulting in ROS accumulation, lipid peroxidation, mitochondrial dysfunction, and tissue injury. For example, cold stress in Takifugu fasciatus has been shown to induce intestinal oxidative stress, activate ROS-mediated MAPK pathways, disrupt lipid metabolism, and cause cellular damage [48]. Similarly, chronic cold stress in hybrid red tilapia resulted in oxidative-stress biomarker changes and severe histopathological alterations in the liver and gill tissues [49]. These findings indicate that cold-induced oxidative stress is directly associated with tissue damage and impaired physiological function in fish.

Cold-acclimated fish often display adjusted antioxidant enzyme activities and enhanced resistance to oxidative damage compared with non-acclimated individuals. Nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signaling is one of the central regulatory pathways that coordinates antioxidant and cytoprotective gene expression under oxidative stress. In zebrafish, cold exposure has been reported to activate Nrf2/ARE-related antioxidant responses [50]. Recent studies further suggest that Nrf2-associated antioxidant regulation is involved in fish responses to cold-related oxidative stress. For example, dietary antioxidant intervention has been reported to improve cold-stress tolerance in juvenile Nile tilapia, and this protective effect may be associated with activation of Nrf2 signaling. However, the magnitude and direction of Nrf2/ARE responses may vary among species, tissues, and cold-exposure regimes, indicating that this pathway should be interpreted within a species- and context-specific framework [51].

4.4. Immune Modulation in Cold-Stressed Fish

Cold stress significantly affects the immune function of fish, as it alters both innate and adaptive immune responses. In particular, cold temperatures can impair the function of immune cells, including macrophages and neutrophils, which are essential for pathogen defense [52]. This immunosuppressive effect increases the susceptibility of cold-stressed fish to bacterial, viral, and parasitic infections. From an aquaculture perspective, reduced immune competence during winter or sudden cold spells may increase the risk of disease outbreaks, compromise the protective efficacy of vaccination, delay immune memory formation, and consequently contribute to increased mortality, reduced production efficiency, and economic losses.

Research indicates that cold temperatures can increase the expression of immune-related genes, such as lysozyme, complement proteins, and antimicrobial peptides (AMPs), which play crucial roles in the fish’s defense against pathogens [53]. However, prolonged cold stress may decrease the efficacy of these immune responses, particularly in species that do not have strong cold-acclimation capabilities [54].

Moreover, studies have demonstrated that the production of stress hormones, such as cortisol, during cold stress can have immunosuppressive effects, further compromising the fish’s ability to mount an effective immune response [55]. Therefore, understanding the dynamic relationship between cold stress, immune function, and disease susceptibility is critical for improving fish health management during cold weather events.

5. Crustacean Response Mechanisms to Cold Stress

5.1. Neuroendocrine Regulation and Behavioral Adaptations

Similar to fish, crustaceans rely on their neuroendocrine system to mediate the stress response to low temperatures. The primary stress hormones in crustaceans include crustacean CHH and octopamine, which regulate metabolic processes, energy mobilization, and cold tolerance [14,56]. Upon cold exposure, CHH is released to increase glucose levels, providing energy for basic metabolic functions and sustaining survival under low-temperature conditions.

Crustaceans exhibit several behavioral adaptations in response to cold stress. For example, species such as the Chinese mitten crab (Eriocheir sinensis) [57] and white shrimp (Litopenaeus vannamei) [58] reduce activity and feeding during cold exposure, conserving energy and reducing the risk of cold-induced damage. Some crustaceans, particularly those in temperate or subarctic regions, are capable of entering a low metabolic state or hibernation, effectively lowering their metabolic demands to survive long-term cold conditions [6].

5.2. Energy Metabolism and Lipid Mobilization

Cold exposure in crustaceans leads to a decrease in metabolic rate, particularly in hepatopancreatic function, which is essential for energy storage and utilization.

Crustaceans rely mainly on glycogen, lipids, and free amino acids as metabolic reserves, and these substrates can be dynamically mobilized to support essential physiological functions during low-temperature exposure. Recent evidence indicates that cold stress can regulate crustacean lipid metabolism through AMPK signaling. For example, in redclaw crayfish (Cherax quadricarinatus), chronic cold stress altered hepatopancreatic and hemolymph lipid stores and modulated AMPK-related lipid metabolic responses, indicating that AMPK acts as an important energy sensor during cold-induced metabolic adjustment [59]. Recent hepatopancreatic transcriptomic and metabolomic analyses in red swamp crayfish (Procambarus clarkii) also indicate that cold stress alters metabolism and innate immune responses [60]. Together, these studies support the view that AMPK-related energy sensing, lipid mobilization, glycolytic adjustment, and hepatopancreatic metabolic remodeling are central components of crustacean cold-stress adaptation.

Crustaceans like the freshwater crayfish (P. clarkii) demonstrate enhanced lipid mobilization during cold stress, and this response is associated with increased activity of lipases and β-oxidation enzymes. This metabolic shift helps crustaceans to utilize stored fats, which are more energy-dense than glycogen, to maintain basic functions during periods of low food availability or extreme cold temperatures [61].

5.3. Antioxidant Defense and Oxidative Stress in Crustaceans

In parallel with fish models, crustaceans experience oxidative stress under cold conditions, which can lead to cellular damage. To counteract oxidative damage, crustaceans activate their antioxidant defense systems, including SOD, CAT, and glutathione. These enzymes play a crucial role in neutralizing ROS and protecting tissues from oxidative damage [62].

Research on species such as L. vannamei [63] and P. clarkii [64] has shown that cold exposure induces the expression of antioxidant enzymes and enhances the glutathione cycle, which is critical for maintaining cellular redox balance. However, prolonged cold exposure can overwhelm the antioxidant system, leading to increased lipid peroxidation and cellular damage. In particular, the hepatopancreas is highly susceptible to oxidative stress, which can impair metabolic and immune functions [6].

5.4. Immune Modulation in Crustaceans

Cold stress negatively impacts the immune responses of crustaceans, leading to immune suppression. Studies have shown that cold exposure can decrease the activity of immune cells such as hemocytes, which are essential for pathogen defense [65]. In addition, cold temperatures can reduce the expression of immune-related genes like Crustin, lysozyme, and complement proteins, which are involved in the crustacean immune response [66].

Furthermore, cold-induced stress hormones like CHH can also suppress the immune system by altering the function of hemocytes and disrupting the production of antimicrobial peptides [67]. This combined effect makes crustaceans more vulnerable to infections during cold stress, and highlights the need for improved immune support in aquaculture management, especially during colder months.

6. Mollusk Response Mechanisms to Cold Stress

6.1. Metabolic Suppression and Energy Reserves

Mollusks exhibit a distinct approach to cold stress compared to fish and crustaceans. The most notable response is metabolic suppression, where mollusks decrease their metabolic rates significantly to conserve energy during cold exposure. This is particularly evident in species like freshwater mussels and marine clams, which rely on stored glycogen and lipids to fuel basic metabolic processes [16].

At the cellular level, this metabolic depression involves a reduction in mitochondrial activity and overall cellular respiration. The suppression of metabolic activity helps mollusks endure prolonged periods of low temperature without exhausting their energy reserves. Research has shown that mollusks such as Pacific oysters (Magallana gigas) can survive in cold environments by utilizing glycogen stored in their tissues, mobilizing these reserves to support essential functions [68].

Additionally, cold-acclimated mollusks exhibit increased lipid mobilization, suggesting that lipids play a key role in energy supply during low-temperature exposure. In some species, like blue mussels, the adjustment of lipid metabolism allows the maintenance of membrane integrity and fluidity, which are critical for cell function in cold conditions [69].

6.2. Oxidative Stress and Antioxidant Defense

Similar to fish and crustaceans, mollusks are also affected by oxidative stress when exposed to cold temperatures. Cold-induced oxidative stress in mollusks can lead to cellular damage, particularly in the liver and gill tissues, where oxidative stress markers such as malondialdehyde (MDA) and ROS are significantly elevated [70].

Mollusks respond to cold-induced oxidative stress by remodeling antioxidant defenses, including SOD, CAT, GSH-Px, glutathione metabolism, and other redox-related pathways. Recent evidence from the warm-water noble scallop Chlamys nobilis showed that chronic cold stress altered tissue structure, antioxidant activity, and the expression of key stress-related genes, indicating that antioxidant remodeling is an important component of cold tolerance in warm-water bivalves [20]. Similarly, a recent study in the bay scallop Argopecten irradians reported that temperature changes induced biochemical and molecular responses associated with antioxidant defense [71].

6.3. Immune System Modulation

Cold stress in mollusks also significantly affects their immune system. Cold temperatures lead to alterations in immune cell function, particularly in hemocytes, which are responsible for pathogen defense. Research indicates that hemocyte activity decreases under cold stress, making mollusks more vulnerable to infections during colder periods [72].

Cold exposure can alter the expression of immune-related genes in mollusks, suggesting that low temperature affects both hemocyte-mediated cellular defense and humoral immune responses. In recent years, increasing attention has been paid to innate immune memory, immune priming, or trained immunity in marine mollusks [73]. Evidence from cultured bivalves, particularly oysters and mussels, indicates that previous immune challenges can enhance subsequent defense responses, and this process may involve immune gene reprogramming, metabolic remodeling, and epigenetic regulation [73,74]. Thermal priming studies further suggest that mollusks can retain a form of physiological stress memory after previous temperature exposure, which may influence later stress tolerance [75]. However, direct evidence that repeated cold exposure alone induces long-lasting immune priming in mollusks remains limited. Therefore, cold-related immune priming should currently be regarded as a promising but insufficiently resolved topic, requiring future studies that integrate repeated cold-challenge experiments, pathogen susceptibility assays, hemocyte functional analysis, and epigenomic profiling.

Similar to crustaceans, cold exposure can lead to changes in the expression of immune-related genes. Interestingly, some species of mollusks, like clams, show signs of immune priming under cold stress, where repeated cold exposure leads to enhanced immune responses upon re-exposure [76]. This phenomenon suggests that mollusks may possess a degree of immune memory for cold stress, allowing them to better respond to future low-temperature challenges.

6.4. Behavioral Adaptations and Microhabitat Selection

Mollusks exhibit a unique set of behavioral adaptations to cope with cold stress. Unlike fish and crustaceans, mollusks rely heavily on their ability to select appropriate microhabitats to avoid extreme temperature fluctuations. Species such as barnacles and mussels can adjust their position within the intertidal zone, moving to deeper waters where temperatures are more stable [77]. This behavior allows mollusks to minimize exposure to cold stress and optimize their chances of survival.

In addition, many infaunal mollusks, such as clams and some juvenile or sediment-associated bivalves, engage in burrowing behavior. By moving into sediments, these animals can reduce direct exposure to cold air or rapidly fluctuating surface water temperatures and occupy a more thermally buffered microhabitat [16]. An experimental study has shown that bivalve burrowing depth and burrowing mode are influenced by sediment properties and geoenvironmental conditions, while freshwater mussel survival and behavior under thermal stress can be modified by the presence of sediment temperature gradients [78].

6.5. Cold Tolerance and Freeze Avoidance

Some mollusks, particularly those inhabiting polar and subpolar regions, exhibit remarkable cold tolerance mechanisms that allow them to survive freezing temperatures. These species, such as certain Antarctic mollusks, have developed the ability to avoid freezing through the production of antifreeze proteins (AFPs), which inhibit the growth of ice crystals in body fluids [79].

In addition to antifreeze proteins (AFPs), some mollusks employ freeze-avoidance or freeze-tolerance strategies that allow them to survive subzero or ice-associated environments [80]. These strategies may involve supercooling of body fluids, control of extracellular ice formation, osmotic adjustment, and protection of critical tissues from lethal intracellular freezing. A representative example is the Antarctic limpet Nacella concinna, which inhabits Antarctic intertidal and shallow subtidal habitats where freezing and ice disturbance are common. Experimental studies have shown that the pedal mucus of N. concinna can delay internal ice formation, while organismal freezing is also influenced by its osmoconforming physiology and tissue-specific ice nucleation characteristics. This case illustrates that freezing-related adaptations in mollusks are not merely general cold-stress responses but can involve specific physiological and extracellular mechanisms that enhance survival in extreme polar habitats [81].

7. Core Regulatory Mechanisms in Cold Stress Response: PGC-1α, DUSP1, and Other Key Players

7.1. Energy and Metabolic Regulation: AMPK, PPAR, and mTOR

At the molecular level, cold stress is primarily regulated through key metabolic sensors and pathways, including AMPK, PPAR, and mTOR pathways. These pathways control energy balance, lipid metabolism, and the overall metabolic reprogramming necessary for cold tolerance (Figure 2).

AMPK acts as a central energy regulator, sensing changes in ATP/AMP ratios and activating metabolic pathways that conserve energy [12]. In fish, AMPK activation has been shown to enhance fatty acid oxidation and glucose production under cold stress, ensuring that energy reserves are utilized efficiently [82]. Similarly, in mollusks and crustaceans, AMPK regulates lipid metabolism during cold exposure, promoting energy conservation and protecting vital functions [59]. PPAR is another critical regulator of cold-induced metabolic changes. PPARα, in particular, is involved in the regulation of fatty acid oxidation and gluconeogenesis [83]. Cold stress activates PPARα expression in fish, crustaceans, and mollusks, helping these organisms mobilize stored fat to meet energy demands during periods of low temperatures. Finally, mTOR is a key integrator of nutrient and energy signals, and its inhibition under cold stress leads to the activation of autophagy, a process that helps cells recycle damaged proteins and organelles [12]. Autophagy is particularly important for maintaining cellular homeostasis under cold conditions and has been shown to enhance cold tolerance in several aquatic species.

7.2. Oxidative Stress and Antioxidant Defense Mechanisms: PGC-1α and DUSP1

In addition to metabolic regulation, cold stress also activates the PGC-1α pathway, which plays a central role in mitochondrial function and oxidative stress management [84]. As shown in Figure 3, PGC-1α is involved in mitochondrial biogenesis and respiration, helping cells maintain energy production and reduce ROS accumulation under cold stress. The activation of PGC-1α has been shown to improve cold tolerance by enhancing the oxidative capacity of cells and preventing cellular damage caused by ROS [85].

DUSP1 (Dual-specificity phosphatase 1), another key regulator in cold stress, plays a critical role in the MAPK signaling pathway, which is involved in inflammation, stress response, and apoptosis. DUSP1 regulates the deactivation of MAPK, thereby reducing ROS generation and promoting cell survival during cold exposure. A large number of studies in fish, such as the Japanese flounder (Paralichthys olivaceus) [86], silver pomfret (Pampus argenteus) [8] and grass carp (Ctenopharyngodon idellus) [87], have all highlighted the importance of DUSP1 in regulating oxidative stress and preventing excessive apoptosis under cold conditions. In contrast to fish, direct evidence for DUSP family involvement in cold-stress responses of mollusks and crustaceans remains limited. Nevertheless, because DUSPs are conserved negative regulators of mitogen-activated protein kinase (MAPK) signaling, they may represent plausible candidates for modulating oxidative stress, inflammation, and apoptosis in invertebrate cold-stress responses. Future studies should therefore identify DUSP family members in representative mollusks and crustaceans and experimentally validate their roles in MAPK dephosphorylation, redox regulation, and cell fate control under low-temperature exposure.

7.3. Protein Homeostasis and Molecular Chaperones: HSPs and Ubiquitin-Proteasome System

Protein misfolding and aggregation are common consequences of cold stress, as low temperatures disrupt the normal folding and function of cellular proteins [88]. To counteract these effects, organisms rely on molecular chaperones, particularly heat shock proteins (HSPs), which help refold damaged proteins and maintain cellular protein homeostasis. In fish and other aquatic ectotherms, cold exposure can induce a coordinated molecular chaperone response involving heat shock protein 70 (HSP70), HSP90, and other heat shock protein families. These chaperones facilitate correct protein folding, prevent aggregation of misfolded proteins, stabilize cellular structures, and support stress recovery under low-temperature conditions [89,90]. However, HSP responses to cold are highly context-dependent; their expression may vary according to species, tissue, thermal history, exposure duration, and whether the stress is acute cold shock, chronic cold stress, or temperature fluctuation [91]. Recent studies further indicate that HSP-related genes are involved in low-temperature tolerance and stress recovery in aquaculture species, while HSP90 has also been functionally linked to reduced cold-induced apoptosis in crustaceans [92]. Additionally, the ubiquitin–proteasome system (UPS) participates in the degradation of irreversibly misfolded or damaged proteins. Together, molecular chaperone induction and UPS-mediated protein turnover form a complementary proteostasis network that helps maintain cellular integrity under cold stress [93,94]. The combined action of HSPs and the UPS ensures that cellular functions are preserved, even under adverse cold conditions (Figure 4).

7.4. Autophagy and Apoptosis Regulation: Cdk1 and p53

Autophagy and apoptosis are two critical processes that regulate cell fate under cold stress. Autophagy, as a protective cellular mechanism, allows the degradation of damaged proteins and organelles, maintaining cellular homeostasis and energy balance during cold exposure [95]. The activation of autophagy is primarily regulated by the AMPK and mTOR signaling pathways. AMPK senses energy depletion or changes, activating fatty acid β-oxidation and glycogen metabolism to support cell function, while mTOR inhibition promotes autophagy. In zebrafish, cold exposure significantly enhanced AMPK activity, promoting fatty acid oxidation and energy metabolism, which helped maintain cellular function and reduce damage from cold stress [96]. Additionally, autophagy helps remove damaged organelles, such as mitochondria, and proteins, preventing further oxidative damage and ensuring long-term cell survival.

However, when the damage exceeds the repair capacity of autophagy, apoptosis is triggered as another cell protection mechanism to eliminate irreparable cells [97]. The p53 signaling pathway plays a crucial role in cold stress response. p53 senses DNA damage and activates downstream apoptotic pathways, such as caspase signaling, leading to cell death and preventing further damage from compromised cells [98]. Research in the large yellow croaker (Larimichthys crocea) has shown that cold exposure induces the activation of p53, enhancing DNA damage repair and initiating apoptosis to clear damaged cells [99]. Recent evidence in zebrafish further supports the involvement of p53 regulation in low-temperature tolerance. Deletion of a novel upstream promoter of p53 impaired cold tolerance at 8 °C, increased reactive oxygen species accumulation, and reduced the expression of antioxidant defense genes such as sod and gsh-px, indicating that p53 also contributes to redox homeostasis and cold adaptation in fish [100]. Similarly, in P. clarkii, cold stress activates both autophagy and apoptosis. The crayfish uses autophagy to remove damaged organelles and proteins, helping to maintain energy balance and prevent cell death [59]. However, when damage exceeds autophagic repair capacity, apoptosis is triggered through the p53 signaling pathway. In crustaceans, Cdk1 plays a critical role in regulating both the cell cycle and apoptosis, ensuring that only healthy cells survive in cold conditions [101]. Cdk1 not only regulates cell cycle progression but also ensures that damaged cells enter apoptosis in a timely manner, preventing their proliferation.

The interplay between these mechanisms plays a crucial role in cold stress, particularly under prolonged cold exposure or extreme cold environments, where the survival and death of cells ultimately depend on the balance between autophagy and apoptosis. The regulatory balance between AMPK/mTOR-mediated autophagy and p53/Cdk1-associated apoptosis is summarized in Figure 5.

8. Cold Stress Adaptation and Breeding Strategies

8.1. Physiological Acclimation and Temperature Control Management

In aquaculture practice, the most direct and widely used method to combat cold stress is through environmental regulation and physiological acclimation to enhance the cold tolerance of aquatic animals. Unlike sudden cold shock, gradual temperature reduction allows the body time to adapt, optimizing metabolic processes, activating antioxidant systems, and adjusting membrane structure for better cold tolerance. This acclimation process significantly reduces mortality rates during cold events. For instance, in E. sinensis [102] and L. vannamei [103], gradual cooling of water temperatures induces a low metabolic state, enhancing long-term cold survival by reducing energy consumption and preventing tissue damage. Recent aquaculture-related evidence from the Manila clam (Ruditapes philippinarum) shows that thermal priming can mitigate the effects of lethal marine heatwaves, supporting the potential application of controlled pre-exposure strategies in farmed aquatic species [75].

Moreover, precise temperature control within aquaculture systems plays a crucial role in maintaining optimal conditions for cold-stressed species. With advancements in Internet of Things (IoT) technologies and smart aquaculture systems, real-time monitoring and temperature regulation have become feasible, providing technological support for managing cold events and reducing associated risks [104].

In summary, temperature control management and physiological acclimation are short-term strategies that can be quickly implemented to manage cold stress. However, these strategies do not rely on genetic modification and are generally not sustainable in the long term.

8.2. Nutritional Regulation and Metabolic Intervention

Nutritional regulation plays an essential role in cold stress management, as it connects basic physiological mechanisms with practical aquaculture applications. As mentioned earlier, cold stress triggers metabolic reprogramming, especially the enhancement of lipid metabolism and the activation of autophagy, which are critical for improving cold tolerance.

Studies have shown that short-term fasting or feeding restrictions can activate AMPK signaling and enhance fatty acid β-oxidation and autophagy, thereby significantly increasing cold tolerance in species like zebrafish [105]. Meanwhile, adjusting the lipid composition of feed by adding unsaturated fatty acids or altering dietary fats can improve membrane fluidity and optimize energy utilization, which is particularly important for cold-acclimated fish [106]. In crustaceans, the mobilization of lipids and glycogen is also essential to support energy demands during cold exposure. Supplementation with antioxidants, such as vitamins C and E, has been shown to alleviate ROS accumulation, thereby reducing oxidative damage and improving immune responses under cold conditions [107]. However, while nutritional regulation has been shown to be an effective short-term strategy, its effects can vary significantly between species and developmental stages. Prolonged nutritional restrictions or excessive modification of nutrient composition may have negative impacts on growth and reproduction [108]. Therefore, more precise, mechanism-driven nutritional interventions are required for effective cold stress management.

8.3. Marker-Assisted Selection (MAS) and Genomic Selection (GS)

With the development of genomic technologies, cold-resistant breeding has gradually shifted from traditional phenotype-based selection to molecular-level strategies, such as Marker-Assisted Selection (MAS) and Genomic Selection (GS).

Currently, numerous studies have identified potential candidate genes linked to cold tolerance through transcriptomic and genomic analyses. Key genes such as PPAR, PGC-1α, CPT1, DUSP1, and Lv-Cdk1 play roles in regulating energy metabolism, mitochondrial function, oxidative stress, and cell fate, making them potential molecular markers for cold resistance [109,110]. Genomic studies in species like pufferfish (Takifugu obscurus) [111], blue tilapia (Oreochromis aureus) [112] and marine mussel (Mytilus galloprovincialis) [113] have shown that genomic selection using high-density molecular markers can significantly improve cold tolerance in breeding programs. However, GS still faces challenges such as high costs, insufficient reference populations, and limited prediction accuracy, especially in species like crustaceans and mollusks. More research is needed to improve the efficiency of GS for cold resistance and to expand its application across different aquatic species.

8.4. Gene Editing and Functional Gene Improvement

With the advent of CRISPR/Cas and other gene editing technologies, directly modifying key functional genes for cold tolerance is now a feasible strategy. For example, by regulating genes involved in lipid metabolism, mitochondrial function, or oxidative stress, it is possible to genetically enhance cold tolerance in aquatic species [114].

In addition, antifreeze proteins found in extreme environment species like Antarctic fish provide a potential resource for gene engineering, although their applicability in most aquaculture species remains to be validated [115]. Nevertheless, the application of gene editing in aquaculture still faces challenges related to ethics, regulation, and ecological safety, requiring cautious assessment before large-scale implementation.

8.5. Hybrid Breeding and Germplasm Utilization

Hybrid breeding is another important traditional method for enhancing cold tolerance. By utilizing genetic differences between populations or species, hybrid offspring can possess superior cold resistance due to hybrid vigor [116].

In fish, hybridization between different geographic populations (e.g., temperate and subtropical groups) has been used to improve cold tolerance, for example, the stronger cold resistance in a hybrid crucian carp “Xiangyun” [117]. In crustaceans, germplasm introduction and hybridization have also been employed to increase survival rates during winter months [118]. Wild populations, particularly those from higher latitudes or higher altitudes, often exhibit enhanced cold resistance and are considered valuable genetic resources for breeding programs [119]. However, hybrid breeding faces challenges such as genetic instability, segregation of traits, and potential growth-cold resistance trade-offs, highlighting the need for integration with molecular breeding technologies.

8.6. Strategy Integration and Future Directions

Overall, current cold resistance strategies can be categorized into three levels: (1) Short-term strategies: Environmental control and physiological acclimation; (2) Mid-term strategies: Nutritional regulation and metabolic interventions; (3) Long-term strategies: Molecular breeding and genetic improvement. Future development should move from a single strategy to an integrated approach, combining physiological acclimation, nutritional interventions, and molecular breeding to enhance cold tolerance. For instance, nutritional adjustments combined with genetic improvement through MAS or GS can help increase cold resistance in species. Moreover, gene editing can be used to precisely modify cold tolerance-related genes, while functional genomics can help identify novel markers for cold stress management. In addition, future cold stress research should focus on the synergistic integration of environmental, nutritional, and genetic management strategies to improve cold resistance in aquaculture systems.

9. Summary and Future Perspectives

9.1. Unified Mechanisms and Cross-Taxon Comparisons

Aquatic animals’ responses to cold stress are complex and multifaceted, involving different physiological, biochemical, and molecular mechanisms. Despite significant differences across taxa, common adaptive pathways such as energy metabolic regulation, oxidative stress response, and protein homeostasis maintenance are essential for survival under cold stress. These common mechanisms are regulated in different ways depending on the species and environmental conditions.

A unifying model of cold stress response suggests that AMPK, PPAR, PGC-1α, and DUSP1 act as key molecular players that regulate energy metabolism, oxidative stress, and cell survival across taxa. These mechanisms are crucial for maintaining cellular function, protecting against oxidative damage, and ensuring that energy supplies are effectively utilized under cold stress. The conservation of these mechanisms across fish, crustaceans, and mollusks underscores the evolutionary stability of cold stress responses.

9.2. Future Research Directions

Despite substantial progress in understanding cold stress mechanisms, several critical gaps remain in current research:

(1)

From Differential Expression to Causal Mechanism Verification

Many studies still focus on differential gene expression and physiological changes without experimentally verifying causal relationships. Future research should incorporate functional validation of candidate genes involved in cold tolerance, such as CPT1b, DUSP1, and Lv-Cdk1. By using CRISPR/Cas9 gene editing, we can determine the direct role of these genes in cold adaptation.

(2)

Integrating Temporal and Tissue-Level Responses

Cold stress responses unfold over time, and different tissues (e.g., liver, brain, immune organs) react at different rates. Future studies should use longitudinal sampling and multi-tissue transcriptomic analysis to provide a comprehensive understanding of the dynamic nature of cold stress and how different organs interact during stress adaptation.

(3)

Studying Combined Stressors, Not Just Cold

Cold stress often co-occurs with other environmental stressors such as hunger, hypoxia, and disease. Research should examine how cold interacts with other environmental factors to more accurately reflect real-world aquaculture conditions and develop effective combined stress mitigation strategies.

(4)

From Stress Markers to Breeding Targets

Antioxidant markers, such as SOD, CAT, and HSP70, are often used to indicate stress, but they may not always serve as suitable genetic selection markers. Instead, PPAR, PGC-1α, DUSP1, and Cdk1 are more promising functional breeding targets for cold tolerance. Future breeding programs should focus on these genes and integrate marker-assisted selection (MAS) and genomic selection (GS) approaches for more efficient cold-tolerance breeding.

(5)

Integrating Physiological Acclimation, Nutritional Regulation, and Genetic Improvement

Future cold stress research should adopt a multidisciplinary approach, combining physiological acclimation, nutritional management, and genetic improvement strategies. Gene editing combined with nutritional interventions will enhance cold resistance while reducing the reliance on environmental control.

(6)

Aquaculture Implications and Practical Applications

From an aquaculture perspective, cold-stress management should be implemented at short-, medium-, and long-term levels. In the short term, acute cold shock can be reduced through temperature buffering, gradual acclimation before predictable cold events, improved overwintering management, and avoidance of additional stressors such as handling, transport, crowding, and poor water quality. In the medium term, nutritional and health-management strategies should support energy metabolism, antioxidant defense, membrane stability, immune competence, and gut microbiota balance, thereby reducing oxidative damage, immunosuppression, and disease susceptibility during low-temperature periods. In the long term, cold-tolerance traits should be incorporated into selective breeding programs, with candidate pathways such as AMPK–mTOR, PPARα/CPT1, Nrf2/ARE, HSP/UPS, and p53/Cdk1 serving as potential molecular targets after functional validation. Therefore, effective aquaculture applications should integrate environmental management, nutritional intervention, health monitoring, and genetic improvement according to species, production stage, and cold-stress risk.

9.3. Conclusions

This review indicates that cold-stress responses in aquatic organisms are organized around several conserved functional modules, including energy redistribution, redox regulation, immune modulation, proteostasis, autophagy, and apoptosis. Key pathways such as AMPK–mTOR, PPARα/CPT1, PGC-1α, Nrf2/ARE, HSP/UPS, DUSP1/MAPK, and p53/Cdk1 repeatedly appear in the literature as important molecular nodes associated with cold tolerance.

However, these mechanisms are not uniform across taxa. Fish generally rely more on neuroendocrine regulation, mitochondrial adjustment, transcriptional remodeling, and lipid metabolism. Crustaceans show stronger dependence on hepatopancreatic energy mobilization, crustacean hyperglycemic hormone (CHH)-related signaling, AMPK-associated metabolism, and innate immune regulation. Mollusks rely more on metabolic depression, glycogen and lipid reserve utilization, burrowing or microhabitat selection, and stress-memory-like responses. Therefore, cold-stress mechanisms should be interpreted according to species-specific thermal windows, habitat background, exposure intensity, duration, and acclimation history.

Current evidence is strongest for descriptive physiological, biochemical, transcriptomic, and metabolomic responses, whereas causal validation remains limited. Future studies should use gene knockdown, gene editing, pharmacological intervention, thermal priming experiments, and standardized cold-challenge models to verify the functional roles of candidate regulators such as CPT1, DUSP1, HSP70/HSP90, Nrf2, p53, and Cdk1.

For aquaculture, cold-stress management should be matched to the time scale of risk. Short-term strategies should include temperature buffering, gradual acclimation, and improved overwintering management. Medium-term strategies should emphasize nutritional support for energy metabolism, antioxidant defense, membrane stability, and immunity. Long-term strategies should focus on cold-tolerance breeding, marker-assisted selection, genomic selection, and functional validation of cold-resistance genes.