A Review on the Involvement of Heat Shock Proteins (Extrinsic Chaperones) in Response to Stress Conditions in Aquatic Organisms

Heat shock proteins (HSPs) encompass both extrinsic chaperones and stress proteins. These proteins, with molecular weights ranging from 14 to 120 kDa, are conserved across all living organisms and are expressed in response to stress. The upregulation of specific genes triggers the synthesis of HSPs, facilitated by the interaction between heat shock factors and gene promoter regions. Notably, HSPs function as chaperones or helper molecules in various cellular processes involving lipids and proteins, and their upregulation is not limited to heat-induced stress but also occurs in response to anoxia, acidosis, hypoxia, toxins, ischemia, protein breakdown, and microbial infection. HSPs play a vital role in regulating protein synthesis in cells. They assist in the folding and assembly of other cellular proteins, primarily through HSP families such as HSP70 and HSP90. Additionally, the process of the folding, translocation, and aggregation of proteins is governed by the dynamic partitioning facilitated by HSPs throughout the cell. Beyond their involvement in protein metabolism, HSPs also exert a significant influence on apoptosis, the immune system, and various characteristics of inflammation. The immunity of aquatic organisms, including shrimp, fish, and shellfish, relies heavily on the development of inflammation, as well as non-specific and specific immune responses to viral and bacterial infections. Recent advancements in aquatic research have demonstrated that the HSP levels in populations of fish, shrimp, and shellfish can be increased through non-traumatic means such as water or oral administration of HSP stimulants, exogenous HSPs, and heat induction. These methods have proven useful in reducing physical stress and trauma, while also facilitating sustainable husbandry practices such as vaccination and transportation, thereby offering health benefits. Hence, the present review discusses the importance of HSPs in different tissues in aquatic organisms (fish, shrimp), and their expression levels during pathogen invasion; this gives new insights into the significance of HSPs in invertebrates.


Introduction
Due to the poikilothermic nature of aquatic animals, minor changes in the environment might lead to stress in fish. Fish are often exposed to various environmental stressors, such as pathogens, toxic gases, trauma, temperature fluctuations, and hypoxia. These factors, often referred to as stressors or stress factors, hold significant importance in determining the sequence of events that unfolds after encountering adverse consequences such as microbial infections, toxic exposure, traumatic injury, radiation, or nutritional deficiencies [1]. According to Selye's [2] original definition from 1950, a normal metabolism is the objective that an animal strives to maintain or restore in the presence of chemical or physical stimuli. response to this type of stress. The influence of stressors such as temperature or salinity on organisms has been studied extensively [24,25]. These stressors can impact the osmoregulation capability of organisms by affecting Na+-K+ ATPase activity or inducing heat shock protein production [26], both of which contribute to maintaining relative osmotic hemolymph homeostasis [27]. Numerous studies have investigated the expression patterns of heat shock proteins (HSPs) under salinity stress. For example, the expression of HSP90 was induced in Crassostrea hongkongensis [28] and Eriocheir sinensis [26,29] under osmotic stress. High salinity stress led to the significant upregulation of HSP70 expression in the hemocytes of Scylla paramamosain [30]. In the hepatopancreas of Portunus trituberculatus, HSP60, HSP70, and HSP90 showed either downregulated or upregulated expression profiles when exposed to low salinity (4 ppt) [31]. These findings suggest that HSPs play a role in mediating the effects of salinity stress in aquatic crustaceans.

Ultraviolet Radiation Stress
Ultraviolet (UV) radiation, an abiotic factor, can have detrimental effects on organisms, both directly and indirectly. Direct exposure to UV radiation can lead to changes in protein synthesis and DNA due to the absorption of high-energy photons. Indirectly, UV radiation can generate reactive oxygen species that cause damage to proteins, nucleic acids, and lipids [32][33][34]. The impact of UV radiation on aquatic organisms has become a significant concern in recent years. Research conducted on calanoid copepods has shown that UVinduced stress can impair feeding mechanisms and digestion and disrupt the entire food chain [35]. UV radiation directly and indirectly influences the survival, growth, and reproduction of organisms, and it led to the increased expression of antioxidant enzymes and heat shock protein (HSP) genes in the copepod Paracyclopina nana [34].

Heavy Metal Stress
Heavy metals pose a significant problem as a cause of pollution in water, soil, and plants. They enter water sources through seepage from household or industrial waste, resulting in serious risks to aquatic ecosystems and aquaculture animals. In laboratory studies focusing on crustaceans, the impact of heavy metals on gene expression changes has been extensively examined. Commonly tested heavy metals include copper (Cu), silver (Ag), zinc (Zn), lead (Pb), manganese (Mn), arsenic (As), and cadmium (Cd) [36][37][38]. Heavy metal stress is closely linked to the induction of oxidative stress. In seawater, heavy metals can trigger oxidative stress in various organisms, including the marine crab Portunus trituberculatus [39]. This type of oxidative stress disrupts the cellular redox balance, prompting a protective stress response. Numerous studies on aquatic organisms, particularly crustaceans, have demonstrated that heavy metal stress significantly stimulates the synthesis of antioxidant enzymes [40] and heat shock proteins [26,38]. Heat shock proteins (HSP) appear to play a crucial role in the innate immune systems and stress responses of crustaceans [36-38].

Effect of Endocrine Disruptor Chemicals in Heat Shock Proteins
Endocrine disruptor chemicals (EDCs) are compounds that imitate natural hormones, inhibiting their activity or altering their normal regulatory function within the immune, nervous, and endocrine systems [41]. These chemicals are ecotoxicologically significant as they have a tendency to be absorbed onto humic material or accumulate in aquatic organisms, persisting in water or the food web for extended periods. Consequently, their effects can induce prolonged stress in aquatic organisms. Various EDCs, including pesticides, bisphenol A, phthalates, dioxins, and phytoestrogens, have been shown to interact with the female reproductive system and cause endocrine disruption [42]. Endosulfan and deltamethrin, commonly used pesticides in shrimp farms [43], are particularly noteworthy. Endosulfan is widely employed as a broad-spectrum insecticide, primarily in agriculture, and is highly toxic to aquatic organisms [44][45][46]. Studies investigating the stress response induced by EDCs have indicated the significant induction of heat shock protein (HSP) family proteins [47][48][49], detoxification enzymes such as glutathione S-transferases [50], and superoxide dismutase. These proteins are considered to potentially contribute to the protection of aquatic organisms against stress.

Other Toxicants
Apart from the previously mentioned primary chemicals, there exist a significant number of other toxic substances in the habitats of aquatic organisms. These toxicants include hydrocarbons, diatom toxins, emamectin benzoate, nitrite, and prooxidant chemical hydrogen peroxide (H 2 O 2 ), among others. They accumulate in aquatic and/or terrestrial environments through the release of household and/or industrial waste. Research studies have demonstrated that these toxicants can have harmful effects on crustaceans [51,52]. In a study conducted by Lauritano et al. [41], it was observed that feeding on a diatom species (Skeletonema marinoi) that produced strong oxylipins for only two days led to the significant downregulation of heat shock proteins (HSP40 and HSP70) in the copepod Calanus helgolandicus. Diatom oxylipins are known to induce the generation of free radicals, including reactive oxygen species, which can cause oxidative stress and cellular damage. Furthermore, nitrite is considered one of the most prevalent pollutants in aquaculture due to its numerous integrated effects. A study on shrimp demonstrated that oxidative stress was one of the mechanisms of nitrite toxicity [53]. Guo et al. [53] confirmed that exposure to nitrite induced the expression of apoptosis-related genes in hemocytes, while also upregulating the expression levels of HSP70 and antioxidant enzymes to protect against nitrite-induced stress.

Immunology and Stress Response
The identification of heat shock proteins (HSP) initially occurred in Drosophila busckii as a response to stress [54]. Since then, their roles as chaperones in protecting cellular proteins from denaturation have garnered significant interest [55,56]. In aquaculture animals, HSPs have been the focus of numerous studies due to their crucial function in mitigating the stress-induced denaturation of client proteins, as well as their involvement in protein folding, assembly, degradation, and gene expression regulation [57,58]. Physiological and environmental stressors, including high thermal shock, heavy metals, free radicals, desiccation, and microbial infection, can induce the synthesis of HSPs. This induction is considered a vital protective response that is conserved across organisms, enabling them to adapt to environmental challenges. Recent research has revealed the involvement of heat shock chaperonins in autoimmune and innate immune responses in various species, including crustaceans. HSPs play a crucial role in mounting protective immune responses against bacterial and viral diseases [59,60]. In the crustacean aquaculture industry, which faces substantial economic losses due to environmental stressors, investigations into heat shock proteins have gained popularity. These proteins play vital roles in conferring resistance to diverse stressors. Extensive research has been conducted to understand the structures, functions, cross-talk, immune response mechanisms, and innate immune pathways of HSPs in crustaceans when exposed to various environmental stressors or xenobiotics. Exploiting HSPs as a means of preventing and treating aquaculture diseases in commercially cultured aquatic organisms is crucial as it provides an alternative to the use of antibiotics and therapeutic drugs [61]. Furthermore, previous studies have aimed to identify effective strategies for the management of environmental stressors in aquaculture settings for aquatic organisms [62].

Crustaceans: Exploring the Link between Environmental Stresses and Disease
Crustacean aquaculture plays a significant role in the economies of several countries worldwide. However, the expansion and intensification of aquaculture farms have led to the emergence of various new diseases in commercially cultivated species. Disease outbreaks caused by viruses, bacteria, and environmental stressors pose a serious threat to the global crustacean aquaculture industry, resulting in substantial economic losses. Unlike ver-tebrates, invertebrates lack true adaptive immunity and have developed defense systems that respond to physiological and environmental stresses [16,63]. During crustacean aquaculture, organisms are constantly exposed to environmental stimuli and a range of natural and anthropogenic stressors (Table 1). Numerous studies have demonstrated that physical stressors such as temperature, salinity, and UV radiation, as well as chemical stressors such as endocrine disruptor chemicals, heavy metals, hydrocarbons, and other toxicants, can be detrimental to crustacean cells. Moreover, in natural ecosystems, multiple environmental forces interact, resulting in situations of combined stress [64,65]. Crustaceans possess an innate immune system, which serves as their first line of defense and responds to natural and anthropogenic stimuli, pollutants, and toxins [41,66]. Studies have indicated that certain metabolic enzymes (such as cytochrome P450, glutathione S-transferase, superoxide dismutase, etc.), heat shock proteins, and immune-related proteins in crustaceans play a role in enhancing disease tolerance and aiding the elimination of harmful compounds from their bodies [41,67]. Upregulation [79] Help medial giant axons to maintain essential structures and functions Hsp70 Upregulation [80]  Shellfish diseases are prevalent and frequently observed in various commercially exploited crustacean species. Currently, a range of pathogens, including Vibrio, chitinoclastic bacteria, Aeromonas, Spiroplasma, Rickettsia-like organisms, Chlamydia-like organisms, Rhodobacteriales-like organisms, white spot syndrome virus (WSSV), yellow head virus (YHV), infectious myonecrosis virus (IMNV), Enterocytozoon hepatopenaei (EHP) microsporidian parasites, and covert mortality nodavirus (CMNV), have been identified as causes of disease in crustaceans [93]. Vibrio species, found in various marine and freshwater crustaceans, are widespread worldwide. Vibrio infections commonly result in bacteremia and shell diseases [94]. For instance, Vibrio parahaemolyticus infection caused acute hepatopancreatic necrosis disease (AHPND) and led to significant mortality in a penaeid shrimp aquaculture [95]. Chitinolytic or chitinoclastic bacteria are often associated with shellfish diseases, leading to unsuccessful molting in crustaceans [96] or septicemic infections caused by opportunistic pathogenic bacteria [97]. Infections by other pathogens, such as Rickettsialike organisms, Chlamydia-like organisms, spiroplasma, and Rhodobacteriales-like organisms, have caused severe stress or fatal diseases in crustaceans. Efforts have been made by numerous researchers to find effective methods to control bacterial diseases. Recent studies have shown that synbiotics can induce penaeid shrimp immunity and promote the growth of aquatic animals [98]. Oxytetracycline has been found to be highly effective in treating spiroplasma disease [99]. Several immune-related genes and proteins, including tachylectinlike genes and proteins and heat shock proteins [67], have been identified as being involved in shrimp tolerance to AHPND-causing strains. Crustacean fibrinogen-related proteins have also been found to participate in the innate immune response during AHPND or other pathogen infections [100]. Additionally, viruses continue to pose a significant challenge to crustacean aquaculture. Recent research has highlighted several new and emerging diseases in shrimp, including hepatopancreatic microsporidiosis, hepatopancreatic haplosporidiosis, aggregated transformed microvilli, covert mortality disease, white spot disease, yellow head disease, infectious myonecrosis, and white tail disease, which represent major viral threats to commercially cultivated shrimp [93].

Expression of Heat Shock Proteins in Fish
The presence of heat shock proteins (HSPs) in fish has been extensively documented, emphasizing their importance in responding to stress and safeguarding cellular integrity. HSPs are a group of highly conserved proteins that serve as molecular chaperones, aiding in the folding, assembly, and breakdown of other proteins. Fish exhibit increased HSP production when exposed to various stressors, such as elevated temperatures, exposure to heavy metals, oxidative stress, and infection by pathogens. Numerous studies have observed the heightened expression of HSPs in different fish species, including zebrafish (Danio rerio), rainbow trout (Oncorhynchus mykiss), and gilthead seabream (Sparus aurata), in response to stressors [101,102] (Table 2). These HSPs play a vital role in maintaining cellular balance, facilitating fish survival, and enabling adaptation to adverse environmental conditions. Furthermore, HSPs have been implicated in fish immune responses, enhancing their ability to defend against bacterial and viral infections [103]. The monitoring of HSP expression in fish serves as a valuable method to assess environmental stress levels and evaluate the overall health of fish populations in aquatic ecosystems ( Figure 1).   Heat-shock-stimulated bacteria Hsps [161] Antioxidants 2023, 12, x FOR PEER REVIEW 8 of 24

Expression of Heat Shock Proteins in Mollusk
The expression of heat shock proteins (HSPs) is associated with important developmental processes in various species, including gametogenesis, embryogenesis, and metamorphosis. In marine invertebrates with a biphasic life cycle, where pelagic larvae undergo settlement and metamorphosis, research has revealed interesting findings. For instance, studies on Eastern oyster C. virginica larvae and early spat have shown the presence of three HSP70 isoforms: HSC77, HSC72, and HSP69. The expression of constitutive and inducible forms of HSP70 differs among the larval and early juvenile stages and in response to thermal stress. Interestingly, the low expression of HSP69 during early larval and spat development may contribute to their vulnerability to environmental stress. In another investigation, Gunter and Degnan examined how the marine gastropod Haliotis asinine expresses HSP90, HSP70, and the heat shock transcription factor (me) during development (Table 3). HSP70, HSP90, and HSF are first expressed in this species by maternal contribution, before being gradually confined to the micromere lineage after cleavage (Figure 2). These proteins are expressed in distinct ways in the prototroch, foot, and mantle during larval morphogenesis. When cells are differentiating and undergoing morphogenesis, their expression is at its highest; however, after morphogenesis is complete, it starts to decline.

Heat Shock Protein Expression in Insects
A group of conserved polypeptides collectively known as heat shock proteins (HSPs) are rapidly increased in synthesis by insects in response to high temperatures and a variety of chemical and physical stimuli. Hspshave molecular-weight-based names, such as Hsp10, Hsp40, Hsp60, Hsp70, Hsp90, and Hsp100. Small Hsps (sHsp) are a subclass of Hsps that play a role in the folding and unfolding of other proteins (Table 4). In the fruit fly Drosophila busckii, Ritossa was the first to note that heat and the metabolic uncoupler dinitrophenol caused a distinctive pattern of puffing in the salivary gland chromosomes [54]. This discovery ultimately helped to identify the Hsps that these puffs were representing. The first observation of the increased production of certain proteins in Drosophila cells in response to stressors such as heat shock was made in 1974 [170]. There is currently a vast body of research that describes the extensive spectrum of action taken by cells in response to a wide range of biotic and abiotic stressors in a variety of insects [171,172].

Heat Shock Proteins in Myxozoan Parasites (Cnidaria)
Heat shock proteins (HSPs) are expressed by parasites as a response to various stimuli, such as heat and oxidative stress. These HSPs provide parasites with resistance to these harsh conditions, which is crucial for their survival. The genes associated with protein refolding, including HSP60, HSP70, and HSP80 family members, express these heat shock proteins. Apart from their role in protein refolding, HSPs also show significant involvement in other processes, such as maintaining protein balance and stability. They have the ability to bind to abnormal forms of proteins and facilitate their folding into their natural conformations. T. bryosalmonae, a parasite, faces the challenge of overcoming the robust immune responses mounted by both brown trout and rainbow trout [185]. This challenge potentially affects various physiological processes of T. bryosalmonae, including protein structure and function. Moreover, HSPs found in several parasites, such as T. cruzi [186] and Schistosomes [187], have been discovered to elicit an immune response in their respective hosts and are immunogenic in nature. In a recent study, it was found that myxozoan parasites such as Ceratonova shasta, Myxobolus cerebralis, and Sphaerospora molnari from the intestine and abdominal cavity (ascitic fluid) of rainbow trout expressed HSP70 when exposed to oxidative stress [188].

Defense Mechanisms of Heat Shock Proteins
The heat shock protein family, such as HSP70, are primarily studied for disease control purposes, but other members, such as small heat shock proteins (sHSPs), including HSP60 and HSP90, alongside HSP40/co-chaperone, have shown potential in treating pathogen infections. sHSPs act as oligomeric platforms, binding structurally perturbed proteins without requiring ATP, thereby preventing their irreversible denaturation under cellular stress. HSP90, HSP70, and HSP60 are stress-induced and provide protection against irreversible protein denaturation. However, their primary function involves binding and folding newly synthesized proteins through allosteric rearrangement, which is driven by ATP, although the mechanisms of action and the molecular structure differ among the chaperone families. In cooperation, these HSPs form intracellular networks with accessory proteins and other co-chaperones. sHSP monomers are a group of conserved α-crystallin domains flanked by carboxyl-and amino-terminal sequences and assemble into oligomers. The α-crystallin domain facilitates monomer dimerization and substrate binding, with the efficiency depending on the terminal region. During stress, sHSP oligomers may undergo structural rearrangement or disassemble, which promotes substrate protein interactions and increases surface hydrophobicity. Upon stress resolution, proteins released from sHSPs have the ability to spontaneously refold with the assistance of HSP70, which is an ATPdependent HSP [189]. The key role of sHSPs is to prevent protein denaturation, which is irreversible during infection and stress.
In aquatic organisms, such as the white shrimp (L. vannamei) and Scrippsiella trochoidea, various HSP genes (e.g., LvHSP40, LvHSP60, LvHSP70, LvHSC70, and LvHSP90) are significantly induced under acute thermal stress, highlighting their sensitivity to temperature fluctuations. Shrimp HSPs are also highly expressed in response to pathogen infections, as demonstrated by the upregulation of LvHsp60 in the gills, hemocytes, and hepatopancreas after challenge with Gram-negative or Gram-positive bacteria. Furthermore, the use of plant-based polyphenolic compounds such as phloroglucinol and carvacrol has been shown to result in the induction of HSP70 and protection against bacterial infection in brine shrimp and freshwater prawns [190]. These findings suggest that HSPs may play a role in crustaceans' immune system regulation, which triggers immune defense against diseases, as evidenced by the modulation of immune-related genes. Overall, the investigation of HSPs in aquatic organisms provides insights into their involvement in combating stress and infection, offering potential avenues for disease control and enhancing the immune responses in these organisms.
Biotic stress factor bacteria induce HSP20 expression in fish [191,192]. Similarly, some sHsp cDNA have been isolated and characterized in an expression analysis performed in fish [117,128,[193][194][195]. Following this, the HSP expression level was detected in Ictalurus punctatus [196], Paralichthys olivaceus [197], and Epinephelus coioides [198]. However, the expression patterns of fish sHsp under environmental stress are still limited with regard to biotic stress factors. Another type of HSP21 transcript was induced after 24 h exposure to Vibrio harveyi in shrimp P. monodon [158]; this was found to be entirely different in WSSV infection with P. monodon [199]. M. rosenbergii showed upregulated expression of HSP37 mRNA in the hepatopancreas under an infectious hypodermal and hematopoietic necrosis virus challenge [200]. In disk abalone Haliotis discus, HSP20 expression reached its highest peak in V. parahemolyticus with the VHSV virus [201]. Although some sHsp cDNA have been isolated and characterized in fish, there is little research on their roles in the immune response [117,128,[193][194][195]. Recently, it was validated that, when infected with Singapore grouper iridovirus (SGIV) and V. alginolyticus, Epinephelus coioides hsp22 mRNA expression was significantly increased, and HSP22 could significantly inhibit the SGIV-induced cell apoptosis [202].
Importantly, abiotic factors also interact with the expression levels of HSPs in aquatic organisms, among which temperature can influence the growth, reproduction, and survival of aquatic organisms (fish and shellfish) and result in serious losses in aquaculture ( Figure 3) [203,204]. In a study, the existing HSP20 gene expression was regulated by heat stress [191,192,200,205]. However, few reports provide information about the temperature regulation of HSP20 in fish, and the HSP expression levels in fish under stress factors are poorly understood. Therefore, it is necessary to discuss the findings regarding HSP expression in a range of aquatic organisms with regard to biotic and abiotic stress factors, as the gene expression profile can reveal the importance of their enhancement against foreign stimuli/invaders. [191,192,200,205]. However, few reports provide information about the temperature regulation of HSP20 in fish, and the HSP expression levels in fish under stress factors are poorly understood. Therefore, it is necessary to discuss the findings regarding HSP expression in a range of aquatic organisms with regard to biotic and abiotic stress factors, as the gene expression profile can reveal the importance of their enhancement against foreign stimuli/invaders.

Conclusions
Our understanding of the chaperone system of HSPs and its significance in farmed aquatic organisms is still limited, but progress is being made in medical and veterinary research. There have been rapid advances in comprehending the fundamental aspects of HSP genes and the effects of their products and their regulation on cell maintenance, as well as cell signaling, inflammation, and the immune response. This knowledge has been applied to various veterinary and human clinical situations, and promising results have

Conclusions
Our understanding of the chaperone system of HSPs and its significance in farmed aquatic organisms is still limited, but progress is being made in medical and veterinary research. There have been rapid advances in comprehending the fundamental aspects of HSP genes and the effects of their products and their regulation on cell maintenance, as well as cell signaling, inflammation, and the immune response. This knowledge has been applied to various veterinary and human clinical situations, and promising results have been obtained during the initial development of HSP vaccines derived from pathogens. These advancements indicate the potential value of HSPs in numerous areas of aquatic science. Further exploration of the HSP chaperone system and its applications could have significant implications for the health and wellbeing of farmed aquatic animals, providing opportunities for advancements in aquaculture practices, disease prevention, and overall aquatic ecosystem management.

Data Availability Statement:
The data in this study will be disclosed upon request.

Conflicts of Interest:
The authors declare no conflict of interest.