Cellular and humoral immunity in shellfish: foundations for sustainable health management in aquaculture

1 Introduction

Shellfish, including crustaceans such as shrimps, prawns, crabs, and lobsters, as well as molluscs, inhabit microbe-rich aquatic environments and are continuously exposed to a broad spectrum of pathogens, including bacteria, viruses, fungi, and parasites (Cerenius et al., 2010; Rowley, 2016). Despite this persistent exposure, only a limited number of microorganisms successfully establish infections, largely due to the presence of a highly efficient and evolutionarily conserved innate immune system in these organisms (Iwanaga and Lee, 2005; Cerenius and Söderhäll, 2004; Rowley and Powell, 2007). Unlike vertebrates, shellfish lack a true adaptive immune system and therefore do not produce antibodies or possess antigen-specific lymphocytes or classical long-term immunological memory (Iwanaga and Lee, 2005; Rowley, 2016). Consequently, their defence mechanisms rely exclusively on innate immune processes, which are broadly categorized into cellular (cell-mediated) and humoral immune responses (Rowley and Powell, 2007).

Shellfish aquaculture has become a major component of global aquaculture production and plays an important role in global food security and economic development. According to the Food and Agriculture Organization (FAO), global aquaculture production reached approximately 130.9 million tonnes in 2022, with an estimated value exceeding USD 313 billion (FAO, 2024). Among cultured aquatic organisms, shellfish—including crustaceans and molluscs—constitute a substantial proportion of this production. In particular, shrimp aquaculture has emerged as one of the most economically valuable sectors, largely driven by global demand for species such as Litopenaeus vannamei and Penaeus monodon (FAO, 2022; Boyd et al., 2018). However, the rapid expansion of shellfish farming has been accompanied by increasing disease outbreaks that significantly affect productivity and profitability. Viral diseases such as White Spot Disease caused by White Spot Syndrome Virus (WSSV), together with bacterial diseases including Acute Hepatopancreatic Necrosis Disease (AHPND), have resulted in severe economic losses in shrimp aquaculture worldwide (Lightner, 2011; Tran et al., 2013; Stentiford et al., 2017). These disease outbreaks are estimated to cause annual economic losses of several billion US dollars, highlighting the urgent need for improved disease management strategies and a deeper understanding of shellfish immune mechanisms (Shinn et al., 2015; Stentiford et al., 2012).

Historically, innate immunity was considered primitive and nonspecific, largely because early immunological research focused predominantly on the adaptive immune system of vertebrates (Iwanaga and Lee, 2005). However, advances in molecular and cellular immunology have revealed that invertebrate innate immunity is far more complex and dynamic than previously assumed, exhibiting pathogen-specific responses and even memory-like immune phenomena, often referred to as immune priming (Kloc et al., 2024). Recent genomic and comparative immunological studies further indicate that immune pathways in invertebrates have evolved independently from vertebrate adaptive immune systems, resulting in a diverse and highly specialized repertoire of defence strategies (Cerenius et al., 2010). Collectively, these findings highlight the importance of shellfish as valuable model organisms for investigating fundamental innate immune mechanisms without the confounding influence of adaptive immune components.

Shellfish rely exclusively on innate immune mechanisms to survive in microbe-rich aquatic environments. The first line of defence consists of physical and physiological barriers, including the external cuticle and the chitin-lined digestive tract, which are further supported by acidic pH, digestive enzymes, and antimicrobial compounds that limit pathogen entry (Vargas-Albores and Yepiz-Plascencia, 2000; Bibby et al., 2008). Once pathogens overcome these barriers and gain access to the haemocoel, rapid cellular immune responses are initiated (Jiravanichpaisal et al., 2006). Haemocytes serve as the primary immune effector cells, recognizing pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs) and executing defence processes such as phagocytosis, encapsulation, nodulation, degranulation, and activation of the prophenoloxidase (proPO)-mediated melanisation cascade (Cerenius and Söderhäll, 2004; Amparyup et al., 2013). For example, haemocytes of Litopenaeus vannamei and Macrobrachium rosenbergii have been reported to play crucial roles in pathogen recognition, phagocytosis, and antimicrobial defence against bacterial and viral infections. In parallel, humoral immune components—including antimicrobial peptides (AMPs), lectins, clotting factors, lysozyme, complement-like molecules, and reactive oxygen species (ROS)—circulate in the haemolymph and contribute to pathogen neutralization (Tassanakajon et al., 2011; Poirier et al., 2014). The coordinated action of cellular and humoral defence mechanisms provides effective protection against invading pathogens. Understanding these innate immune strategies is essential not only for elucidating the evolutionary basis of immunity in invertebrates but also for developing sustainable disease management approaches in shellfish aquaculture, where disease outbreaks pose significant economic challenges.

1.1 Literature search methodology

A comprehensive literature search was conducted to gather relevant information on cellular and humoral immunity in shellfish. Scientific articles were retrieved from major databases including PubMed, Scopus, Web of Science, and Google Scholar. The search was performed using combinations of keywords such as “shellfish immunity,” “innate immunity in shellfish,” “haemocytes,” “prophenoloxidase system,” “antimicrobial peptides,” and “shellfish diseases in aquaculture.” Only peer-reviewed research articles and review papers published in English were considered for inclusion. Publications from 2000 to 2024 were primarily selected to incorporate both foundational studies and recent advances in shellfish immunology. Additional relevant studies were identified by screening the reference lists of selected articles. The collected literature was critically evaluated and synthesized to summarize current knowledge on cellular and humoral immune mechanisms in shellfish and to highlight their implications for disease prevention and sustainable health management in aquaculture systems.

2 Overview of major components of shellfish innate immunity

Shellfish inhabit aquatic ecosystems characterized by dense and diverse microbial communities, including bacteria, viruses, fungi, protozoa, and parasites. Despite constant exposure to such pathogen-rich environments, these organisms maintain physiological homeostasis and health primarily through a highly efficient and evolutionarily ancient innate immune system (Bouallegui, 2021; Vazquez et al., 2009). Unlike vertebrates, shellfish lack an adaptive immune system and therefore do not produce antibodies, antigen-specific lymphocytes, or long-term immunological memory (Kloc et al., 2024). Consequently, immune defence in shellfish relies exclusively on innate mechanisms that operate rapidly and broadly to provide effective, non-specific protection against a wide spectrum of invading pathogens (Cerenius and Söderhäll, 2004; Vargas-Albores and Yepiz-Plascencia, 2000). The innate immune system of shellfish is structured around three major components (Cerenius et al., 2010; Amparyup et al., 2013): 1. Physical and physiological barriers, 2. Cell-mediated immunity, and 3. Humoral immunity. These components operate in a coordinated and sequential manner to detect, contain, and eliminate invading microorganisms (Amparyup et al., 2013; Söderhäll and Cerenius, 1998). An overall schematic representation of these components is shown in Figure 1.

2.1 Physical and physiological barriers

Physical and physiological barriers constitute the first line of innate defence in shellfish, preventing pathogen entry, adhesion, and colonization. In crustaceans, the rigid chitinous exoskeleton, and in bivalves, the calcareous shell, act as strong mechanical barriers that limit microbial penetration and physical injury (Bouallegui, 2021). Beyond mechanical protection, these structures may also host antimicrobial compounds and beneficial microbial communities that help restrict pathogen settlement (Allam and Raftos, 2015). Mucus secreted by epithelial tissues lining the integument, gills, and digestive tract forms an important protective interface. This mucus layer traps microorganisms, reduces their adhesion to epithelial surfaces, and facilitates their removal through ciliary movement or water flow (Bibby et al., 2008). It is also enriched with antimicrobial molecules such as lectins, lysozyme, and other immune-related proteins that contribute to pathogen neutralization (Tassanakajon et al., 2013). Recent studies suggest that mucus-associated microbiota can further enhance protection by competitively excluding opportunistic pathogens and modulating local immune responses (Lokmer and Wegner, 2015).

Within the digestive tract, the chitin–protein peritrophic membrane provides an additional barrier by separating ingested microorganisms from the midgut epithelium, thereby minimizing epithelial damage and microbial translocation (Hellio et al., 2007). This semi-permeable structure permits nutrient absorption while limiting direct pathogen contact. Epithelial tissues further contribute to defence by secreting antimicrobial peptides, lysozyme, proteases, and other bioactive molecules that inhibit microbial growth or disrupt pathogen integrity (Cerenius et al., 2010). Filtration and respiratory organs such as the gills also contribute to immune protection. By continuously filtering large volumes of water, gills remove suspended particles and microorganisms while maintaining physiological homeostasis (Vazquez et al., 2009). Increasing evidence indicates that gill epithelia are immunologically active tissues capable of pathogen detection and localized immune signalling, thereby linking barrier defence with haemocyte-mediated immune responses (Allam and Raftos, 2015). Collectively, these physical and physiological barriers form a multilayered defence system that reduces pathogen exposure at the host–environment interface. By limiting pathogen entry and initiating early defensive interactions, these barriers provide the foundation upon which cellular and humoral immune responses are activated.

2.2 Cell-mediated immunity

Cell-mediated immunity represents the principal internal defence mechanism in shellfish and is primarily mediated by haemocytes, which constitute the central effector and regulatory cells of the innate immune system. Once invading pathogens overcome external physical and physiological barriers, haemocytes are rapidly activated and initiate a broad range of defensive responses, including phagocytosis, encapsulation, nodule formation, cytotoxic reactions, and haemolymph clotting (Cerenius and Söderhäll, 2004; Bouallegui, 2021; Vazquez et al., 2009). These processes enable efficient recognition, sequestration, and elimination of microorganisms and foreign particles from the haemocoel, providing rapid and broad-spectrum protection against bacteria, viruses, fungi, protozoans, and metazoan parasites (Jiravanichpaisal et al., 2006; Amparyup et al., 2013). In contrast to vertebrates, shellfish lack adaptive immune cells such as lymphocytes; consequently, their cellular immune defence relies exclusively on haemocytes, which function as specialized circulating and tissue-resident immune cells.

In addition to direct cellular interactions, haemocytes generate reactive oxygen and nitrogen species, secrete antimicrobial molecules and hydrolytic enzymes, and actively participate in haemolymph coagulation, thereby limiting pathogen dissemination and preventing haemolymph loss at sites of injury or infection (Jiravanichpaisal et al., 2006; Perazzolo and Barracco, 1997). Haemocytes also play a pivotal role in immune signalling by activating key immune cascades, most notably the prophenoloxidase (proPO) system, through tightly regulated serine protease pathways (Cerenius and Söderhäll, 2004). Activation of this system results in melanisation, production of cytotoxic intermediates, and reinforcement of encapsulation responses, effectively linking cellular defences with humoral immune mechanisms (Perazzolo and Barracco, 1997; Johansson et al., 2000; Jiravanichpaisal et al., 2006; Amparyup et al., 2013). Recent advances further highlight haemocytes as highly dynamic and functionally plastic cells capable of modulating immune responses in a context-dependent manner, rather than acting solely as simple phagocytes (Hauton, 2012; Smith et al., 2003). Collectively, these coordinated haemocyte-mediated processes constitute a highly efficient and adaptable cellular immune network that is essential for shellfish survival in pathogen-rich aquatic environments. A schematic overview of the major haemocyte-mediated defence mechanisms is presented in Figure 2.

The production and maintenance of haemocytes are fundamental to shellfish immunocompetence, as effective cell-mediated immunity depends on the continuous availability of functional immune cells. Haematopoietic mechanisms vary considerably among shellfish taxa, reflecting distinct evolutionary adaptations. In crustaceans, particularly decapods, haematopoiesis occurs in specialized haematopoietic tissues (HPT) located near major arteries (Johansson et al., 2000; Jiravanichpaisal et al., 2006). These tissues consist of organized lobules containing undifferentiated progenitor cells and differentiating haemocytes, ensuring a regulated supply of immune cells. Cytokine-like molecules such as astakines stimulate haemocyte proliferation and release, while transglutaminases regulate extracellular matrix remodelling and haemocyte mobilization (Söderhäll and Cerenius, 1998; Bouallegui, 2021). In contrast, many bivalve molluscs lack a distinct haematopoietic organ. Instead, haemocytes are generated through mitotic division of circulating or tissue-resident cells within haemolymph sinuses and connective tissues, enabling localized immune cell production in response to infection or injury (Mao et al., 2020). These contrasting haematopoietic strategies highlight the evolutionary diversity of shellfish immune systems and provide the cellular basis for effective haemocyte-mediated defence responses.

Haemocytes are the principal immune effector cells in shellfish and are broadly classified into three functional groups based on morphology, granule content, and immunological activity: hyaline cells, semigranular cells, and granular cells (Johansson et al., 2000; Cerenius et al., 2010).

2.2.1 Hyaline cells

Hyaline cells are the smallest and least granulated haemocytes, characterized by a high nucleus-to-cytoplasm ratio and few or no cytoplasmic granules (Tassanakajon et al., 2013; Jiravanichpaisal et al., 2006). They are highly motile and function as the primary phagocytes in shellfish, efficiently engulfing bacteria, fungi, and small foreign particles (Liu et al., 2020). Hyaline cells also play an important role in early pathogen recognition through pattern-recognition receptors (PRRs), including peptidoglycan recognition proteins (PGRPs) and β-glucan-binding proteins (β-GBPs), which detect pathogen-associated molecular patterns (PAMPs) (Bouallegui, 2021; Liu et al., 2020). In addition to phagocytosis, they contribute to immune regulation by releasing cytokine-like signalling molecules and chemotactic factors that recruit and activate other haemocyte populations, particularly semigranular and granular cells (Johansson et al., 2000; Bouallegui, 2021).

2.2.2 Semigranular cells

Semigranular cells are intermediate in size and contain a moderate number of cytoplasmic granules that store immune effector molecules (Johansson et al., 2000; Söderhäll and Cerenius, 1998). These cells act as important coordinators within the haemocyte system by linking early pathogen recognition to downstream immune responses (Amparyup et al., 2013). Their major function is the encapsulation of large pathogens or foreign bodies that cannot be removed by phagocytosis. Semigranular cells also play a central role in activating the prophenoloxidase (proPO) system, promoting melanisation and strengthening encapsulation responses (Amparyup et al., 2013; Cerenius and Söderhäll, 2004). In addition, they contribute to cytotoxic defence through the release of antimicrobial molecules and enzymes via regulated degranulation (Bouallegui, 2021; Liu et al., 2020).

2.2.3 Granular cells

Granular cells are the largest haemocytes and contain abundant cytoplasmic granules rich in hydrolytic enzymes, antimicrobial peptides (AMPs), and components of the prophenoloxidase (proPO) system (Bouallegui, 2021). Ultrastructurally, these cells exhibit electron-dense granules that distinguish them from semigranular and hyaline haemocytes, as reported in transmission electron microscopy studies of crustacean haemocytes (Johansson et al., 2000). Functionally, granular cells act as major cytotoxic effector cells. Upon immune activation, they undergo degranulation, releasing reactive oxygen species (ROS), antimicrobial peptides, and lytic enzymes that directly eliminate invading microorganisms and promote proPO-mediated melanisation (Liu et al., 2020). Although they are not the primary phagocytes, granular cells support both cellular and humoral immunity by participating in encapsulation, reinforcing melanisation responses, and contributing to immune homeostasis through controlled release of effector molecules (Bouallegui, 2021).

3 Major cell-mediated immune mechanisms

3.1 Phagocytosis

Phagocytosis is a primary cell-mediated defence mechanism in shellfish and represents one of the earliest immune responses against invading microorganisms. In the absence of adaptive immunity, it serves as a key strategy for eliminating bacterial, fungal, and viral pathogens (Cerenius et al., 2010; Liu et al., 2020; Bouallegui, 2021). Two main haemocyte populations participate in this process: fixed phagocytes, located in tissues such as gill lacunae, pericardial sinuses, and the hepatopancreas, and mobile phagocytes, primarily hyaline haemocytes, which circulate in the haemolymph and migrate rapidly to infection sites (Iwanaga, 2002; González García et al., 2020; Liu et al., 2020; Rathour et al., 2025).

Phagocytosis begins with pathogen recognition by haemocyte pattern-recognition receptors (PRRs), including peptidoglycan recognition proteins (PGRPs), β-glucan-binding proteins (β-GBPs), Gram-negative binding proteins (GNBPs), and C-type lectins that detect pathogen-associated molecular patterns (PAMPs) such as peptidoglycan, lipopolysaccharides (LPS), β-1,3-glucans, and viral glycoproteins (Bouallegui, 2021; Betancourt et al., 2024; González García et al., 2020). Following recognition, haemocytes extend actin-driven pseudopodia to engulf pathogens and form phagosomes. These fuse with lysosomal granules to produce phagolysosomes, where pathogens are degraded by hydrolytic enzymes, reactive oxygen species (ROS), and antimicrobial peptides (Ding et al., 2024).

Hyaline haemocytes function as the primary phagocytes, while semigranular and granular cells enhance microbial killing by activating the prophenoloxidase (proPO) system and releasing antimicrobial factors into the haemolymph (Johansson et al., 2000; Amparyup et al., 2013). In some cases, phagocytosed material aggregates into melanized nodules visible in tissues such as gills and appendages (Rowley and Powell, 2007; Smith et al., 2003). In shrimp, this response may appear as “black gill” lesions that are often eliminated during moulting (Bao et al., 2025). Beyond pathogen removal, phagocytosis also triggers downstream immune signalling that links cellular recognition to humoral defence mechanisms (Liu et al., 2020).

3.2 Encapsulation

Encapsulation is a defence response used against pathogens or parasites too large to be removed by phagocytosis. During this process, haemocytes—primarily semigranular cells—adhere to the foreign surface and form multilayered cellular capsules that isolate the invader from surrounding tissues (Ratcliffe et al., 1985; Smith et al., 2003; Amparyup et al., 2013). The encapsulated target subsequently undergoes melanisation through activation of the prophenoloxidase (proPO) system, producing reactive quinones and melanin that immobilize and kill the pathogen (Amparyup et al., 2013; Betancourt et al., 2024). Opsonin-mediated interactions, including the action of a ~76 kDa multifunctional opsonin, enhance haemocyte adhesion and spreading during this process (Bao et al., 2025). Encapsulation therefore represents a coordinated immune response integrating haemocyte aggregation, enzymatic activation, and melanin deposition.

3.3 Nodule formation

Nodule formation occurs when moderately sized aggregates of pathogens or foreign particles enter the haemocoel (Smith et al., 2003; Cerenius and Söderhäll, 2004). Haemocytes rapidly aggregate around these particles, forming compact nodules that sequester pathogens and prevent their systemic dissemination (González García et al., 2020). These nodules are subsequently melanized through activation of the prophenoloxidase system or degraded enzymatically, leading to localized pathogen neutralization. By confining infection sites, this mechanism helps limit systemic spread.

3.4 Cytotoxicity

Cytotoxicity enables haemocytes to eliminate virus-infected cells, tumour-like cells, and large targets that cannot be phagocytosed, functionally resembling natural killer (NK) cell activity in vertebrates (Destoumieux‐Garzón et al., 2020; Xin and Zhang, 2023). Granular and semigranular haemocytes serve as the primary cytotoxic cells and contain granules rich in reactive oxygen species (ROS), antimicrobial peptides (AMPs), and hydrolytic enzymes (De la Ballina et al., 2022; Sokolnikova et al., 2023). Upon target recognition, haemocytes undergo controlled degranulation, releasing cytotoxic molecules that induce membrane damage, lysis, or apoptosis-like cell death (Donaghy et al., 2012; Vidya et al., 2007). After target destruction, haemocytes may participate in additional immune responses such as encapsulation and pathogen clearance.

3.5 Clotting reaction

Clotting is a rapid defence response that seals wounds, prevents haemolymph loss, and restricts pathogen dissemination (Iwanaga, 2007; Jiravanichpaisal et al., 2006). Haemocytes release clotting proteins and enzymes that promote cellular aggregation and plasma protein coagulation at sites of injury. Two main clotting mechanisms occur in shellfish. In horseshoe crabs (Tachypleus tridentatus), lipopolysaccharides trigger a proteolytic cascade involving factor C, factor B, and a proclotting enzyme, converting coagulogen into insoluble coagulin that forms a stable clot (Wu et al., 2019; Iwanaga, 2007). In crustaceans such as shrimp, crabs, and lobsters, clotting occurs mainly through a transglutaminase (TGase)-dependent pathway in which clottable proteins are cross-linked to form insoluble clot matrices (Kawabata and Shibata, 2022; Lin et al., 2012). These clots not only seal wounds but also help trap pathogens and enhance subsequent immune responses (Elbahnaswy et al., 2025).

4 Humoral immunity

Humoral immunity represents a major component of the innate defence system in shellfish and is mediated by a variety of soluble immune molecules circulating in the haemolymph that function independently or in coordination with haemocytes (Bouallegui, 2021; Rowley, 2016; Söderhäll et al., 2022). These factors, largely produced and secreted by haemocytes, provide rapid and broad-spectrum protection against microbial pathogens including bacteria, fungi, viruses, and parasites (Destoumieux-Garzón et al., 2016). A key feature of humoral immunity is the recognition of pathogen-associated molecular patterns (PAMPs) by soluble pattern-recognition proteins (PRPs), which detect conserved microbial components such as peptidoglycans, lipopolysaccharides (LPS), and β-1,3-glucans (Iwanaga and Lee, 2005; Amparyup et al., 2013). Major PRP families identified in shellfish include peptidoglycan-recognition proteins (PGRPs), Gram-negative binding proteins (GNBPs), β-glucan-binding proteins (β-GBPs), lipopolysaccharide/β-1,3-glucan-binding proteins (LGBPs), and various C-type lectins (Bouallegui, 2021; Rowley, 2016).

Important humoral effectors also include antimicrobial peptides (AMPs) such as penaeidins, anti-lipopolysaccharide factors (ALFs), and crustins, which exhibit broad-spectrum antibacterial, antifungal, and antiviral activities (Tassanakajon et al., 2011). Lectins and other soluble immune molecules facilitate pathogen agglutination and opsonization, thereby enhancing haemocyte-mediated clearance (Wang and Wang, 2013). Lysozyme contributes to defence by hydrolysing bacterial cell wall peptidoglycan, while clotting proteins released into the haemolymph promote wound repair and pathogen containment by sealing breaches and entrapping invading microbes (Cerenius and Söderhäll, 2004; Smith et al., 2003).

An important pathway linking cellular and humoral immunity in shellfish is the prophenoloxidase (proPO) system. ProPO is synthesized and stored in haemocytes—particularly semigranular and granular cells—but remains inactive until immune stimulation occurs. Following pathogen recognition, haemocytes release proPO and activating serine proteases into the haemolymph, where a regulated proteolytic cascade converts proPO into active phenoloxidase (PO) (Jiravanichpaisal et al., 2006). Activated PO catalyses the oxidation of phenolic substrates, leading to melanin formation and the production of reactive cytotoxic intermediates that contribute to pathogen immobilization, encapsulation, and elimination. Although proPO originates from haemocytes, its activation and function occur in the haemolymph, illustrating the close integration between cellular and humoral immune responses in shellfish. However, the efficiency of the proPO cascade may vary with species, environmental conditions, and pathogen load, indicating that its immune effectiveness is context dependent and warrants further investigation.

4.1 Lectins

Lectins are an important class of humoral immune molecules in shellfish and play multifunctional roles in innate defence. These carbohydrate-binding proteins recognize specific glycan residues on the surfaces of bacteria, fungi, viruses, and parasites, resulting in pathogen agglutination that facilitates immobilization and clearance (Bouallegui, 2021; Destoumieux-Garzón et al., 2016). Lectins also function as opsonins, coating microbial surfaces to enhance recognition and phagocytosis by haemocytes (Bouallegui, 2021; Rowley, 2016). A wide diversity of lectins has been identified in crustaceans and molluscs, highlighting their importance in pathogen recognition (Smith et al., 2003). For example, monodin from Penaeus monodon is a 27 kDa lectin with specificity for particular glycan residues (Pereyra et al., 2004; Tassanakajon et al., 2013). Lectins have also been described in freshwater prawns; in Macrobrachium rosenbergii, a haemolymph lectin with a ~9.6 kDa subunit exhibits haemagglutinating activity and specificity toward sialic acid residues (Pereyra et al., 2004), while in Macrobrachium malcolmsonii, a high-molecular-weight haemagglutinin (~413 kDa) specific for N-acetyl-galactosamine has been isolated from serum (Acharya et al., 2004). In addition to pathogen recognition and opsonisation, lectins can modulate immune responses by participating in activation of the prophenoloxidase (proPO) system, thereby linking extracellular pathogen sensing with melanisation and haemocyte-mediated defence mechanisms (Bouallegui, 2021; Söderhäll et al., 2022).

4.2 Antimicrobial peptides

Antimicrobial peptides (AMPs) are among the most rapid and potent humoral defence molecules in shellfish. These small, cationic peptides are mainly synthesized and stored in the cytoplasmic granules of semigranular and granular haemocytes and are rapidly released into the haemolymph following microbial challenge (Tassanakajon et al., 2011; Destoumieux-Garzón et al., 2016; Bouallegui, 2021). AMPs exert antimicrobial activity primarily through electrostatic interactions with negatively charged microbial membranes, leading to membrane disruption, pore formation, and cell death (Tassanakajon et al., 2011; Destoumieux-Garzón et al., 2016; Guryanova et al., 2023). Some AMPs also interfere with intracellular processes such as nucleic acid synthesis or protein translation, providing multi-target antimicrobial activity (Destoumieux-Garzón et al., 2016; Guryanova et al., 2023). Major AMP families reported in shellfish include penaeidins, crustins, anti-lipopolysaccharide factors (ALFs), and several molluscan peptides.

4.2.1 Penaeidins

Penaeidins are antimicrobial peptides unique to penaeid shrimp and possess a characteristic structure consisting of a proline-rich N-terminal domain and a cysteine-rich C-terminal domain (Tassanakajon et al., 2013; Destoumieux-Garzón et al., 2016). This structure enables broad antimicrobial activity, particularly against Gram-positive bacteria and fungi (Somboonwiwat et al., 2008; Tassanakajon et al., 2011). In addition to direct antimicrobial effects, penaeidins contribute to immune regulation by promoting haemocyte recruitment and influencing activation of the prophenoloxidase (proPO) system during infection (Tassanakajon et al., 2011; Destoumieux-Garzón et al., 2016).

4.2.2 Crustins

Crustins are cysteine-rich antimicrobial peptides widely identified in shrimp, crabs, and crayfish (Tassanakajon et al., 2011; Destoumieux-Garzón et al., 2016; Guryanova et al., 2023). They display strong antibacterial activity, particularly against Gram-positive bacteria, and moderate antifungal effects (Li et al., 2025; Guryanova et al., 2023). Their cysteine-rich structure provides stability and resistance to proteolytic degradation in the haemolymph. In addition to direct antimicrobial activity, crustins may contribute to immune regulation by promoting pathogen agglutination and indirectly influencing activation of the prophenoloxidase (proPO) system (Bouallegui, 2021; Destoumieux-Garzón et al., 2016).

4.2.3 Anti-lipopolysaccharide factors

Anti-lipopolysaccharide factors (ALFs) are potent antimicrobial peptides in crustaceans that exhibit strong activity against Gram-negative bacteria and enveloped viruses (Tassanakajon et al., 2013; Guryanova et al., 2023). Their antimicrobial effect primarily results from high-affinity binding to lipopolysaccharides (LPS) on bacterial outer membranes, leading to endotoxin neutralization and membrane disruption (Li et al., 2014; Destoumieux-Garzón et al., 2016; Jiang et al., 2022). In addition to direct antimicrobial activity, ALFs can act as opsonins that enhance haemocyte-mediated phagocytosis. Multiple ALF isoforms have been reported in species such as Penaeus monodon, Litopenaeus vannamei, and Macrobrachium spp., with expression regulated by immune signalling pathways including STAT, Toll (Dorsal), and IMD (Relish) (Li et al., 2014; Jiang et al., 2022).

4.2.4 Molluscan AMPs

In molluscs such as oysters and mussels, several AMP families have been identified, including mytilins, myticins, and defensins. These peptides exhibit strong antibacterial, antiviral, and antifungal activities and play an important role in defence against marine pathogens, particularly Vibrio species (Guryanova et al., 2023; Jeon et al., 2025). Their expression is highly inducible and can be rapidly upregulated in response to infection, environmental stress, tissue damage, or pathogen-associated molecular patterns (PAMPs). Regulation of molluscan AMPs involves conserved immune pathways such as Toll, IMD, and JAK/STAT (Rolandelli et al., 2021; Ran et al., 2022). Once released, these peptides act synergistically with lectins, pattern-recognition proteins, reactive oxygen species, and the prophenoloxidase system to ensure efficient pathogen neutralization (Destoumieux-Garzón et al., 2016; Bouallegui, 2021; Guryanova et al., 2023).

4.3 Lysozyme and hydrolytic enzymes

Lysozymes are among the most ancient and evolutionarily conserved antimicrobial effectors of humoral immunity in shellfish. In both crustaceans and molluscs, these enzymes hydrolyse the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in bacterial peptidoglycan, leading to rapid lysis of Gram-positive bacteria (Bouallegui, 2021; Li et al., 2025). Although the outer membrane of Gram-negative bacteria restricts direct lysozyme access, these enzymes still contribute to bacterial clearance by acting synergistically with antimicrobial peptides (AMPs), reactive oxygen species (ROS), and other membrane-disrupting immune factors (Destoumieux-Garzón et al., 2016; Bouallegui, 2021).

Haemocytes represent the main source of lysozyme production in shellfish. Following stimulation by invading microorganisms or pathogen-associated molecular patterns (PAMPs), semigranular and granular haemocytes undergo degranulation and release lysozyme into the haemolymph (Li et al., 2025; Söderhäll et al., 2022; Bouallegui, 2021). Consequently, increased lysozyme activity is widely considered an indicator of infection, environmental stress, moulting-associated stress, and general immune activation in both crustaceans and bivalves (Bouallegui, 2021; Söderhäll and Cerenius, 1998). In addition to lysozymes, shellfish haemolymph contains a range of hydrolytic enzymes that contribute to antimicrobial defence and immune regulation. These include acid and alkaline phosphatases, proteases and serine proteases, β-glucuronidase, β-galactosidase, esterases, and arylsulfatases (Bouallegui, 2021; Söderhäll et al., 2022). Together, these enzymes create a chemically hostile environment that works synergistically with AMPs, lectins, and oxidative molecules to degrade and eliminate invading pathogens, thereby strengthening humoral immunity in shellfish (Destoumieux-Garzón et al., 2016; Söderhäll et al., 2022).

4.3 Prophenoloxidase system

The prophenoloxidase (proPO) system is a central innate immune pathway in shellfish and crustaceans, contributing to melanisation, pathogen encapsulation, cytotoxic defence, and wound healing (Cerenius and Söderhäll, 2004; Amparyup et al., 2013). Activation begins when pattern-recognition proteins detect pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and β-1,3-glucans. This recognition triggers a serine protease cascade that converts inactive prophenoloxidase (proPO) into active phenoloxidase (PO) (Arockiaraj et al., 2012; Jearaphunt et al., 2015). Regulatory molecules such as pacifastin-type protease inhibitors help control this cascade and prevent excessive tissue damage (Wang et al., 2025).

Activated PO catalyses the oxidation of phenolic compounds into quinones that polymerize to form melanin. Melanin deposition around pathogens restricts their spread and generates cytotoxic intermediates and reactive oxygen species with antimicrobial activity (Cerenius and Söderhäll, 2004; Amparyup et al., 2013). The proPO system also strengthens encapsulation and nodule formation by promoting haemocyte aggregation and localized melanisation (Sritunyalucksana and Söderhäll, 2000). Through this proteolytic cascade, the proPO pathway integrates pathogen recognition with haemocyte-mediated cellular responses and humoral immune defence. An overview of the proPO activation cascade is presented in Figure 3.

5 Immune responses of shellfish to major aquaculture diseases

Shellfish aquaculture is increasingly challenged by the emergence and persistence of virulent viral, bacterial, and protozoan pathogens that cause large-scale mortality, growth impairment, and recurrent disease outbreaks, leading to significant socio-economic and ecological impacts on global aquaculture production. In the absence of antibody-mediated adaptive immunity, shellfish relies entirely on innate immune mechanisms for pathogen recognition and defence (Iwanaga and Lee, 2005; Rowley and Powell, 2007). Consequently, disease resistance depends largely on the stability of haemocyte-mediated cellular responses, the efficiency of humoral immune pathways, and the regulation of the prophenoloxidase (proPO) cascade (Cerenius and Söderhäll, 2013).

At the molecular level, this defence system involves coordinated interactions among pattern-recognition receptors (PRRs), immune signalling pathways such as Toll, IMD, and JAK/STAT-like pathways, antimicrobial effectors, and oxidative defence mechanisms. Together, these components form an integrated innate immune network that enables shellfish to recognize and respond rapidly to invading pathogens. However, immune competence in aquaculture species such as Litopenaeus vannamei, Penaeus monodon, Crassostrea gigas, Crassostrea virginica, and Mytilus galloprovincialis is strongly influenced by farming practices, environmental conditions, nutritional status, and culture intensity.

Recent studies indicate that immune dysregulation—including immune suppression, exhaustion, or excessive immune activation—can significantly influence disease susceptibility and outbreak dynamics in intensive shrimp and bivalve farming systems. Pathogen-induced immune responses often interact with environmental stressors such as temperature fluctuations, hypoxia, ammonia accumulation, poor water quality, and high stocking densities, resulting in complex immunopathological outcomes that affect survival, growth performance, and overall production stability.

Therefore, understanding disease-specific immune responses in major aquaculture species provides an important framework for applying shellfish immunology to practical aquaculture management. This includes disease risk prediction, immune biomarker-based health monitoring, development of functional feeds and immunostimulants, selective breeding for disease resistance, and improved health management strategies. The following sections summarize current knowledge on major shellfish diseases, focusing on pathogen-specific immune responses, haemocyte dynamics, proPO regulation, and their relevance to sustainable aquaculture practices.

5.1 White spot syndrome virus

White Spot Syndrome Virus (WSSV) is one of the most destructive viral pathogens in shrimp aquaculture, causing rapid epizootics and severe production losses in cultured populations of Litopenaeus vannamei and Penaeus monodon worldwide (Lightner, 2011). Following infection, haemocytes act as the primary immune effector cells, initiating phagocytic activity and antiviral responses (Liu et al., 2020). Viral components are recognized by pattern-recognition receptors (PRRs) such as β-glucan-binding proteins, scavenger receptors, and C-type lectins, which activate immune signalling pathways and stimulate the production of antimicrobial peptides (AMPs), anti-lipopolysaccharide factors (ALFs), interferon-like antiviral molecules, and RNA interference (RNAi)-mediated defence mechanisms (Li et al., 2018, 2013).

The prophenoloxidase (proPO) system is also activated during WSSV infection, leading to melanisation and the production of cytotoxic intermediates that contribute to viral containment (Cerenius and Söderhäll, 2013). However, excessive proPO activation may cause oxidative stress and host tissue damage, aggravating disease pathology (Li et al., 2018). WSSV infection further disrupts haemocyte dynamics through the induction of apoptosis mediated by caspase activation, mitochondrial dysfunction, and cytoskeletal disorganization, resulting in reduced total haemocyte count (THC), impaired proliferation, and decreased phagocytic capacity (Wang et al., 2019). Notably, the magnitude of haemocyte depletion and immune suppression varies between species, with Litopenaeus vannamei often showing more pronounced reductions in THC compared to Penaeus monodon, suggesting species-specific differences in antiviral resilience (Tang et al., 2019; Hameed et al., 2006). However, haemocyte depletion does not always correlate with mortality, indicating the influence of viral load and environmental stressors. The precise regulatory mechanisms governing haemocyte recovery and turnover during WSSV infection remain poorly understood, representing a key limitation in current research (Cerenius et al., 2010; Stentiford et al., 2017; Elbahnaswy et al., 2025).

At the molecular level, WSSV employs immune evasion strategies, including suppression of NF-κB signalling to inhibit AMP production and manipulation of host reactive oxygen species (ROS) pathways to facilitate viral replication (Xiao et al., 2025; He et al., 2025). Transcriptomic studies also reveal extensive reprogramming of host metabolic and immune genes during infection (Sun et al., 2024). Consequently, monitoring immune biomarkers such as total haemocyte count (THC), phenoloxidase activity, and phagocytic capacity may provide early indicators of WSSV infection and support improved disease surveillance in shrimp farming systems.

5.2 Acute hepatopancreatic necrosis disease

Acute Hepatopancreatic Necrosis Disease (AHPND), caused by toxigenic strains of Vibrio parahaemolyticus, is a major bacterial disease affecting shrimp aquaculture, particularly in Litopenaeus vannamei and Penaeus monodon culture systems (López-Landavery et al., 2024). Infection triggers rapid activation of haemocyte-mediated phagocytosis and increased expression of antimicrobial peptides, lysozyme, and other bactericidal factors. Recognition of bacterial pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) activates immune pathways, including the prophenoloxidase (proPO) system; however, this process is highly species-specific (Amparyup et al., 2013; Wang et al., 2018). In crustaceans such as Litopenaeus vannamei, diverse PRRs (e.g., lipopolysaccharide- and β-glucan-binding proteins) efficiently trigger proPO activation and melanisation (Amparyup et al., 2013), whereas in bivalves such as Crassostrea gigas, PRR engagement more often leads to phagocytosis and cellular responses rather than strong melanisation (Allam and Raftos, 2015). This indicates that PAMP recognition and downstream immune outcomes vary across taxa, particularly in response to lipopolysaccharides (LPS), reflecting differences in receptor composition and immune strategy (Wang et al., 2018).

However, PirAB toxins produced by AHPND-causing strains directly damage hepatopancreatic tissues and haemocytes in L. vannamei, leading to epithelial disruption, haemocyte apoptosis, and impaired immune surveillance (Zheng et al., 2021). These effects result in reduced haemocyte viability, altered haemocyte populations, decreased phagocytic activity, and suppression of humoral immune responses, ultimately contributing to rapid disease progression in intensive shrimp farming systems. Transcriptomic studies further indicate significant alterations in immune and metabolic gene expression during infection, reflecting coordinated metabolic–immune responses (Aldersey et al., 2026). Monitoring immune indicators such as haemocyte viability, phenoloxidase activity, and antibacterial responses may therefore assist in early detection and management of AHPND outbreaks.

5.3 Enterocytozoon hepatopenaei

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that causes chronic growth retardation and significant production losses in cultured Litopenaeus vannamei and Penaeus monodon (Ma et al., 2024). Unlike acute viral infections, EHP primarily induces long-term immune modulation rather than rapid mortality, resulting in persistent metabolic and immune dysregulation (Haridevamuthu et al., 2025).

In infected shrimp, sustained haemocyte activation and chronic stimulation of humoral immune pathways are observed. Sub-maximal activation of the prophenoloxidase (proPO) system corresponds to a measurable 2–3 fold increase in phenoloxidase activity relative to basal levels, and varies with species, haemocyte subtype, and pathogen load (Cerenius and Söderhäll, 2004; Amparyup et al., 2013; Wang et al., 2018; Lu et al., 2025). Enterocytozoon hepatopenaei (EHP) infection additionally disrupts haemocyte differentiation, reduces phagocytic activity, and induces chronic inflammatory-like responses, while simultaneously altering glucose and lipid metabolism (Cao et al., 2023; Ma et al., 2024). These observations show that “moderate” activation is a quantifiable, species- and context-dependent immune response that balances pathogen clearance with potential chronic immune stress.

These immune and metabolic alterations divert energy from growth to immune maintenance and stress responses, explaining the growth suppression commonly observed in infected shrimp populations (Haridevamuthu et al., 2025). In addition, EHP infection may alter gut microbiota composition and impair digestive function, further contributing to physiological stress and reduced productivity (Lu et al., 2025). Monitoring indicators such as haemocyte function, proPO activity, and metabolic–immune biomarkers may therefore assist in early detection and management of EHP in shrimp aquaculture systems.

5.4 Infectious myonecrosis virus

Infectious Myonecrosis Virus (IMNV) primarily infects muscle tissues in cultured shrimp, particularly Litopenaeus vannamei, causing extensive muscle necrosis, reduced mobility, and high mortality (Lightner, 2011). Host defence involves haemocyte-mediated antiviral responses, including phagocytosis of viral particles, induction of antiviral effector molecules, RNA interference (RNAi)-mediated viral suppression, and activation of humoral immune pathways.

The prophenoloxidase (proPO) system contributes to melanisation and localized containment of infected tissues. However, persistent viral replication can trigger excessive immune activation, oxidative stress, and mitochondrial dysfunction, leading to tissue damage. Progressive haemocyte apoptosis and immune exhaustion further reduce immune surveillance and host resistance, facilitating disease progression in intensive farming systems. Monitoring immune indicators such as total haemocyte count, oxidative stress markers, and antiviral responses may therefore assist in early detection and management of IMNV infections in shrimp aquaculture.

5.5 Vibrio infections in bivalves

Vibrio species are major bacterial pathogens in bivalve aquaculture and are responsible for recurrent mass mortality events in oysters, mussels, and clams, particularly in Crassostrea gigas, Crassostrea virginica, and Mytilus galloprovincialis (Elston et al., 2008; Martín-Gómez et al., 2014). Host defence primarily involves haemocyte-mediated phagocytosis, lectin-based pathogen recognition, complement-like pathways, and humoral antimicrobial responses. Infection typically induces activation of the prophenoloxidase (proPO) system, leading to increased phenoloxidase activity and melanisation responses (Allam and Raftos, 2015). Haemocyte activation also enhances phagocytosis, reactive oxygen species (ROS) production, antimicrobial peptide release, and extracellular trap-like responses. However, virulent Vibrio strains possess immune evasion mechanisms such as quorum-sensing regulation, toxin production, and interference with host immune signalling, which suppress haemocyte activity and reduce phagocytic efficiency. Monitoring immune indicators including haemocyte phagocytic activity, ROS production, and phenoloxidase activity may therefore help assess Vibrio-associated disease risks in bivalve aquaculture systems.

5.6 Perkinsus infections

Perkinsus spp., particularly Perkinsus marinus and P. olseni, are persistent protozoan parasites that cause chronic infections in oysters and clams, especially Crassostrea virginica and C. gigas, leading to tissue damage, reduced physiological performance, and long-term stock decline (Romero et al., 2015; Villalba et al., 2004). Following infection, haemocytes infiltrate affected tissues and initiate phagocytosis, encapsulation, and oxidative burst responses. Virulent strains of P. marinus stimulate increased haemocyte production of reactive oxygen species (ROS) and nitric oxide (NO), although parasite persistence is often associated with incomplete clearance and progressive host damage (Kim et al., 2025). Pathogen recognition through pattern-recognition receptors such as C-type lectins and scavenger receptors activates immune signalling pathways, humoral effectors, and the prophenoloxidase (proPO) system, which contributes to melanotic encapsulation and localized cytotoxicity (Cerenius and Söderhäll, 2013). However, Perkinsus can evade host defence by modulating apoptosis and oxidative responses; for example, P. marinus has been reported to suppress apoptotic haemocyte responses after initial activation, allowing intracellular persistence (Hughes et al., 2010). Chronic infection imposes metabolic strain and oxidative stress on haemocytes, contributing to immune exhaustion and reduced host resilience. Monitoring haemocyte activity, oxidative burst responses, and proPO activity may therefore help assess infection pressure and immune status in oyster aquaculture systems.

6 Immune biomarkers in health monitoring of shellfishes

The practical application of shellfish immunology in aquaculture relies on identifying reliable immune biomarkers that reflect physiological status, environmental stress, and disease susceptibility. In penaeid shrimp and bivalve molluscs, which lack adaptive immunity, health monitoring largely depends on assessing innate immune parameters (Rowley and Powell, 2007; Wang and Wang, 2013). Both cellular and humoral indicators are therefore used for disease surveillance, evaluation of nutritional immunostimulants, and farm-level health assessment. Commonly applied biomarkers include total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst, lysozyme activity, and the phagocytic index. These parameters represent interconnected components of the innate immune system, and their diagnostic value is highest when interpreted collectively within environmental and pathological contexts.

6.1 Total haemocyte count

Total haemocyte count (THC) is a fundamental indicator of immune status in crustaceans. Haemocytes circulating in the haemolymph function as key effector cells involved in phagocytosis, nodulation, encapsulation, coagulation, cytotoxic responses, and activation of the prophenoloxidase (proPO) cascade (Cerenius and Söderhäll, 2004). Because these cells participate in both cellular and humoral immunity, variations in haemocyte abundance provide a sensitive indicator of physiological and immune condition.

In cultured Litopenaeus vannamei, THC typically remains within a stable range under optimal farming conditions but declines during infections such as White Spot Syndrome Virus and pathogenic Vibrio parahaemolyticus, reflecting haemocyte apoptosis, tissue sequestration, and immune exhaustion (Ji et al., 2009, 2011). However, total counts alone may not fully represent immune competence. Differential haemocyte counts (DHC), which distinguish hyaline, semigranular, and granular cells, often reveal changes in specific subpopulations during infection or stress. Environmental stressors such as temperature fluctuation, salinity variation, hypoxia, and ammonia accumulation can also reduce THC, indicating stress-induced immunosuppression rather than infection (Le Moullac and Haffner, 2000). Therefore, THC is most informative when combined with functional immune biomarkers.

6.2 Phenoloxidase activity

Phenoloxidase (PO) activity reflects the functional state of the prophenoloxidase (proPO) system, a key humoral defence pathway in crustaceans. Upon recognition of pathogen-associated molecular patterns (PAMPs), proPO is activated through a serine protease cascade, leading to melanisation and production of reactive quinones with antimicrobial activity (Cerenius and Söderhäll, 2004). In shrimp aquaculture, elevated PO activity is commonly observed during early stages of bacterial or viral infection, indicating activation of melanisation responses (Ji et al., 2009). However, excessive activation can cause oxidative tissue damage, while suppressed PO activity during advanced infection may indicate pathogen-mediated immune evasion. Consequently, interpretation of PO activity requires consideration of infection stage and environmental conditions.

6.3 Respiratory burst activity

Respiratory burst refers to the rapid production of reactive oxygen species (ROS) by activated haemocytes via NADPH oxidase complexes. These molecules, including superoxide anions and hydrogen peroxide, contribute to microbial killing and immune signalling (Wang and Wang, 2013). Increased respiratory burst activity has been reported following bacterial challenge or administration of immunostimulants in penaeid shrimp (Ji et al., 2011). Although ROS production supports pathogen elimination, excessive or prolonged oxidative activity may cause cellular damage, lipid peroxidation, and apoptosis. In intensive aquaculture systems, environmental stress can also elevate ROS levels independently of infection. Therefore, respiratory burst measurements are best interpreted alongside antioxidant markers to distinguish protective immune activation from oxidative stress.

6.4 Lysozyme activity

Lysozyme is a humoral antimicrobial enzyme that hydrolyses β-1,4-glycosidic linkages in bacterial peptidoglycan. Produced by haemocytes and released into haemolymph, it contributes to basal antibacterial defence. Lysozyme activity is commonly measured using turbidimetric assays with Micrococcus lysodeikticus cell walls as substrate. Elevated lysozyme activity has been reported during bacterial infection and dietary immunostimulation in shrimp (Ji et al., 2009). However, its activity is primarily directed against Gram-positive bacteria, and its antiviral relevance is limited. Therefore, lysozyme is considered an indicator of antibacterial defence rather than a standalone immune predictor and is most useful when evaluated within a broader biomarker panel.

6.5 Phagocytic index

Phagocytosis is a central cellular immune mechanism in crustaceans. The phagocytic index measures the proportion of haemocytes capable of engulfing foreign particles or the number of particles internalized per cell. This parameter is typically assessed using fluorescent bacteria or latex beads combined with microscopy or flow cytometry (Iunes et al., 2021). In L. vannamei, hyaline haemocytes are primarily responsible for phagocytosis, while granular cells contribute to enzymatic defence pathways. Reduced phagocytic activity is commonly associated with viral infection, environmental stress, and immune exhaustion, whereas immunostimulants such as probiotics or β-glucans often enhance phagocytic efficiency (Chiu et al., 2007). Because it directly reflects functional immune competence, the phagocytic index provides a more mechanistic indicator of immune status than haemocyte abundance alone.

6.6 Integration of molecular and functional immune biomarkers for disease monitoring in shellfish aquaculture

Recent advances in molecular immunology have improved understanding of innate immune signalling pathways in shellfish, particularly the Toll, immune deficiency (IMD), and JAK/STAT pathways, which regulate genes involved in pathogen recognition, signalling, and effector responses (Li et al., 2018; Tassanakajon et al., 2013). However, their practical application in aquaculture requires integration with functional immune biomarkers such as total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst, and phagocytic activity to enable effective disease monitoring and health management.

Gene expression profiling using quantitative real-time PCR (qPCR) provides early indication of pathogen exposure and immune activation, typically quantified using the ΔΔCt method with housekeeping genes such as β-actin and elongation factor 1-alpha (EF1α). In penaeid shrimp (Litopenaeus vannamei, Penaeus monodon), disease-specific gene panels enable targeted monitoring. Viral infections such as White Spot Syndrome Virus (WSSV) are associated with Toll pathway genes (LvToll, MyD88), JAK/STAT pathway components (PmJAK, STAT), and RNA interference-related genes such as Dicer-2 and Argonaute (Li et al., 2018; Wang et al., 2019; Xiao et al., 2025). In contrast, bacterial diseases such as Acute Hepatopancreatic Necrosis Disease (AHPND) are better monitored using antibacterial markers including β-glucan-binding proteins, C-type lectins, anti-lipopolysaccharide factors (ALFs), crustins, lysozyme, and prophenoloxidase-associated enzymes (Amparyup et al., 2013; Wang et al., 2019; He et al., 2025). In bivalves such as Crassostrea gigas and Mytilus galloprovincialis, lectins, complement-like molecules, and heat shock proteins (HSP70, HSP90) serve as indicators of immune activation and environmental stress (Allam and Raftos, 2015; Cocci et al., 2020).

Similar integrative approaches can be applied to other diseases. Infectious Myonecrosis Virus (IMNV) infection is associated with early upregulation of RNA interference-related genes and JAK/STAT components followed by reduced THC and impaired phagocytosis. Enterocytozoon hepatopenaei (EHP) infection shows sustained activation of stress-related and proPO-associated genes along with reduced haemocyte efficiency, indicating chronic immune stimulation. In bivalves, infections caused by Vibrio spp. and Perkinsus spp. are associated with increased lectin expression and oxidative stress markers, including elevated reactive oxygen species (ROS) production. However, during severe infection, functional immune parameters may decline despite elevated gene expression, indicating immune exhaustion or pathogen-mediated immune evasion (Cerenius and Söderhäll, 2004; Rowley and Powell, 2007).

Therefore, molecular biomarkers must be interpreted together with functional immune parameters. Functional indicators such as THC, PO activity, and phagocytic index represent physiological outcomes of molecular activation and help distinguish between effective immune responses and pathological immune dysregulation. For example, transient immune gene upregulation with stable THC and PO activity indicates effective immune activation, whereas sustained gene upregulation with declining THC and PO activity indicates haemocyte depletion and disease progression.

For field application, a stepwise biomarker-based monitoring protocol is recommended: (i) periodic haemolymph sampling from representative individuals; (ii) qPCR analysis targeting disease-specific gene panels; and (iii) simultaneous measurement of functional immune parameters. Regular monitoring, particularly during environmental stress or disease outbreaks, enables early warning and timely management interventions. Although molecular diagnostics currently face limitations such as cost, lack of standardized thresholds, and interspecific variability, the development of portable qPCR platforms, simplified gene panels, and biosensors shows strong potential for integration into precision aquaculture health management (Allam and Raftos, 2015; Tassanakajon et al., 2013).

7 Environmental stress and immunity in shellfish

Environmental conditions strongly influence immune competence in shellfish aquaculture. Because shellfish relies solely on innate immune mechanisms—including haemocyte-mediated phagocytosis, phenoloxidase activity, antimicrobial peptides, and oxidative responses—environmental stress can disrupt these processes and increase susceptibility to disease (Rowley and Powell, 2007; Ellis et al., 2011). Key stressors affecting shellfish immunity include temperature fluctuations, ammonia accumulation, hypoxia, and salinity changes, all of which influence haemocyte activity, oxidative defence, and overall physiological stability.

7.1 Temperature

Temperature is a critical factor regulating immune responses in shellfish. Moderate increases may enhance metabolic activity and transiently stimulate immunity, but excessive or abrupt thermal stress impairs haemocyte function, reduces phagocytosis, suppresses phenoloxidase activity, and lowers reactive oxygen species (ROS) production (Mydlarz et al., 2006; Ellis et al., 2011). Elevated temperatures also favour the proliferation of opportunistic pathogens. In Penaeus monodon, outbreaks of Vibrio and Aeromonas species have been associated with thermal stress and shifts in pond microbial communities (Sung et al., 2001). Similarly, Litopenaeus vannamei exposed to high temperatures (32–34 °C) show reduced haemocyte counts, phenoloxidase activity, respiratory burst, and phagocytic capacity, leading to increased susceptibility to Vibrio alginolyticus infection (Cheng et al., 2005). These findings highlight the importance of thermal stability for maintaining immune competence.

7.1.1 Practical implications for farm management

Under commercial aquaculture conditions, thermal stress events require immediate management interventions to prevent immune suppression. In penaeid shrimp such as Litopenaeus vannamei and Penaeus monodon, water temperatures exceeding 32–34 °C are associated with reduced total haemocyte counts, impaired phagocytic activity, and suppressed phenoloxidase responses. During such periods, farm managers should reduce feeding rates to minimize metabolic stress and uneaten feed accumulation, while simultaneously increasing aeration to maintain adequate dissolved oxygen levels. Handling practices such as grading or transfer should be avoided during peak thermal stress to prevent additional physiological burden. Furthermore, maintaining optimal pond depth and minimizing diurnal temperature fluctuations can buffer abrupt thermal changes and help preserve immune competence in cultured shrimp species (Cheng et al., 2005).

7.2 Ammonia

Ammonia accumulation from uneaten feed and metabolic waste is a common stressor in intensive aquaculture. Elevated ammonia levels disrupt cellular metabolism, induce oxidative stress, and impair haemocyte activity. This results in reduced phagocytosis and suppression of key immune enzymes such as lysozyme and phenoloxidase (Zhang et al., 2024; Zhao et al., 2020). Chronic exposure may also damage epithelial tissues and disturb hepatopancreatic metabolism, further weakening host defence. Effective ammonia management is therefore essential to maintain immune stability in shellfish culture systems.

7.2.1 Practical implications for farm management

In intensive aquaculture systems, ammonia accumulation represents a critical threshold beyond which immune function becomes compromised. In shrimp species such as Litopenaeus vannamei and Penaeus monodon, total ammonia nitrogen levels exceeding approximately 0.5 mg/L have been associated with reduced haemocyte activity, suppressed phenoloxidase function, and weakened immune responsiveness. Under such conditions, immediate corrective actions, including reduction in feeding, partial water exchange, and the application of probiotics or biofloc-based carbon sources, should be implemented to restore water quality. Prolonged ammonia exposure induces oxidative stress, damages epithelial and hepatopancreatic tissues, and increases susceptibility to opportunistic pathogens, thereby necessitating the concurrent use of dietary immunostimulants to support host defence mechanisms (Liu and Chen, 2004; Li et al., 2018).

7.3 Hypoxia

Hypoxia, or low dissolved oxygen (DO), is another major environmental stressor. In Litopenaeus vannamei, DO levels below approximately 3 mg/L significantly reduce phenoloxidase activity, phagocytosis, and antibacterial responses, increasing susceptibility to pathogens such as Vibrio parahaemolyticus (He et al., 2024). Hypoxia also alters immune gene expression and induces physiological stress responses. Similar effects have been observed in Marsupenaeus japonicus, where low oxygen conditions cause tissue damage and immune modulation (Wang et al., 2022; Masuda, 2021). Reduced haemocyte counts and limited metabolic energy under hypoxia divert resources from immune defence to survival, increasing disease risk in intensive aquaculture systems.

7.3.1 Practical implications for farm management

Dissolved oxygen depletion is a frequent and often acute stressor in shrimp aquaculture systems, particularly in high-density culture of species such as Litopenaeus vannamei and Marsupenaeus japonicus. When dissolved oxygen levels fall below 3 mg/L, significant reductions in phagocytosis, phenoloxidase activity, and antibacterial defence responses have been reported. In such scenarios, emergency aeration should be initiated immediately, and feeding should be temporarily suspended to reduce oxygen demand. Chronic exposure to hypoxic conditions necessitates improved aeration infrastructure, optimized stocking density, and better biomass management, as sustained oxygen limitation diverts metabolic energy away from immune processes and increases vulnerability to pathogens. Preventive strategies, including continuous monitoring and maintenance of phytoplankton balance, are essential to minimize recurrent hypoxic stress in intensive shrimp farming systems (Cheng and Combes, 1990; Cheng et al., 2005).

7.4 Salinity

Salinity fluctuations are common in estuarine and coastal aquaculture environments. Because shellfish are osmoconformers, sudden changes in salinity disrupt cellular homeostasis and affect haemocyte function. Abnormal salinity levels have been shown to alter haemocyte counts, reduce phagocytic activity, and affect antimicrobial enzyme production, thereby weakening immune defence and increasing susceptibility to infection (Gagnaire et al., 2006). Maintaining stable salinity conditions is therefore essential for preserving immune function and promoting disease resistance.

7.4.1 Practical implications for farm management

Salinity fluctuations, particularly in coastal and estuarine aquaculture environments, must be carefully managed to prevent osmotic stress and immune dysfunction in cultured species such as Litopenaeus vannamei, Penaeus monodon, and other euryhaline crustaceans. Rapid salinity changes exceeding approximately 5 ppt per day can disrupt haemocyte stability, reduce phagocytic activity, and impair antimicrobial defence mechanisms. During environmental disturbances such as heavy rainfall, gradual salinity adjustment through controlled water exchange or the addition of higher-salinity water is recommended to avoid abrupt osmotic imbalance. The application of lime and mineral supplements can further stabilize ionic composition and support physiological homeostasis. Maintaining salinity within species-specific optimal ranges is therefore essential for sustaining immune efficiency and reducing disease susceptibility in shrimp aquaculture systems (Lin et al., 2012).

7.5 Integrated effects of environmental stress

In aquaculture systems, environmental stressors often occur simultaneously and may interact synergistically. For example, elevated temperature combined with hypoxia or poor water quality can significantly amplify physiological stress and suppress immune responses. Effective farm management—including water quality monitoring, aeration, and appropriate stocking densities—is therefore essential to minimize stress and prevent disease outbreaks. Integrating environmental monitoring with immune biomarker assessment can provide an early warning system for disease risk and support sustainable shellfish health management.

8 Immunostimulants in aquaculture health management

The intensification of aquaculture has increased the vulnerability of cultured organisms to infectious diseases, which remain a major constraint on sustainable production. In shellfish such as shrimp and bivalves, disease resistance depends entirely on innate immunity because adaptive immune responses are absent. Consequently, the use of immunostimulants has become an important strategy to enhance disease resistance. Immunostimulants are natural or synthetic compounds that stimulate cellular and humoral immune pathways, improving the host’s ability to resist pathogens. In shrimp species such as Litopenaeus vannamei and Penaeus monodon, immunostimulants are commonly administered through dietary supplementation or immersion to enhance immune readiness before pathogen exposure. Studies have shown that these compounds increase key immune indicators, including total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst, lysozyme activity, and phagocytic capacity (Sakai, 1999; Smith et al., 2003). By enhancing multiple innate defence mechanisms, immunostimulants improve pathogen recognition, clearance, and overall disease resistance while reducing reliance on antibiotics and chemotherapeutics.

8.1 Mechanisms of immune activation

Most immunostimulants act by activating innate immune recognition systems. Many of these compounds resemble pathogen-associated molecular patterns (PAMPs) and interact with pattern-recognition receptors (PRRs) on haemocytes and other immune cells. This interaction triggers intracellular signalling pathways that activate key immune responses. One major pathway stimulated by immunostimulants is the prophenoloxidase (proPO) system, which promotes melanisation, pathogen encapsulation, and production of cytotoxic intermediates. Immunostimulants also enhance haemocyte phagocytosis and stimulate respiratory burst activity, resulting in increased production of reactive oxygen species that facilitate microbial killing. Additionally, they may regulate the expression of immune-related genes encoding antimicrobial peptides, lectins, and PRRs, strengthening pathogen detection and defence mechanisms (Vazquez et al., 2009).

8.2 Major categories of immunostimulants in aquaculture

β-glucans derived from yeast, fungi, and marine microalgae (e.g., diatoms) are among the most widely studied immunostimulants in shrimp aquaculture. These β-1,3/1,6-linked polysaccharides function as pathogen-associated molecular pattern (PAMP)-like molecules that interact with haemocyte receptors and activate innate immune pathways, promoting haemocyte proliferation, enhancing phenoloxidase (PO) activity via the prophenoloxidase (proPO) system, and stimulating phagocytosis and respiratory burst responses (Sakai, 1999; Vazquez et al., 2009). In penaeid shrimp such as Litopenaeus vannamei and Penaeus monodon, dietary β-glucan supplementation has been associated with improved antioxidant capacity, immune enzyme activity, and resistance to bacterial and viral pathogens, particularly under environmental stress. For example, in Penaeus merguiensis, dietary β-glucans (2 g kg^-¹ feed) increased survival from 51.1% to 77.8–82.2% following Vibrio parahaemolyticus infection (Pooljun et al., 2022).

However, the efficacy of β-glucans varies depending on dose, species, source, and feeding strategy. In L. vannamei, dietary inclusion at 0.1–0.2% improves growth, antioxidant capacity, and immune responses, with approximately 0.2% identified as an optimal level, whereas higher inclusion levels (e.g., 0.4%) may reduce growth performance, indicating a non-linear dose–response relationship (Qiao et al., 2022). Interspecific variation is also evident, as P. monodon exhibits different optimal inclusion ranges (Sakai, 1999). Feeding strategy further influences outcomes; intermittent supplementation can modulate immune gene expression and gut microbiota composition but does not consistently improve growth compared to continuous feeding (Qiao et al., 2023). Additionally, β-glucan activity varies with source and structural characteristics, with glucans derived from marine diatoms such as Chaetoceros muelleri and Thalassiosira weissflogii showing different immunological effects (Pooljun et al., 2022). Overall, β-glucans are effective immunostimulants, but their performance is context-dependent and requires optimization of dose, formulation, and administration strategy.

8.2.1 β-Glucans

8.2.1.1 Practical implications for farm management

For Litopenaeus vannamei, dietary β-glucan inclusion at 0.1–0.2% is recommended to enhance total haemocyte count (THC), phenoloxidase activity, antioxidant defenses, and overall immune competence, with ~0.2% considered optimal (Qiao et al., 2022). Inclusion levels above 0.4% may negatively affect growth due to the non-linear dose–response relationship (Qiao et al., 2022). In Penaeus monodon, optimal inclusion levels differ and require species-specific adjustment (Sakai, 1999). From a management perspective, β-glucans are most effective when applied as short-term prophylactic supplements before predictable stress events such as handling, temperature fluctuations, salinity changes, or disease outbreaks, rather than as continuous feed additives (Sakai, 1999; Qiao et al., 2022). Their use should be integrated with water quality management, aeration, biosecurity, and disease surveillance for consistent and sustainable outcomes (Pooljun et al., 2022; Qiao et al., 2022).

8.2.2 Probiotics

Probiotics are beneficial microorganisms that improve host health by modulating gut microbiota and enhancing immune responses. In shrimp aquaculture, probiotic strains such as Lactobacillus acidophilus, Rhodopseudomonas palustris, and Bacillus coagulans have been shown to improve growth performance and increase immune parameters including phenoloxidase activity and antioxidant enzyme levels (Wang and Gu, 2010; Lakshmi et al., 2013). Probiotics function through multiple mechanisms, including competitive exclusion of pathogens, production of antimicrobial substances, and stimulation of host immune pathways. They also enhance gut microbial balance and activate innate immune signalling pathways, improving resistance against bacterial infections such as Vibrio and Photobacterium species (Naiel et al., 2021; Tamilselvan and Raja, 2024). As environmentally friendly alternatives to antibiotics, probiotics play an important role in sustainable aquaculture health management.

8.2.2.1 Practical implications for farm management

Probiotics provide an environmentally friendly approach to enhancing shrimp immunity, but their effectiveness is strain- and species-specific. In L. vannamei, supplementation with strains such as Lactobacillus acidophilus and Bacillus coagulans improves antioxidant enzyme activity, PO function, and disease resistance (Wang and Gu, 2010; Naiel et al., 2021). P. monodon exhibits similar benefits with compatible strains but requires careful monitoring of gut colonization efficiency. Limitations include variable survival of probiotic strains under pond conditions and dependency on water quality and feeding regimes, emphasizing the need for continuous monitoring of growth, water quality, and immune biomarkers for consistent benefits.

8.2.3 Plant-derived immunostimulants

Plant-derived immunostimulants are increasingly used as natural alternatives to synthetic immune modulators (Citarasu, 2010). Many medicinal plants contain bioactive compounds such as flavonoids, polysaccharides, and phenolics that possess immunomodulatory and antioxidant properties. Dietary supplementation with plant extracts can enhance haemocyte proliferation, phenoloxidase activity, and respiratory burst responses in shrimp. For example, Phyllanthus niruri extract has been shown to increase haemocyte counts and improve immune responses in shrimp challenged with vibriosis (Wachid et al., 2022). Similarly, Andrographis paniculata supplementation improves growth performance and resistance to Vibrio alginolyticus infection (Yin et al., 2023). Seaweed extracts such as Sargassum horneri also stimulate immune gene expression and phenoloxidase activity in shrimp (Lee et al., 2020). These phytogenic additives therefore represent promising eco-friendly immunostimulants for aquaculture.

8.2.3.1 Practical implications for farm management

Plant-derived immunostimulants, including Phyllanthus niruri, Andrographis paniculata, and seaweed extracts like Sargassum horneri, have demonstrated immunomodulatory effects in both L. vannamei and P. monodon, enhancing haemocyte proliferation, PO activity, and respiratory burst (Wachid et al., 2022; Yin et al., 2023; Lee et al., 2020). However, variability in phytochemical composition across plant sources and extraction methods can lead to inconsistent immune responses. Therefore, plant-based immunostimulants are best applied as complementary preventive measures during periods of predictable stress or disease challenge, rather than as a primary immune booster.

8.2.4 Nucleotide-based immunostimulants

Dietary nucleotides have been recognized as effective immunostimulants in shrimp aquaculture. Supplementation enhances haemocyte proliferation, activates the prophenoloxidase system, increases respiratory burst activity, and improves lysozyme activity and antioxidant defence (Guo et al., 2016; Andrino et al., 2012). Optimal inclusion levels (approximately 90–120 mg kg^-¹ or 0.2–0.4% of feed) improve growth performance, feed utilization, and intestinal morphology in Litopenaeus vannamei. Nucleotide supplementation also enhances resistance to bacterial pathogens such as Vibrio parahaemolyticus and viral infections including WSSV (Andrino et al., 2012). By supporting rapid haemocyte renewal and immune function, nucleotide-based supplements represent a sustainable strategy to improve shrimp health under intensive culture conditions.

8.2.4.1 Practical implications for farm management

Nucleotide-based supplements support rapid haemocyte renewal, enhance PO and lysozyme activity, and improve intestinal morphology, particularly in L. vannamei (Guo et al., 2016; Andrino et al., 2020). Optimal inclusion levels (0.2–0.4% of feed) improve growth, feed utilization, and pathogen resistance. In P. monodon, similar benefits are observed, but environmental conditions and baseline health status can modulate effectiveness. Over-supplementation is generally unnecessary and may increase cost without additional immune benefits. Strategically timed supplementation, such as 1–2 weeks prior to anticipated stress events, provides the most reliable protection.

8.3 Cost–benefit analysis of immunostimulants

Although immunostimulants can enhance innate immunity and disease resistance in shellfish, their practical use in aquaculture depends on a clear cost–benefit balance. Economic considerations include supplement cost, optimal dosage, and duration of application (continuous vs short-term), which must be evaluated against measurable benefits such as improved survival, growth performance, immune enhancement, and disease resistance. These benefits are not always proportional to input costs, emphasizing the need for system-specific evaluation rather than generalized application.

Among immunostimulants, β-glucans represent a moderately priced but effective option when applied at optimal inclusion levels (0.1–0.2%), where they enhance haemocyte activity, phenoloxidase function, and pathogen resistance (Qiao et al., 2022; Pooljun et al., 2022). However, cost-effectiveness declines at higher inclusion levels (>0.4%), where additional benefits are limited and growth performance may be negatively affected, indicating a non-linear dose–response relationship (Qiao et al., 2022). Their efficacy also varies with species, environmental conditions, and glucan source, requiring precise optimization.

Probiotics generally provide the most favourable cost–benefit profile due to their relatively low cost and multifunctional effects on gut health, nutrient utilization, immune response, and disease resistance (Wang and Gu, 2010; Naiel et al., 2021). However, their effectiveness depends on strain selection, environmental conditions, and successful colonization, and inconsistent results may occur under suboptimal culture conditions.

Plant-derived immunostimulants show variable cost–benefit performance due to differences in plant source, extraction methods, and species-specific responses (Yin et al., 2023; Lee et al., 2020; Wachid et al., 2022). While they possess immunomodulatory and antioxidant properties, inconsistent outcomes and higher processing costs may limit their large-scale economic feasibility. Similarly, nucleotide-based immunostimulants can improve immune function and growth performance, but their relatively high cost requires targeted application and controlled dosing to maintain cost-effectiveness (Guo et al., 2016; Andrino et al., 2012).

From a farm management perspective, immunostimulants are most cost-effective when used strategically during periods of environmental stress, disease risk, or prior to pathogen exposure rather than as continuous feed additives. Overall, probiotics provide the most consistent economic benefit, β-glucans are effective when properly optimized, and plant- and nucleotide-based immunostimulants are best used as targeted supplements. Thus, immunostimulants can yield positive economic outcomes when applied judiciously, whereas indiscriminate use may increase production costs without proportional benefits.

9 Immune priming in shellfish

Immune priming in invertebrates describes enhanced immune responsiveness following prior exposure to pathogens or pathogen-associated molecular patterns (PAMPs). Although shellfish lack classical adaptive immunity, increasing evidence indicates that their innate immune system can exhibit memory-like characteristics that improve resistance to subsequent infections (Kurtz, 2005; Milutinović and Kurtz, 2016). However, the generality, durability, and mechanistic basis of immune priming in shellfish remain incompletely resolved and, in some cases, controversial.

In crustaceans, several studies report enhanced haemocyte-mediated responses following initial exposure, including increased phagocytosis, prophenoloxidase activity, and antimicrobial responses. For example, Litopenaeus vannamei exposed to inactivated Vibrio harveyi showed increased haemocyte phagocytic activity and improved survival upon secondary challenge (Pope et al., 2011), and similar immune enhancement has been reported in Penaeus monodon (Cerenius and Söderhäll, 2013). However, these responses are often transient and variable, with some studies reporting limited or inconsistent priming effects, indicating strong influence of pathogen load, exposure regime, and environmental conditions (Milutinović and Kurtz, 2016). This variability raises concerns regarding the reproducibility and practical application of immune priming in aquaculture systems.

In bivalve molluscs such as Crassostrea gigas and Mytilus galloprovincialis, evidence for immune priming is less consistent and often indirect. Prior exposure to pathogens or PAMPs has been associated with increased haemocyte activity, lectin expression, and oxidative responses; however, these responses do not always translate into improved survival (Allam and Raftos, 2015). In some cases, chronic exposure to pathogens such as Vibrio spp. or Perkinsus spp. leads to prolonged immune activation followed by immunosuppression, complicating interpretation of priming responses (Allam and Raftos, 2015). These findings suggest that immune priming in bivalves is highly context-dependent and may not always confer adaptive benefits.

At the molecular level, priming responses are associated with modulation of pattern-recognition receptors (PRRs), including C-type lectins, β-glucan-binding proteins, and scavenger receptors, which activate downstream signalling pathways such as Toll, IMD, and JAK/STAT, leading to increased production of antimicrobial peptides, anti-lipopolysaccharide factors, lysozyme, and activation of the prophenoloxidase system (Low and Chong, 2020). Antiviral defence mechanisms involving RNA interference (RNAi), including Dicer and Argonaute proteins, have also been implicated in priming responses (He et al., 2025). However, distinguishing true memory-like responses from prolonged immune activation remains challenging, particularly in the absence of standardized experimental frameworks (Milutinović and Kurtz, 2016). Epigenetic mechanisms such as DNA methylation and histone modification have been proposed as potential mechanisms underlying priming, although direct causal evidence in shellfish remains limited (Bassim et al., 2015).

The concept of transgenerational immune priming (TGIP) further extends this phenomenon, whereby parental pathogen exposure enhances immune competence in offspring. Experimental evidence in Crassostrea gigas demonstrates that parental exposure to Vibrio splendidus results in enhanced immune gene expression and improved resistance in offspring (Song et al., 2025). Nevertheless, the mechanisms underlying TGIP, including the relative contributions of epigenetic inheritance and maternal provisioning, remain poorly understood and require further investigation.

10 Molecular immune pathways in shellfish

Innate immune responses in shellfish are regulated by conserved intracellular signalling pathways that connect pathogen recognition with downstream defence mechanisms. Because shellfish lack antibody-mediated adaptive immunity, these pathways serve as the primary regulators of host defence. Pattern-recognition receptors (PRRs) located on haemocytes and epithelial cells detect pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharides, peptidoglycans, viral nucleic acids, and fungal β-glucans. Recognition of these molecules activates intracellular signalling cascades that regulate transcription of immune genes involved in antimicrobial peptide production, inflammatory responses, and antiviral defence (Cerenius and Söderhäll, 2013; Tassanakajon et al., 2013).

Among the most studied immune pathways in crustaceans and molluscs are the Toll, immune deficiency (IMD), and Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathways. These signalling systems regulate haemocyte activity, antimicrobial peptide synthesis, oxidative responses, and other immune processes required for pathogen clearance. Increasing evidence indicates that these pathways operate in interconnected networks that determine the efficiency of immune responses in shellfish (Li et al., 2018). Disruption of these pathways by pathogens or environmental stress can impair immune competence and increase disease susceptibility in aquaculture systems.

10.1 Toll signalling pathway

The Toll signalling pathway is a key innate immune pathway involved in pathogen recognition and antimicrobial defence. Toll-like receptors detect conserved microbial molecules such as lipopolysaccharides, peptidoglycans, and β-glucans. Following activation, intracellular adaptor proteins including MyD88, Tube, and Pelle initiate signalling cascades that activate the transcription factor NF-κB. This factor translocates to the nucleus and induces transcription of immune effector genes (Tassanakajon et al., 2013). Activation of the Toll pathway stimulates production of antimicrobial peptides such as crustins, penaeidins, anti-lipopolysaccharide factors (ALFs), and lysozymes. In penaeid shrimp such as Litopenaeus vannamei, silencing Toll pathway components significantly reduces antimicrobial peptide expression and increases susceptibility to pathogens including White Spot Syndrome Virus (WSSV) (Li et al., 2018). The Toll pathway also interacts with other immune mechanisms, including activation of the prophenoloxidase (proPO) system and reactive oxygen species production.

10.2 IMD signalling pathway

The immune deficiency (IMD) pathway plays a central role in defence against Gram-negative bacteria and contributes to antiviral immunity in crustaceans. IMD homologues have been identified in species such as Fenneropenaeus chinensis and Procambarus clarkii, where they regulate antimicrobial peptide production in tissues including haemocytes, gills, and hepatopancreas (Jiang et al., 2013). In Litopenaeus vannamei, the IMD pathway interacts with the Toll pathway, forming coordinated transcriptional networks that regulate immune responses (Liu et al., 2016). Activation of IMD upregulates signalling molecules such as Pelle, TRAF6, and IAP1 and enhances antimicrobial peptide expression, improving bacterial clearance. Epigenetic regulation also influences this pathway; for example, suppression of the m6A reader PmYTHDF2 in Penaeus monodon increases expression of IMD pathway genes and antimicrobial peptides, improving resistance to White Spot Syndrome Virus (Jiang et al., 2025).

10.3 JAK–STAT signalling pathway

The Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway is a conserved immune signalling system involved in antiviral defence, immune modulation, and stress responses. Activation occurs when cytokine-like molecules bind to cell surface receptors, triggering phosphorylation of JAK proteins and activation of STAT transcription factors. Activated STAT dimers translocate to the nucleus to regulate immune gene expression. In crustaceans, the JAK–STAT pathway plays an important role in antiviral immunity. Studies in Litopenaeus vannamei have shown that silencing STAT alters viral infection dynamics, demonstrating its involvement in host–virus interactions during White Spot Syndrome Virus infection (Huang et al., 2016; Wen et al., 2014). In addition to antiviral defence, this pathway contributes to regulation of apoptosis, inflammatory signalling, and cellular stress responses.

10.4 Integration of signalling pathways in aquaculture health

The Toll, IMD, and JAK–STAT pathways collectively regulate haemocyte activation, antimicrobial peptide production, prophenoloxidase-mediated melanisation, and reactive oxygen species generation. Disruption of these pathways by environmental stress or pathogen immune evasion can compromise host immunity and increase disease susceptibility. In aquaculture, transcriptional profiling of key signalling molecules such as Toll, MyD88, Relish, STAT, and antimicrobial peptide genes can serve as molecular biomarkers for early detection of immune stress and pathogen exposure. Combining molecular pathway analysis with functional immune indicators—such as total haemocyte count, phenoloxidase activity, and phagocytosis—can improve health monitoring and disease management in shellfish culture systems.

11 Significance of shellfish immunology in sustainable health management

Understanding the cellular and molecular mechanisms of shellfish immunity provides a scientific foundation for developing sustainable health management strategies in aquaculture. Unlike finfish, shellfish lack adaptive immunity and cannot benefit from conventional vaccination programs. Therefore, disease prevention relies primarily on strengthening innate immune responses and minimizing environmental stress that predisposes animals to infection. Insights into cell-mediated immunity—particularly haemocyte functions such as phagocytosis, encapsulation, nodulation, and prophenoloxidase activation—enable the development of targeted interventions that enhance pathogen clearance and host resistance. Knowledge of these immune mechanisms has practical applications in aquaculture health management. For example, immunostimulants such as β-glucans, probiotics, plant-derived compounds, and nucleotide supplements can enhance haemocyte activity, antimicrobial peptide production, and oxidative defense responses. Similarly, immune biomarkers including total haemocyte count, phenoloxidase activity, and phagocytic capacity can be used for early detection of stress or infection in farming systems. Integrating immune-based tools with environmental management, selective breeding for disease resistance, and biosecurity practices provides a comprehensive and sustainable approach to disease prevention in shellfish aquaculture.

12 Comparative immune strategies of crustaceans and molluscs: evolutionary adaptations and functional divergence

The immune differences between crustaceans and molluscs reflect distinct evolutionary strategies shaped by ecological pressures and life-history traits rather than simple taxonomic variation (Rowley and Powell, 2007; Gerdol et al., 2020; Gerdol and Venier, 2015). Crustaceans such as shrimp (Litopenaeus vannamei, Penaeus monodon) possess a highly developed prophenoloxidase (proPO) activation system that enables rapid melanisation, pathogen encapsulation, and localized cytotoxic responses, providing an effective defence against acute infections (Cerenius and Söderhäll, 2004; Amparyup et al., 2013). The presence of abundant semigranular and granular haemocytes supports efficient storage and release of proPO and other immune effectors (Söderhäll and Cerenius, 1998; Sun et al., 2024).

In contrast, bivalve molluscs such as oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis) rely less on melanisation and more on phagocytosis, reactive oxygen species (ROS), and antimicrobial peptides as primary defence mechanisms (Allam and Raftos, 2015). This strategy is consistent with their filter-feeding lifestyle, which exposes them continuously to high microbial loads, making sustained and energy-efficient immune responses more advantageous than rapid but metabolically expensive melanisation responses (Allam and Raftos, 2015; Gerdol et al., 2020). Consequently, bivalves possess haemocytes with strong phagocytic capacity and continuous humoral immune activity (Donaghy et al., 2012; Wang et al., 2018).

The comparatively lower reliance on the proPO system in bivalves should therefore be interpreted as an adaptive trade-off rather than a weaker immune system. Melanisation is metabolically costly and generates cytotoxic intermediates that may damage host tissues if not tightly regulated; thus, reduced dependence on proPO likely reflects selection for energy-efficient immune strategies compatible with continuous filtration and long-term environmental exposure (Cerenius and Söderhäll, 2004; Allam and Raftos, 2015; Gerdol et al., 2020). Differences in antimicrobial peptide repertoires further illustrate functional divergence: crustaceans predominantly produce penaeidins, crustins, and anti-lipopolysaccharide factors (ALFs), which are effective against acute infections, whereas bivalves produce defensins, mytilins, and myticins associated with broad-spectrum microbial regulation and immune homeostasis (Destoumieux-Garzón et al., 2016; Amparyup et al., 2013; Mitta et al., 2000; Gerdol and Venier, 2015).

WSSV infection further disrupts haemocyte dynamics through the induction of apoptosis mediated by caspase activation, mitochondrial dysfunction, and cytoskeletal disorganization, resulting in reduced total haemocyte count (THC), impaired proliferation, and decreased phagocytic capacity (Wang et al., 2019). Notably, the magnitude of haemocyte depletion and immune suppression may vary between species, with Litopenaeus vannamei often showing more pronounced reductions in total haemocyte count (THC) compared to Penaeus monodon, suggesting potential differences in antiviral resilience (Tang et al., 2019; Hameed et al., 2006). However, the precise regulatory mechanisms governing haemocyte recovery and turnover during WSSV infection remain poorly understood, representing a key limitation in current research (Stentiford et al., 2017; Elbahnaswy et al., 2025).

To synthesise these functional and evolutionary differences, a comparative overview of key immune parameters across major shellfish groups is presented in Table 1.

Table 1. Comparative immune characteristics of major shellfish groups.

13 Future research directions

Despite substantial advances in understanding shellfish innate immunity, several key areas require further investigation to support sustainable disease management in aquaculture. Future research should integrate molecular, genomic, and ecological approaches to better elucidate immune regulation and host–pathogen interactions in shellfish. Recent developments in high-throughput technologies, including transcriptomics, proteomics, and single-cell RNA sequencing, provide powerful tools to investigate immune cell heterogeneity and gene regulation. These approaches may facilitate the discovery of novel immune-related genes, signalling pathways, and regulatory networks involved in pathogen recognition and immune activation.

Further characterization of major immune signalling pathways—particularly the Toll, IMD, and JAK–STAT pathways—is also necessary to understand how shellfish respond to different classes of pathogens. Detailed investigation of these molecular interactions will enhance knowledge of immune regulation and pathogen defence mechanisms. In applied aquaculture, identifying reliable immune biomarkers remains an important priority. Biomarkers based on immune gene expression, haemocyte function, and enzymatic activities could support early disease detection and routine health monitoring in shrimp and molluscan farming systems. In parallel, selective breeding programmes aimed at improving disease resistance in economically important species such as Litopenaeus vannamei and Macrobrachium rosenbergii can directly benefit from genomic tools. Immune traits such as prophenoloxidase activity, haemocyte counts, and antimicrobial peptide expression show measurable heritability, providing targets for selective breeding. Marker-assisted selection (MAS) enables breeders to choose individuals with superior immune responses based on molecular markers, while genomic selection approaches leverage genome-wide data to predict disease resistance more accurately. Practical breeding programs have demonstrated improved survival against viral and bacterial pathogens, illustrating how fundamental immunological research can be applied to sustainable aquaculture.

Future studies should also examine how environmental stressors—such as temperature fluctuations, hypoxia, ammonia accumulation, and salinity variation—affect immune competence and disease susceptibility. Integrating immunological research with environmental monitoring will be critical for developing adaptive aquaculture management strategies, particularly under changing climate conditions. Finally, the development of novel immunostimulants derived from probiotics, plant extracts, and microbial products represents a promising strategy for improving shellfish health while reducing reliance on antibiotics. Understanding the mechanisms through which these compounds modulate immune responses will facilitate their effective use in sustainable aquaculture systems.

14 Conclusion

Shellfish rely entirely on innate immune mechanisms to defend against the wide range of pathogens present in aquatic environments. Cellular components such as haemocytes, together with humoral factors including antimicrobial peptides, lectins, the prophenoloxidase (proPO) system, and reactive oxygen species, play essential roles in pathogen recognition, phagocytosis, encapsulation, and melanization. Despite the absence of classical adaptive immunity and antibody production, shellfish possess sophisticated defence strategies, including immune priming and diverse pattern-recognition systems, that enable rapid responses to microbial challenges. Understanding the cellular and molecular basis of shellfish immunity has important implications for sustainable aquaculture. Immune-related biomarkers such as total haemocyte count, phenoloxidase activity, respiratory burst, and lysozyme activity are increasingly used to assess health status and disease resistance in cultured shrimp and molluscs. Monitoring these parameters can facilitate early detection of stress and infection, enabling timely management interventions. Advances in shellfish immunology have also supported the development of immunostimulant-based strategies to enhance disease resistance in farmed species. Compounds such as probiotics, β-glucans, plant-derived extracts, and microbial products have shown potential to stimulate haemocyte activity and antimicrobial defence mechanisms. Integrating these approaches with improved environmental management, biosecurity practices, and selective breeding for disease-resistant strains can help reduce disease outbreaks and improve aquaculture productivity. Overall, continued progress in shellfish immunology provides a strong scientific foundation for developing innovative and sustainable health management strategies. Future integration of molecular immunology, genomics, and environmental monitoring will further improve disease control and support the long-term sustainability of global shellfish aquaculture.

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Footnotes

Edited by: Neelesh Kumar, Rani Lakshmi Bai Central Agricultural University, India

Reviewed by: Niti Sharma, Central Inland Fisheries Research Institute (ICAR), India

Bismay Sahoo, Central Institute of Freshwater Aquaculture (ICAR), India

Novi Rosmala Dewi, National Taiwan Ocean University, Taiwan