Traditionally, the immune system is divided into the innate or nonspecific immune system and the adaptive or specific immune system. The fish immune system has both types of immunity, innate immunity, and adaptive immunity.

It is essential to keep the fish immune system in an optimal condition, as it is responsible for defending the body from external threats, such as viruses, bacteria, or protozoa and, therefore, it allows to prevent infections.

Effects of stress and the use of immunostimulants in aquaculture

During their culture fishes face a wide variety of stressors, associated with environmental, social and management factors. There are several immunostimulant products available that boost the immune system of fish, counteracting the negative effects of stress and ensuring their productivity.

Primary and secondary lymphoid organs

One noteworthy difference between the immune system of fish and mammals are the organs that act as primary and secondary lymphoid organs. In the case of teleost or bony fish, such as the Atlantic salmon (Salmo salar), which lack bone marrow, the anterior kidney and thymus act as primary lymphoid organs, responsible for the hematopoiesis of immune cells, while the spleen and lymphoid tissue associated with mucous membranes act as secondary lymphoid organs, where immune system cells interact and where the immune response takes place.

The anterior kidney is the main hematopoietic organ of fish and has great similarities to the bone marrow of vertebrates. It produces the different immune cells that participate in the cell immune response: macrophages, granulocytes, lymphocytes, etc.

This anterior region of the kidney lacks nephrons and, therefore, it lacks renal function, while the mid and distal regions of that organ have both functions: hematopoietic and renal function. The anterior kidney of teleosts showed to act, at the same time, as a secondary lymphoid organ, involving immune response induction.

The anatomical structure of the anterior kidney varies between the different species of teleosts and is bilobed in the case of cyprinids and salmonids (figure 1).

In these aquatic animals without lymph nodes, the liver plays a fundamental role in antigenic presentation and in the onset of adaptive immune response. The liver has a high concentration of melanomacrophages and ellipsoids, which capture the antigens and immune complexes present in the blood. In addition, melanomacrophages retain antigens for long periods of time.

Types of immunity

Like upper vertebrates, fish have two types of immunity, innate or nonspecific immunity and adaptive or specific immunity (table 1).

Innate or nonspecific immunity

Innate or non-specific immunity is the first line of defense that the fish’s immune system has to deal with the different pathogens that threaten their homeostasis. This defensive system can be divided into three main components: mucosal immunity, humoral components, and cellular components.

Mucosal immunity plays a key role in the fish immune system, acting as a barrier and preventing pathogens from reaching the body. In addition to the mechanical and physical protection provided by the mucus that coats the body surface of these animals, as well as gills and other tissues, it contains a large number of immune components such as antimicrobial peptides, complement factors and immunoglobulins.

The main difference between innate immunity and adaptive immunity is the specificity with which they are able to recognize different pathogens. Such specificity is determined by the type of receptors they can recognize.

The cells and humoral components that constitute nonspecific immunity recognize these types of molecular patterns:

• Pathogen Associated Molecular Patterns (PAMPs): which constitute a set of highly conserved substances/structures in different types of pathogens that are not present in eukaryotic cells, such as cell wall lipopolysaccharides (LPS). In the case of teleostswei, TLR (Toll-like receptors), of which more than 20 different types have been described, play a primary role.
• Damage/Danger Associated Molecular Patterns (DAMPs): signals emitted by cells of the body itself, for example, in case of thermal stress.

As in mammals, the innate fish immune system has different humoral and cellular components. Humoral components include antimicrobial peptides, the complement system, lectins, TNF-α and interleukins.

Antimicrobial peptides have been described in multiple aquatic species and are present in the mucus, covering the skin and gills. For example, in the case of the Atlantic cod (Gadus morhua), polypeptides present in the mucus have been described to be effective against Gram + and Gram – bacteria.

One of the defensive mechanisms of the innate immunity is the complement system. This system consists of a set of serum proteins that circulate inactively and are activated in the form of a cascade. As in the case of upper vertebrates, the complement system is activated by the 3-way, the classic, the alternative, and the lectins pathways. All of them lead to the formation of pores in the cell membrane of the pathogen and the consequent death by osmotic shock. One noteworthy difference between the mammalian and the fish immune system is the optimal temperature for this immune complex. In the case of teleost, its activity is maximum between 15-25°C and can remain active at temperatures between 0-4C°, while, in mammals, its optimal temperature is 35°C.

The innate immune system of fish has, at the same time, different cell types that participate in the nonspecific cellular response, including cells with phagocytic activity, such as monocytes and macrophages, and cells with cytotoxic activity, such as granulocytes and non-specific cytotoxic cells.

In the presence of an antigen, neutrophils are mobilized first from the anterior kidney and then the macrophages that mobilize from nearby tissues are attended. Thanks to the phagocytic and cytotoxic activity of these cells, pathogens are eliminated.

Adaptive or specific immunity

The specific or adaptive immune system is present in cartilaginous fish and has been described in all jawed fish. Like mammals, this specific response in fish starts from the detection of the molecules of the major histocompatibility complex (MHC) by T cells. The MHC molecules are a superfamily of immunoglobulins that act as receptors. Unlike higher mammals, where MHC genes are joined into a single chromosome, in the fish immune system these genes are not connected and can be found in different chromosomes.

The two types of MHC receptors described in upper vertebrates are present in teleosts. The MHC I molecules, which are related to endogenous antigens, are present in all nucleated cells and are recognized by CD8 or cytotoxic T cells. The MHC II molecules are related to exogenous antigens and are only found in antigen-presenting cells (dendritic cells, macrophages, and B lymphocytes).

B lymphocytes are responsible for the production of immunoglobulins, which allow this immune system to have a certain “memory”, causing a more intense and lasting response in a second exposure to the same antigen. Activation of B lymphocytes requires two steps (figure 3). On the one hand, they must recognize external antigens through type II MHC, and, at the same time, such antigens have to be presented by a CD4 T lymphocyte or helper. Once activated, B lymphocytes are transformed into plasma cells capable of secreting the different types of immunoglobulins.

Teleosts are able to synthesize 3 types of immunoglobulins: IgM, IgD and IgT/IgZ, unlike upper vertebrates, where 5 types of immunoglobulins are described.

IgM is the most abundant immunoglobulin in the fish immune system and, as in mammals, is able to pass through epithelial membranes. The IgM of teleosts is a tetramer consisting of two heavy chains and two light chains (2H:2L). This immunoglobulin plays a key role in the innate immune system against bacteria and viruses, as it is responsible for fixing the complement, opsonization and activation of the cytotoxic response, among other functions.

The location of immunoglobulins is specific in certain parasitic infections, for example, in trouts affected by Ceratomixa shasta, a greater increase in IgT/IgZ was observed in the intestinal mucosa, while the IgM title increased significantly in the serum. These assays seem to indicate some affinity in the location of immunoglobulins, as IgM are more abundant in serum and IgT/IgZ are the most abundant in mucous-associated immunity.

Stressors and stress response

Stress, especially chronic stress, is known to have a negative effect on different productive species, including aquaculture species, because the immune response and disease resistance decrease.

Stressors in fish can be divided into three large groups, environmental factors, social and reproductive factors and physical or management factors.

Environmental factors include sudden changes in temperature and oxygen, as well as changes in water quality and high culture densities. Social factors refer to the establishment of a dominance hierarchy within cages or ponds. The reproductive season has also proven to be a very stressful period for aquaculture species and should certainly be taken into account in production systems.

There are physical or handling factors that can affect fish immune system, including the transport of animals, both within the same facility and between different facilities, and tasks such as tank cleaning, vaccinations, and animal classifications.

The stress state can be divided into three stages: initial stage, endurance stage and exhaustion stage. When an animal is exposed to a stressful agent, different signals are sent from the sensitive organs that reach the central nervous system, activating the hypothalamus-hypophysis axis. Activation of this nervous axis causes the release of catecholamines and cortisol, secreted by chromaffin cells and interrenal cells, respectively, located in the kidney of these aquatic animals.

Catecholamines and cortisol are responsible for the immune system’s response to stress. The secretion of these molecules prepares the animal to face a possible challenge by increasing heart rate, blood flow, energy availability (glucose levels) and stimulating the immune response.

If the stressor persists, we enter the resistance phase, during which changes occur in metabolism and enzymatic secretion that allow to maintain the alarm state. However, if the stressful stimulus continues for a long time, the animal is not able to maintain this stage, and its immune response decreases, which is known as the exhaustion stage. At this stage, the animal is not able to continue producing high levels of the molecules involved in defensive mechanisms (lysozyme, complement system, IgM, leukocytes, etc.).

In Table 2, we can see how the number of leukocytes increases when animals are subjected to acute stress and, if stress is sustained, the number of white blood cells decreases, making animals more susceptible to pathogens.

These stressors have a negative impact on aquaculture production. On one hand, they decrease animal growth rate, because stressed animals ingest a smaller amount of food. On the other hand, stress states generate immunosuppression in fish, making them more susceptible to the microorganisms present in the environment.

Use of immunostimulants in aquaculture

Fish are subjected to different stressors during their production. Even if we can prevent some of them, especially environmental factors such as sudden changes in temperature or poor water quality, and maintain appropriate culture densities, other factors, especially those associated with management practices, are difficult to prevent.

There are routine tasks, such as cleaning tanks, fish classification, vaccination, and transportation, necessary within farms, but, at the same time, they generate great stress in animals and affect their productive performance. There are immunostimulant products capable of improving the immune response of fish during stressful periods.

They are products based on active molecules of a botanical origin, such as immunostimulant pronutrients that, added in feed, are able to improve the immune response, both innate and adaptive, and reduce the effects of stress.

Immunostimulant pronutrients promote the activity of macrophages and neutrophils, which increase the elimination of pathogens and, at the same time, stimulate the secretion of various molecules involved in the immune response, such as the gamma-interferon. Improving the fish immune system has a direct effect on the productive parameters, because they are more prepared to fight infections and, thus, avoid their negative effect on growth and feed utilization. The use of immunostimulant pronutrients allows to improve feed conversion rate by 31% and increase weight gain by 61%, thanks to their direct effect on the immune system (table 4).

At the same time, it these active molecules of botanical origin improve the antibody titers after vaccination. Therefore, the use of this type of molecules is of great interest in aquaculture, where a large number of vaccines are used as a prevention measure.

Conclusion

Fish immune system has both types of immunity, non-specific immunity, and specific immunity, similarly to upper vertebrates, but with some characteristic differences, such as the immune organs and the functioning of some effectors.

During the productive cycle, animals are subjected to environmental and management-related stressors capable of affecting their productive performance and the functioning of the fish immune system.

The use of natural immunostimulants, such as immunostimulant pronutrients, helps to prevent the negative effects during stressful periods, as they ensure a proper functioning of the fish immune system and, at the same time, improve the response to vaccination.