THE IMMUNE SYSTEM OF SHRIMP By : Franklin S. Martínez (fmartinezt@alicorp.com.pe) Nicovita-ALICORP SAA Technical Service Introduction Penaeid shrimp aquaculture is an important economic activity worldwide. Nevertheless, shrimp production has been seriously affected by diseases, mostly those caused by viruses (Flegel, 2006) and Vibrio bacteria (Bachère, 2000). Shrimp resistance to invading organisms is strongly influenced by its immune status. The defense mechanisms of crustaceans is less developed than that in finfish and other vertebrates (Figure 1). More specifically, crustaceans have no adaptive memory. In other words, they do not have the ability of producing immunoglobulins, so that they apparently depend only on innate defense systems (Roch, 1999). Understanding shrimp defense mechanisms in combination with different strategies can contribute to improve disease management. In fact, several different methods exist to learn about the health/disease status of these animals, including the evaluation of immune/physiological parameters such as hemolymph protein concentration, total hemocyte counts, phenoloxidase activity, free radical production, phagocytic activity, and field variables including stress tests, survival, and growth rates, which can be used as shrimp health status indicators (Rodríguez y Le Moullac, 2000). The purpose of this bulletin is to review some major defense mechanisms of shrimp.

Figure 1. The immune system of (A) vertebrates, and (B) invertebrates

Vertebrates

Adaptive Memory

Non specific

A

Invertebrates

Non specific

B

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Shrimp defense mechanisms The innate defense system – also known as natural or non-specific defense system – includes both cellular and humoral components (Figure 2) which work in jointly coordination for the detection/elimination of all foreign organisms potentially hazardous for the host (Jiravanichpaisal et al., 2006). Cellular defense components include all those reactions performed directly by hemocytes (phagocytosis, encapsulation, nodule formation). On the other hand, humoral components include the activation and release of molecules stored within hemocytes, such as anticoagulant proteins, agglutinins, phenoloxidase enzyme, antimicrobial peptides, protease inhibitors, etc. (Jiravanichpaisal et al., 2006; Holmblad and Söderhäll, 1999.) Function of crustacean immune system The cuticle works as the first physical barrier, and it contains antimicrobial substances (Söderhäll and Cerenius, 1992). Once the pathogen has crossed the outer defense barriers, hemocytes play an important role in the crustacean immune response. In addition to participating in the inactivation of invading organisms, hemocytes are also involved in the regulation of different physiological functions i.e., exoskeleton hardening, cuticle damage healing, coagulation, carbohydrate metabolism, and protein/amino acid transportation and storage (Jiravanichpaisal et al., 2006.)

Figure 2. Cellular and humoral components of crustacean immune system

Innate Immunity (Natural or Non-specific)

Cellular Components

Humoral Components

- Phagocytosis

- Encapsulation

- Formation of nodules

- Anticoagulant proteins

- Agglutinins

- Phenoloxidase enzyme

- Antimicrobial peptides

- Free radicals

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Hemocyte classification is based on the presence and size of 3 types of cytoplasmic granules: hyaline, semi-granular, and granular hemocytes (Figure 3). Even though the proportion and function of hemocytes can vary among species, it is generally considered that granular and semi-granular hemocytes have the ability of producing melanin by the pro-phenoloxidase system (Johansson y Söderhäll, 1989). On the other hand, hyaline hemocytes and – in a lesser extent – semi-granular hemocytes are responsible for the phagocytosis process (Giulianini et al., 2007.)

Pathogen recognition The first step in the immune process is the recognition of microorganisms. This process is carried out by hemocytes through molecules that have the ability of recognizing structures in the cell walls of invading organisms, such as attachment proteins, and by the recognition of β-1,3-glucans, lipopolysaccharides, and peptidoglycans (Lin et al., 2006; Vargas-Albores and Yepiz-Plascencia, 2000). Once invading organisms are detected, hemocytes get activated then a whole series of mechanisms is triggered to control or remove the intruders. Phenoloxidase activity The phenoloxidase system has been recognized as an efficient defense mechanism against the non-self. This system is stored and produced by semi-granular and granular hemocytes, and it can be activated by a minimum presence of microbes. Activation of the pro-phenoloxidase system results in the production of melanin, a dark-brown pigment responsible – among other processes – for inactivating foreign particles, and preventing their spread throughout the host body, as well as for healing cuticle damages (Sritunyalucksana and Söderhäll, 2000).

Figure 3. Classification of hemocytes: (A) Hyaline, (B) Semi-granular, (C) Granular

(Modified photos from Giulianini et al., 2007)

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A C

12.4 × 7.8 14.8 × 8.3 13.6 × 9.5

B

Free radicals and antioxidant mechanisms Destroying the phagocytized materials involves the intracellular production of free radicals. During contact with and recognition of the pathogen, host enzymes like NADPH-oxidase are activated, which in turn increase oxygen consumption, resulting in the production of free radicals such as superoxide anions (O2

-) and hydrogen peroxide (H2O2), among others (Muñoz et al., 2000; Rodríguez and Le Moullac, 2000). These free radicals can directly kill the invading organism or work in combination with nitrogen compounds (nitric oxide), or exert a synergistic effect with lysozymes (Roch, 1999). Nevertheless, free radicals do not discriminate host-self cells from microbes, so that they can be deleterious in the event of acting in the extra-cellular spaces. Under normal conditions, the potential damage caused by free radicals is regulated by mechanisms such as antioxidant molecules i.e., ascorbic acid, poly-unsaturated fatty acids, and antioxidant enzymes (superoxide dismutase, various peroxidases) (Dandapat et al., 2003; Campa-Córdova et al., 2002.)

Phagocytosis, encapsulation and nodule formation Phagocytosis is the most common reaction of defense cell mechanisms. By this process, cells (hemocytes) ingest and destroy invading pathogens, foreign particles or modified (aged) cells of the body itself (Secombes, 1996.) Encapsulation and nodule formation (Figure 4) are processes by which several hemocytes cooperate with each other aiming to stop the action of invading organisms, when the host is attacked by either extremely-large particles or numerous tiny particles, to be ingested then destroyed by individual cells (Söderhäll y Cerenius, 1992).

Figure 4. Defense cell processes include: (A) Phagocytosis, (B) Encapsulation, and (C)

Nodule formation. Hemocytes are represented in green, while the invading organisms appear in red

A B C

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References

Bachère, E. 2000. Shrimp immunity and disease control. Aquaculture, 191:3-11.

Campa-Córdova, A. I., N. Y. Hernández-Saavedra, R. De Philippis and F. Ascencio. 2002. Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to β-glucan and sulphated polysaccharide. Fish & Shellfish Immunology, 12:353-366.

Dandapat, J., G. B. N. Chainy and K. J. Rao. 2003. Lipid peroxidation and antioxidant defense status during larval development and metamorphosis of giant prawn, Macrobrachium rosenbergii. Comparative Biochemestry and Physiology Part C, 135:221-233.

Flegel, T. W. 2006. Detection of major penaeid shrimp viruses in Asia, a historical perspective with emphasis on Thailand. Aquaculture, 258:1-33.

Giulianini, P. G., M. Bierti, S. Lorenzon, S. Battistella and E. A. Ferrero. 2007. Ultrastructural and functional characterization of circulating hemocytes from the freshwater crayfish Astacus leptodactylus: Cell types and their role after in vivo artificial non-self challenge. Micron, 38:49-57.

Holmblad, T. and Söderhäll, K. 1999. Cell adhesion molecules and antioxidative enzymes in a crustaceans, possible role in immunity. Aquaculture, 172:111-123.

Jiravanichpaisal, P., B. L. Lee and K. Söderhäll. 2006. Cell-mediated immunity in arthropods: Hematopoiesis, coagulation, melanization and opsonization. Immuno-biology, 211:213-236.

Johansson, M. W. and Söderhäll, K. 1989. Cellular immunity in crustaceans and the proPO system. Parasitology Today, 5:171-176.

Lin, C. Y., K. Y. Hu, S. H. Ho and Y. L. Song. 2006. Cloning and characterization of a shrimp clip domain serine protease homolog (c-SPH) as a cell adhesion molecule. Developmental and Comparative Immunology, 30:1132-1144.

Muñoz, M., R. Cedeño, J. Rodríguez, W. P. W. van der Knaap, E. Mialhe and E. Bachère. 2000. Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp, Penaeus vannamei. Aquaculture, 191:89-107.

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Roch, P. 1999. Defense mechanisms and disease prevention in farmed marine invertebrates. Aquaculture, 172:125-145.

Rodríguez, J. and Le Moullac, G. 2000. State of the art of immunological tools and health control of penaeid shrimp. Aquaculture, 191:109-119.

Secombes, C. J. 1996. The Nonspecific Immune System: Cellular Defenses, In The Fish Immune System: Organism, Pathogen, and Environment, Iwama, G. and Nakanishi, T., Academic Press, San Diego, USA, pp. 63-103.

Söderhäll, K. and Cerenius, L. 1992. Crustacean immunity. Annual Review of Fish Diseases, 3-23.

Sritunyalucksana, K. and Söderhäll, K. 2000. The proPo and clotting system in crustaceans. Aquaculture, 191:53-69.

Vargas-Albores, F. and Yepiz-Plascencia, G. 2000. Beta glucan binding protein and its role in shrimp immune response. Aquaculture, 191:13-21.

Tumpis Edition Editors: Dagoberto Sánchez dsanchezc@alicorp.com.pe Carlos Ching cchingm@alicorp.com.pe Máximo Quispe mquispec@alicorp.com.pe

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