Modulation of penaeid shrimp immune system as a strategy to control disease

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FOCUS Feeding shrimp

Modulation of penaeid shrimp immune system as a strategy to control disease By Àlvaro Ortiz, Product Manager, Norel

One of the biggest problems related to intensification in animal production has been disease. Farm animals suffer from a great variety of infectious diseases that have been traditionally controlled by the use of pharmaceuticals. But as zootechnology has advanced, prevention has gained more importance, moving from a reactive to a preventive strategy.

One of the best proven ways to prevent pathologies is to create immunity in the animal. With more than 200 years to back up their efficacy, vaccines have helped to prevent and in some cases eliminate many disorders in humans, farm animals and family pets.

Regarding aquaculture, the evolution has been a bit slower. If we look at fish, the first vaccine for salmonids was licensed in the US in 1976. Since then, the use of immunoprophylaxis in fish has expanded to other places and species.

In the case of penaeid shrimp things are a bit more complicated. Unlike more complex vertebrates, whose immune systems depend heavily on adaptive, antigen-specific responses such as antibodies and T cell receptors, invertebrate defenses use generic, innate immunity.

Therefore, they lack any form of immunological memory similar to that found in jawed vertebrates. Vaccinating invertebrates, such as shrimp, should consequently be an ineffective management strategy resulting in no more than shortlived, non-specific immune stimulation.

Photo by courtesy of CSIRO

The shrimp innate defense system consists of cellular and humoral components working in coordination for the detection/elimination of pathogens potentially hazardous to the host. In both cases hemocytes play an important role. In cellular defense hemocytes are responsible for phagocytosis, encapsulation and nodule formation. On the other hand, humoral response consists on the activation and release of molecules stored within hemocytes, such as anticoagulant proteins, agglutinins, phenoloxidase enzymes, antimicrobial peptides, pattern recognition proteins, lectins, toll recepAQUAFEED::ADVANCES IN PROCESSING & FORMULATION from Aquafeed.com

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29 tors, protease inhibitors, etc.

The first organ involved in crustacean immune system is the cuticle, it works as the first physical barrier, and it contains antimicrobial substances.

If the pathogens cross the outer defense, hemocytes participate in the inactivation of invading organisms, and regulate different physiological functions i.e., exoskeleton hardening, cuticle damage healing, coagulation, carbohydrate metabolism, and protein/amino acid transportation and storage.

Three different types of hemocytes are present in the shrimp: hyaline, semigranular, and granular.

Hyalinocytes (5-15% of circulating hemocytes or CE) are small non-refractive cells, with a small nucleus relative to their cytoplasm, which have few or no cytoplasmic granules. The primary role of these cells is related to clotting and phagocytosis.

Fig1: Light microscopy of hemocytes from spiny lobster, Panulirus argus: Hyalinocytes (H), semigranulocytes (SG), and granulocytes (G). Scale bar=10µm (C. Li, J.D. Shields / Journal of Invertebrate Pathology 94 (2007) 48–55)

pigment responsible (among other processes) for inactivating foreign particles, and preventing their spread throughout the host body, as well in as for healing cuticle damages.

“ Many different additives exist the market claiming to have an immunostimulant effect. In the FAO Fisheries and Aquaculture Report No. 1053, probiotics were recommended as a tool in the control of AHPNS ...”

Granulocytes (10-20% of CE) have the smallest nucleus and a high number of cytoplasmic granules. Granulocytes display phagocytic activity and store enzymes as prophenoloxidase (proPo), lysozyme, phosphatase, esterase, phospholipase, peroxidase, protease, etc. These cells may be stimulated by glucans, peptidoglycans and lipopolysaccharides to provoke exocytosis and enzyme release.

Activation of the proPo system results in the production of melanin, a dark-brown

Semi-granulocytes (75% CE) have a large numbers of small granules similar to vertebrate granulocytes.

These cells posses β-1,3-glucans receptors and their principal function involves phagocytosis, encapsulation and clotting.

The modulation of the shrimp immunology is a key element in establishing strategies for the control of diseases in shrimp aquaculture.

Several different methods exist to evaluate the immune system status such as hemolymph protein concentration, total hemocyte counts, phenoloxidase activity, free radical production, phagocytic activity, etc.

Many different additives exist in the market claiming to have an immunostimulant effect.

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In the FAO Fisheries and Aquaculture Report No. 1053, probiotics were recommended as a tool in the control of AHPNS, a devastating penaeid disease causing massive mortalities of cultivated shrimp all over the globe.

Probiotics are known to have a positive impact on vertebrates’ immune system.

To elucidate, if the use of a commercial probiotic ECOBIOL AQUA® (Bacillus amyloliquefaciens CECT 5940) a study was conducted by the Federal University of Rio Grande, Brazil.

The study was conducted in 12 tanks of 1,000 L, in a greenhouse at the Estação Marinha de Aquacultura (Aquaculture research Center), belonging to the Federal University of Rio Grande (FURG), in the town of Rio Grande-RS-Brazil.

Liptopenaeus vannamei shrimp juveniles (2 g) were used. The tanks were prepared with biofloc from other shrimp farms, with a density of 300 individuals per m3. Shrimp were randomly distributed to four different treatments, with

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30 three replicates each, the probiotic was applied directly to water. The probiotic dose varied in each treatment: 0, 20, 40 and 80 g in 1,000 L of water per week (CO, TA, TB and TC). The probiotic application was performed daily.

CONTROL

TA

TB

TC

P

TPC (mg/ml)

104 ± 7a

128 ± 4b

128 ± 4 b

124 ± 6 b

<0.05

HG (%)

51 ± 7a

79 ± 5 b

81 ± 5 b

67 ± 5 b

<0.05

Apoptotic cells/5µl

3 ± 1a

1 ± 1b

1 ± 1b

2 ± 1b

<0.05

gen, can regulate the binding of pathogens to hemocytes, thereby controlling its phagocytic capacity. Therefore a higher TPC is related to a higher capacity to deTotal protein in hemolymph (TPC) was tect and control pathogens. The higher higher in probiotic treated shrimp. Also granulocyte percentage also indicates a granulocyte percentage (HG) was higher in better prepared immune system as these ECOBIOL AQUA® treated animals. are responsible for phagocytic and prophenoloxidase activity.

Additionally, apoptosis index, a way to evaluate infection, was higher in the con- In conclusion, the addition of ECOBIOL trol group.

AQUA® helped shrimp’s immune system

AFΩ

Significant differences among treatments were found in all immune system indicators.

to be better prepared to fight a potential infection. These results are consistent The proteins present in the hemolymph, with other effects observed in fish (Tilapia) are the mechanism by which the immune as an increased number of intraepithelial system identifies shrimp pathogens, these lymphocytes or higher concentration of in addition to identify the type of patholysozyme in blood.

Results:

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More information

Àlvaro Ortiz, Product Manager, Norel E: info@norel.net

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