Characterisation of Some Immune Genes in the Black Tiger Shrimp, Penaeus monodon

Sammanfattning: The molecular mechanisms of the immune system in shrimp, Penaeus monodon, are completely unknown, despite its economic importance as an aquaculture species, especially in Asia and Latin America. The genes and their gene products involved in the prophenoloxidase activating system, which is considered to be a non-self recognition and defence system in many invertebrates, have been isolated and characterised in shrimp. These include a zymogen of this cascade, prophenoloxidase (proPO); a cell adhesion protein, peroxinectin and a pattern recognition protein, β-1,3-glucan binding protein (GBP). All proteins are synthesised in shrimp hemocytes, not in the hepatopancreas. The shrimp proPO cDNA clone has 3,002 bp and contains an open reading frame of 2,121 bp encoding a putative polypeptide of 688 amino acids, with a molecular mass of 78.7 kDa. Comparison of amino acids sequences showed that this shrimp proPO was more closely to that of another crustacean, the freshwater crayfish, Pacifastacus leniusculus, than to insect proPOs. Upon activation of the proPO system in shrimp, a cell adhesion activity in the hemolymph is generated. Inhibition of adhesion by an antiserum against the crayfish cell adhesion protein, peroxinectin, revealed that the cell adhesion activity detected in shrimp hemolymph might result from a peroxinectin in shrimp. Indeed, a cDNA clone which encoded shrimp peroxinectin was isolated with an open reading frame of 2,337 bp encoding a putative protein of 778 amino acids including a signal peptide. Two putative integrin-binding motifs (RGD and KGD) are present suggesting that integrin is involved in the adhesion activity. The peroxinectin transcript was slightly reduced in shrimp injected with a β-1,3-glucan or laminarin. Also found in shrimp hemolymph was a 31 kDa-GBP that could bind to β-1,3-glucan polymers such as curdlan and zymosan, but not to LPS. The cDNA sequence of shrimp GBP showed high similarity to that of crayfish LGBP, other insect recognition proteins as well as bacterial and sea urchin glucanases. Shrimp injected with an insoluble β-1,3-glucan, curdlan or heat-killled Vibrio harveyi did not show any significant changes in relevant mRNA levels. An attempt to knock out the LGBP expression by its exogeneous dsRNA was done in a proliferating blood cell culture from the hematopoietic tissue of crayfish. We found that the expression of endogeneous LGBP mRNA could be substantially inhibited by incubation of dsRNA-LGBP in the cell culture. The effect is quick, specific, and also affects the cell behaviours.