Abstract
Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity.
Highlights
Like other crustaceans, shrimp rely on innate immunity in which circulating hemocytes play an important role in defending against microbial invasion [1]
Normal cells have fully intact nuclei, cells proceeding to necrosis have lysed nuclei, and dead cells have no apparent nuclei or DNA
The percentage of cell necrosis in hemocytes incubated with LPS, bG, or PG increased directly with dosage and was significantly higher than in hemocytes incubated with shrimp salt solution (SSS) (Fig. 1E)
Summary
Shrimp rely on innate immunity in which circulating hemocytes play an important role in defending against microbial invasion [1]. Hemocytes are involved in cellular immune reactions like phagocytosis, nodule formation, and encapsulation and are involved in humoral immune reactions like the prophenoloxidase (proPO) cascade and releases of antimicrobial peptide and lysozymes [4]. HCs and SGCs are involved in phagocytosis, during which a respiratory burst occurs and leads to the release of the superoxide anion and other reactive oxygen species (ROS) that plays a crucial role in microbicidal activity [9,10,11]. The proPO activating system and phagocytosis are important immune responses in crustaceans [2, 3]. Several immune molecules and their functions in shrimp immunity have been recently reported [12]
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