Molecular and functional characterization of an astakine cDNA from the giant freshwater prawn Macrobrachium rosenbergii

Astakine is a cytokine of invertebrates that promotes the proliferation and differentiation of hematopoietic stem cells, which regulates hematopoietic and immune responses. In this study, we have identified an astakine homolog (MrAst) from the freshwater prawn Macrobrachium rosenbergii. The MrAst cDNA consists of 408 bp with a 390 bp open reading frame (ORF) encoding 129 amino acids. MrAst contains a prokineticin domain, wherein the mature peptide consist of 10 conservative cysteine residues. Multiple sequence and phylogenetic analyses revealed that MrAst shares a close evolutionary relationship with astakines from crustaceans. MrAst was highly expressed in the hemocytes and nerve tissues. Both Vibrio parahemolyticus, lipopolysaccharide (LPS), and Decapod iridescent virus 1 (DIV1) infection upregulated MrAst expression in hemocytes and gills. Shrimps were more vulnerable to V. parahemolyticus and DIV1 infection upon MrAst knockdown, resulting in a lower survival rate. Additionally, inhibition of MrAst resulted in a significant reduction in total hemocyte count (THC) and phenoloxidase (PO) activity. However the lysozyme activity was increased and the superoxide dismutase (SOD) activity was unaffected. Moreover, knockdown of MrAst significantly inhibited the expression of hemocyanin, STAT, prophenoloxidase (proPO), crustacean hematopoietic factor (CHF), and Crustin and enhanced the expression of Toll-like receptor (TLR), IMD, Relish, and anti-lipopolysaccharide factor (ALF). The reduction in MrAst also increased the level of hemocytes apoptosis and significantly upregulated the apoptosis-related genes, such as Caspase 9, Caspase 3, p53, cytochrome C (Cyt C), Endonuclease G (Endo-G), and Cathepsin B. The results indicated that MrAst may play an important role in response to V. parahemolyticus and DIV1 infection and involved in the apoptosis process to avoid DNA damage for maintenance of hemocytes homeostasis.