For 24-well culture plates, 4 mL of PULSin, 100 mL 20 mM Hepes and 1 mg rCqTRP1 were used to prepare mixture of solution. For 96-well culture plates, 1 mL PULSin, 20 mL 20 mM Hepes and 0.3 mg of rCqTRP1 were used.

The beta-thymosins are a group of structurally related, highly conserved intracellular small peptides in vertebrates with various biological functions, including cytoskeletal remodeling, neuronal development, cell migration, cell survival, tissue repair and inhibition of inflammation. In contrast to vertebrates, the function of beta-thymosin is not fully understood in crustaceans. Previously, we found that a thymosin-repeated protein1 (CqTRP1) gene was up-regulated after white spot syndrome virus (WSSV) challenge in hematopoietic tissue (Hpt) cells from the red claw crayfish Cherax quadricarinatus. To further identify the effect of CqTRP1 on WSSV infection, a full length cDNA sequence of beta-thymosin homologue was cloned and analyzed from red claw crayfish followed by functional study. The CqTRP1 cDNA contains an open reading frame of 387 nucleotides encoding a protein of 129 amino acids with a putative molecular mass of 14.3kDa. The amino acid sequence showed high identity with other beta-thymosins and contained three characteristic thymosin beta actin-binding motifs, suggesting that CqTRP1 was a member of the beta-thymosin family. Tissue distribution analysis revealed a ubiquitous presence of CqTRP1 in all the examined tissues with the highest expression in hemocytes, Hpt and gonad at the transcriptional level. Interestingly, the gene silencing of endogenous CqTRP1 by RNAi enhanced the WSSV replication in Hpt cells. Meanwhile, the WSSV replication was significantly reduced in the Hpt cell cultures if overloaded with a recombinant CqTRP1. Taken together, these data clearly indicated that CqTRP1 was likely to be associated with the anti-WSSV response in a crustacean C. quadricarinatus, which provides new strategy against white spot disease in crustacean aquaculture.