Abstract
A novel viral responsive protein, namely hemocyte homeostasis-associated protein (HHAP), was characterized for its role in the response of shrimp to white spot syndrome virus infection. The full-length cDNAs of HHAP from the black tiger shrimp (PmHHAP), Penaeus monodon, and the fresh water crayfish (PlHHAP), Pacifastacus leniusculus, were obtained and showed high sequence identity to a hypothetical protein from various organisms, with the highest identity to the hypothetical protein TcasGA2_TC006773 from the red flour beetle, Tribolium castaneum (54% amino acid sequence identity). Transcripts of PmHHAP were expressed in various shrimp tissues with the highest expression in hematopoietic tissue, whereas the transcripts of PlHHAP were found in the hematopoietic and nerve tissues. Upon white spot syndrome virus infection, a high up-regulation level of shrimp hemocytic HHAP mRNA and protein was observed by real-time reverse transcription-PCR and immunofluorescence microscopy, respectively. Gene silencing of PmHHAP by RNA interference resulted in a significant decrease in the number of circulating hemocytes and 100% shrimp mortality within 30 h of the double-stranded PmHHAP RNA injection (but not in control shrimp), indicating that HHAP is essential for shrimp survival. Interestingly, severe damage of hemocytes was observed in vivo in the PmHHAP knockdown shrimp and in vitro in shrimp primary hemocyte cell culture, suggesting that PmHHAP plays an important role in hemocyte homeostasis. Thus, it is speculated that the up-regulation of PmHHAP is an important mechanism to control circulating hemocyte levels in crustaceans during viral infection.