In this study, we provided the full-length sequence of the aP2X7R gene. The deduced protein possessed several common features shared by other P2X7R homologues 
. Sequence comparison and phylogenetic tree analysis also confirmed aP2X7R as a distinct member of the fish purinergic subtype receptor. These data indicated that aP2X7R is homologue to mammalian P2X7R and may play a role in ayu innate immunity. Multiple alignment of fish, amphibian (clawed frog), and mammalian P2X7R sequences revealed a high level of conservation of the P2X7R family signature, the two transmembrane domains and the long C-terminal domain. Five residues important for nucleotide binding in mammalian P2X7R were also present in a conserved position in the fish and amphibian proteins. The conserved structures of P2X7R in evolution indicated that P2X7R may play important roles in both fish and mammals.
P2X7R has a ubiquitous tissue distribution, and its expression levels may vary over orders of magnitude in mammals 
. It is predominantly expressed in immune cells from myeloid lineages, such as macrophages, monocytes, and dendritic cells 
. The head kidney, thymus, spleen, and mucosa-associated lymphoid tissues are known to be the major lymphoid tissues in teleost fish 
. In this study, aP2X7R transcripts were widely distributed in ayu tissues and were especially abundant in the head kidney, spleen, and liver. Similarly, aP2X7R mRNA was abundant in ayu head kidney-derived macrophages. These results suggest that aP2X7R is also mainly expressed in immune organs and cells. Mammalian P2X7R has been found upregulated after infection 
. A significant increase in the expression of P2X7R has been observed on human macrophages infected with M. tuberculosis
or mouse macrophages infected with Leishmania amazonensis
. A significant release of ATP has also been detected by M. tuberculosis
-infected macrophages 
. In our study, aP2X7R was upregulated in all examined ayu tissues after bacterial infection, suggesting that aP2X7R is implicated in ayu infection response.
In mammals, it has been demonstrated that prolonged stimulation of macrophages using high concentrations of ATP causes cell death via P2X7R-induced apoptosis 
. Cell death by ATP stimulation proceeds through apoptotic nuclear alterations, such as chromatin condensation and DNA fragmentation, but not cytolytic or membrane damage and occurs concomitant with a decrease in bacterial viability 
. However, it is still unclear whether ATP activates P2X7R to induce cell death in fish. In the current study, ayu macrophage death was observed following treatment with ATP. The EC50
concentration of ATP needed to induce aP2X7R-dependent cell death was 1.5 mM, which seemed higher than the concentration of ATP required in their mammalian counterparts 
. We also found that ATP-induced cell death was suppressed after aP2X7R blockage via RNAi, aEPAb, and oATP. Our data suggest ATP activates aP2X7R to induce the macrophage cell death in ayu, a teleost.
P2X7R has also been reported to be involved in phagocytosis and clearance of bacteria by macrophages 
. Phagocytosis is inhibited after ATP dissociates myosin IIA from P2X7R complex 
. However, in the absence of ATP, P2X7R may function in phagocytosis 
. Furthermore, the mechanisms underlying P2X7R and phagocytosis in the absence of ATP have also been defined 
. A peptide mimicking the extracellular domain of P2X7R can bind phagocytosed particles, suggesting that P2X7R mediates phagocytosis via direct recognition of the particles 
. In the present study, we observed that siRNA, specially designed to knockdown the expression of aP2X7R, aEPAb (the anti-aP2X7R extracellular domain antibody), and oATP (a P2X7R antagonist), could significantly attenuate the phagocytic activity of ayu macrophages. Therefore, aP2X7R may mediate phagocytosis as a scavenger receptor in ayu macrophages.
After infection, intracellular bacterial viability is reduced after ATP is released into the extracellular media 
. Here, we found that the survival of bacteria was downregulated in ATP induced macrophages compared with negative control. Moreover, the number of CFUs was confirmed to be reduced after the cells were treated with ATP for different times. This result suggests that bacteria may actually be killed in macrophages treated with ATP rather than just exhibit halted replication. As shown in multiple studies, P2X7R mediates this process of ATP-induced bacterial killing of macrophages 
_ENREF_30. Many studies have also suggested that killing of bacteria and other pathogens via the P2X7R-mediate pathway is independent of nitric oxide (NO) 
. The death of host cells may explain this method of bacterial killing 
. We also found that ATP induced cell death in ayu macrophages, suggesting that ATP-induced bacterial killing may also result from cell death. Furthermore, ATP-induced bacterial killing was inhibited by aP2X7R knockdown by siRNA or blockage by aEPAb and oATP. Thus, we speculate that aP2X7R may be involved in bacterial killing in ayu macrophages.
In conclusion, we found that aP2X7R was mainly distributed in immune tissues of the ayu. Moreover, upon bacterial challenge, aP2X7R mRNA was significantly upregulated in all tested tissues. The mRNA and protein levels of aP2X7R were also significantly increased in macrophages in response to bacterial infection. In the seabream, a teleost fish, activation of P2X7R regulates phosphatidylserine externalization and cell permeabilization, but fails to induce IL-1β release in leukocytes 
. However, the function of P2X7R in other fish species remains poorly understood. Our data further demonstrate that aP2X7R may regulate cell death, phagocytosis, and bacterial killing in ayu macrophages in response to bacterial infection, suggesting a conserved function for P2X7R in macrophage modulation.