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MPO is a cationic leukocyte peroxidase that plays an important role in the host defense system. It has been cloned in zebrafish (Danio rerio), turbot, channel catfish (Ictalurus puncyatus), rock bream (Oplegnathus fasciatus) and crucian carp (Carassius auratus gibelio) (15-22). Despite these findings, the function and evolution of MPO remains largely unknown.

The aim of this study is to identify the gene and deduced protein sequences of MPO in flounder Paralichthys olivaceus. MPO expression was analyzed by quantitative real-time PCR in peritoneal leukocytes, head kidney leukocytes, gill, skin, and muscle cells of flounder. PoMPO was found to be expressed in MHCII+ macrophages and GCSFR+ neutrophils. The gene encoding PoMPO was identified by a 2313 bp open reading frame and predicted to encode a protein with 770 amino acids. Compared with MPO from other fish species, the deduced protein sequence of flounder MPO formed a distinct clade and shared highest similarity with turbot MPO (Figure 1 ).

Flow cytometric analysis showed that PoMPO was mainly expressed in peritoneal leukocytes and head kidney cell (Figure 2 ). HKLs isolated from the peritoneal cavity and head kidney were stimulated with PMA to induce the formation of extracellular traps (ETs). The presence of MPO in ETs was confirmed by the entrapment of E. coli and S. aureus (Figure 3 ). The entrapped bacteria were quantified at different time points after PMA stimulation. Interestingly, the viability of the entrapped bacteria was significantly decreased in the presence of anti-PoMPO antibodies (Figure 4 ).

In order to determine whether PoMPO contributes to the antimicrobial effect of ETs, HKLs incubated with or without anti-rPoMPO Abs were incubated with E. coli, E. tarda or S. aureus for 0 h, 2 h, 4 h and 8 h. The enzymatic activity of MPO was measured with an MPO colorimetric assay kit (Nanjing Jiancheng Co., Ltd, China). PoMPO exhibited an activity that was reduced in the presence of anti-PoMPO Abs. https://heylink.me/mpo99bet suggests that PoMPO contributes to the formation of ETs and has an antimicrobial activity. This is consistent with the results of other studies on the effect of MPO on extracellular trap formation (26-28). Moreover, a high activity of PoMPO in ETs may be associated with the rapid bacterial entrapment and killing.