Although defensins have been isolated from a variety of metazoan, their role in cellular immunity has not been answered. In the study, we found that the hemocytes of the Manila clams Ruditapes philippinarum release defensin (designated as Rpdef3) in response to Vibrio parahaemolyticus challenge. The antimicrobial Rpdef3 was proved to be involved in the extracellular traps (ETs) that hemocytes released in response to Vibrio challenge. Scanning electron microscopy observation proved the patterns how ETs eliminate invading bacteria. Furthermore, Rpdef3 involved in ETs had broad-spectrum antimicrobial effect on both Gram-negative bacteria and Gram-positive bacteria. ELISA assay revealed that Rpdef3 could bind lipopolysaccharides and peptidoglycan in a dose-dependent manner. As concerned to the antibacterial mechanisms, Rpdef3 can cause bacterial membrane permeabilization, leading to cell death. As a result, Rpdef3 might contribute to the trap and the elimination of invading Vibrio in clam ETs. Taken together, our study suggest that the formation of ETs is a defense mechanism triggered by bacterial stimulation, coupled with antibacterial defensin.

Fas-associated protein with death domain (FADD) is an essential element in cell death, and also implicates in cell cycle progression, inflammation and innate immunity. In the study, an FADD (designated as RpFADD) was identified and characterized from manila clam, Ruditapes philippinarum. Multiple alignments and phylogenetic analysis strongly suggested that RpFADD was a new member of the FADD family. The RpFADD transcripts were constitutively expressed in a wide range of tissues, and dominantly expressed in hemocytes. After challenged with Vibrio anguillarum or Micrococcus luteus, the expression level of RpFADD transcripts was significantly induced and reached the maximum level at 72 h and 48 h, respectively. Knockdown of RpFADD down-regulated the transcript levels of RpIKK, RpTAK1 and RpNF-κB with the exception of RpIκB. Moreover, RpFADD primarily localized in the cell cytoplasm, and its over-expression promoted the apoptosis of HeLa cells. These results revealed that RpFADD perhaps regulated the NF-κB signaling pathways positively, which provided a better understanding of RpFADD in innate immunity.