## 4 Discussion
As mentioned in previous studies, when common carp (_Cyprinus carpio_ L.) was injected intraperitoneally with _V.__anguillarum_, the expression level of LEAP-2A was quickly upregulated in the liver, spleen, head kidney, skin, gills, foregut, and hindgut, while the expression level of LEAP-2B was similarly upregulated in the spleen, skin, gills, and hindgut, but not in the liver, head kidney, and foregut [49]. As Antarctic fishes live in a cold environment below 2 \({}^{\circ}\)C [50], and AMPs from Antarctic ice-fish have been known to show temperature-dependent antibacterial activity [51], we investigated the effect of low temperature on the antibacterial activity of LEAPs from Antarctic notothenioid and eelout. We could identify effective antibacterial activity at low temperatures. With MIC values of 0.25 \(\upmu\)M at 5 \({}^{\circ}\)C and 1 \(\upmu\)M at 15 \({}^{\circ}\)C, nLEAP-2 and eLEAP-2 were more effective than nLEAP-1 and eLEAP-1, respectively. To investigate the possible mechanism behind the antibacterial activity, we performed the SYTOX green uptake assay using _S. aureus_[5; 27; 31]. The LEAPs showed an initial increase, followed by an immediate decrease in the fluorescence intensity of SYTOX green dye, to a level below that of the negative control [5]. As described in a previous study, the trout hepcidin inhibits the bacterial cell growth but does not show cell permeabilization. Further confocal microscopic analysis of trout hepcidin had revealed the intracellular localization of hepcidin peptides [5]. In other studies, human \(\beta\)-defensin peptide HBD4 does not permeabilize the model lipid membranes but damage the inner membrane of _E. coli_ cells, and showed the SYTOX green dye accumulation inside the cells by confocal microscopy studies [52]. Therefore, it is possible that LEAPs permeabilized the cells without causing lysis [5]. Another possibility is that LEAPs have a higher DNA binding affinity than the SYTOX green dye. Therefore, initially, the SYTOX green dye bound to DNA, leading to an increase in fluorescence intensity, after which, the LEAPs replace the DNA-bound SYTOX green dye, resulting in a decrease in fluorescence intensity. In previous studies, a similar quenching effect was noticed in the fluorescent intercalator displacement assay [53]. In this assay, the EB binds to DNA, thereby leading to an increase in fluorescence. However, the addition of a DNA-binding compound displaces the DNA-bound fluorescent dye, resulting in a decrease in the fluorescence signal [53]. We performed the competitive displacement assay to investigate whether LEAPs displace the SYTOX green dye from the DNA. It has been shown that cationic AMPs work by entering the cells and interfering with the metabolic functions, hampering the intracellular activities such as DNA and protein synthesis, post-translational folding, enzymatic activity, and cell wall synthesis [54]. LEAPs are \(\beta\)-sheet peptides which may form ion channels in lipid bilayers through aggregation. A short beta-sheet peptide forms porin-like voltage-gated channels in lipid bilayers [55]. Some studies show that the beta-hairpin form barrel-like structures in the lipid bilayer responsible for the peptides lytic action [56].The cationic antimicrobial peptides such as cecropin A2 [57] (+10), buforin II [58] (+7), human lactoferrin1-11 [59] (+4), P7 [60] (+7), APP [61] (+5), human \(\alpha\) defensin 5 [62](+4), Human Neutrophil Peptide-I [63] (+3), and CP-1 [64] (+3) show DNA binding as a mechanism of action, as revealed by gel retardation assays and competitive EB displacement assays. Cecropin A2 [57], an \(\alpha\)-helix peptide with 36 amino acids residues, isolated from mosquito _Aedes aegypti_, has been shown to inhibit DNA migration at a concentration 128 \(\upmu\)M. Similarly, buforin II [58], a random coil peptide with 21 amino acid residues, derived from a natural peptide isolated from Asian load, inhibits DNA migration at a DNA: peptide ratio of 1:50.