1 Antibacterial activity by hemolymph defensin from 2 the hard tick Haemaphysalis longicornis 3

Ticks are key vectors of some important diseases of humans and animals. Although they 21 are carriers of disease agents, the viability and development of ticks are not harmed by the infectious 22 agents due to their innate immunity. Antimicrobial peptides directly protect hosts against 23 pathogenic agents such as viruses, bacteria, and parasites. Among the identified and characterized 24 antimicrobial peptides, defensins have been considerably well studied. Defensins, which contain 25 intramolecular disulfide bridges between cysteine residues, are commonly found among fungi, 26 plants, invertebrates, and vertebrates. The sequence of the tick hemolymph defensin (HEdefensin) 27 gene from the hard tick Haemaphysalis longicornis was analyzed after identification and cloning from 28 a cDNA library. HEdefensin has a predicted molecular mass of 8.15 kDa and a theoretical isoelectric 29 point of 9.48. Six cysteine residues were also identified in the amino acids. The synthetic HEdefensin 30 peptide only showed antibacterial activity against Gram-positive bacteria such as Micrococcus luteus. 31 A fluorescence propidium iodide exclusion assay also showed that HEdefensin increased the 32 membrane permeability of M. luteus. Additionally, an indirect fluorescent antibody test showed that 33 HEdefensin binds to M. luteus. These results suggested that HEdefensin strongly affects the innate 34 immunity of ticks against Gram-positive bacteria. 35