ARTHROPOD/HOST INTERACTION,IMMUNITY Antibacterial Hemoglobin Fragments from the Midgut of the Soft Tick, Ornithodoros moubata (Acari: Argasidae) YOSHIRO NAKAJIMA, KAZUMASA OGIHARA, DEMAR TAYLOR, AND MINORU YAMAKAWA 1 Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki, 305-8572, Japan J. Med. Entomol. 40(1): 78Ð81 (2003) ABSTRACT Midgut contents of Ornithodoros moubata showed strong antibacterial activity against Staphylococcus aureus. A combination of reversed-phase chromatography and mass spectrometric analysis was used to isolate two antibacterial peptides from the tick midgut lumen. Partial amino acid sequences by Edman degradation of these two peptides showed they are identical with the 1Ð11 and 3Ð19portionsofrabbit  hemoglobin.Hostrabbit  hemoglobinappearstobecleavedbetweenMet 32 andPhe 33 toproducethesetwoantibacterialpeptides.Isolationofahostbovinehemoglobinfragment with antimicrobial activity has been demonstrated in the Ixodid tick, Boophilus microplus (Fogaca et al. 1999). Similar immune mechanisms in the two major families of ticks, Ixodidae and Argasidae, appear to use the hemoglobin of the host as an antimicrobial agent in midgut defense. KEY WORDS Ornithodoros moubata, midgut, hemoglobin fragment, antibacterial activity TICKS ARE IMPORTANT VECTORS of human and animal diseases, transmitting viruses, bacteria, rickettsia, pro- tozoa, and fungi. Microorganisms transmitted by ticks apparently evade the tick immune system. Therefore, vector immunity is an important factor for under- standing vector-pathogen interactions. Ticks, like other arthropods, have cellular and humoral immune mechanisms against septic injury. Phagocytic activity in Ornithodoros moubata sensuWaltonhemocyteshas been reported by Inoue et al. (2001). Production of reactive oxygen species, a well-known mechanism for microorganism killing by vertebrate phagocytes, has been shown in Boophilus microplus (Canestrini) he- mocytes(Pereiraetal.2001).Borreliacidalactivityhas been demonstrated with hemolymph plasma from Dermacentor variabilis (Say) (Johns et al. 2000, Johns et al. 2001b). Antibacterial peptide defensin and de- fensin-likepeptidewereisolatedfromthehemolymph of D. variabilis (Johns et al. 2001a) and O. moubata (van der Goes van Naters-Yasui et al. 2000), respec- tively. However, little is known about tick defense mechanisms against microorganisms ingested by bloodfeeding. The tick midgut is a highly favorable environment for survival of ingested microorganisms because of the absence of proteolytic enzymes (Sonenshine1991).Recently,lysozymeof O. moubata has been puriÞed from the gut (Kopacek et al. 1999). Our previous studies showed that Ornithodoros de- fensin genes are predominantly expressed in the mid- gutandup-regulatedafterbloodfeeding(Nakajimaet al.2001,Nakajimaetal.2003).Inaddition, Ornithodo- ros defensin A was puriÞed from the midgut lumen (Nakajimaetal.2002).Moreover,Fogacaetal.(1999) reported that the antimicrobial activity present in the gut contents of B. microplus is a fragment of the host bovine  hemoglobin.TheseÞndingsstronglysupport the presence of a midgut defense mechanism against ingested microorganisms in ticks. In this study, we report the puriÞcation of two rabbit  hemoglobin fragments with anti-Staphylococ- cus aureus activity from the midgut lumen of O. mou- bata. This study emphasizes the possibility that ticks usethehosthemoglobinfragmentsasimmunefactors against ingested microorganisms. Materials and Methods Ticks. Softticks, O. moubata sensuWalton(Walton 1962) (Acari: Argasidae), were obtained in 1994 from Dr. Yasuo Chinzei, School of Medicine, Mie Univer- sity,Tsu,Japan,andhavesincebeenmaintainedinour laboratory. Ticks were fed on rabbits (Oryctolagus cunniculus)andmaintainedat30°C  1°C,70%  10% RH, and total darkness. Feeding and rearing of ticks were as described by Chinzei (1983). Extraction and Purification of Antibacterial Pep- tides. The content of midguts from 15 engorged adult females was collected and washed in phosphate-buff- ered saline containing protease inhibitors (Complete; Roche Diagnostics, Tokyo, Japan). Equal volumes of 0.1 M sodium acetate buffer were added, and samples were incubated at 4°C for 1 h to extract the peptides. The acid extract was heated at 100°C for 10 min and centrifuged at 10,000  g for 10 min. The supernatant was concentrated in a vacuum centrifuge, and the 1 InnateImmunityLaboratory,NationalInstituteofAgrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan. 0022-2585/03/0078Ð0081$04.00/0  2003 Entomological Society of America Downloaded from https://academic.oup.com/jme/article/40/1/78/903737 by guest on 02 March 2022