Control of Bacterial Infections in the Hard Tick Dermacentor variabilis (Acari: Ixodidae): Evidence for the Existence of Antimicrobial Proteins in Tick Hemolymph ROBERT JOHNS, DANIEL E. SONENSHINE, AND WAYNE L. HYNES Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529 J. Med. Entomol. 35(4): 458-464 (1998) ABSTRACT The ability of hard ticks to prevent infection by bacteria was investigated. During a 72-h period, virtually all Dermacentor variabilis females survived inoculation with Bacillus subtilis, Escherichia coli, and Staphyloccocus aureus but few survived infection with Pseudomonas aeruginosa. The hemocyte population increased to peak abundance at 48 h to «*6 times that of the uninfected controls. In contrast, the soluble hemolymph protein content decreased inversely as the hemocytes increased. D. variabilis hemolymph was found to be constitutively antimicrobial (i.e., hemolymph from bacteria-naive individuals inhibited bacterial growth). Infection with various bacterial species enhanced this innate capability. When hemolymph fractions separated by high-pressure liquid chromatography were tested for their ability to inhibit microbial growth, activity against the gram-positive bacterium, B. subtilis, was found in 2 polar fractions. Antimicrobial activity was lost when the fractions were incubated with protease. The least polar fraction contained 1 major protein, M r 14.5 kDa, that comigrated with human lysozyme. This protein, tentatively identified as tick lysozyme, was abundant in bacteria-naive ticks but increased greatly (43%) following challenge with B. subtilis. The identity of the other, more polar protein is unknown. Studies to characterize these antimicrobial proteins are planned. KEY WORDS Dermacentor variabilis, Bacillus subtilis, antimicrobial proteins, hemolymph, phago- cytosis, lysozyme THE ABILITY OF arthropods to control bacterial infec- tions is well known (Gillespie et al. 1997). Insects respond to microbial challenge by phagocytosis, en- capsulation, secretion of lectins and lysozyme, or the production of inducible antimicrobial polypeptides and proteins. The latter may exhibit bacteriocidal/ bacteriostatic activity against either gram-positive or gram-negative bacteria or both. These antibacterial peptides have been categorized into several major groupings, among the most important of which are the defensins, cercropins, attacins, and sarcotoxins (Hoff- man and Hetru 1992, Lowenberger et al. 1995, Co- ciancich et al. 1994). Other proteins that play an im- portant role in insect defense against bacteria are constitutive (e.g., lysozymes and lectins). Defensins and cercropins are small peptides ( Sa! 4 kDa) that at- tack gram-positive bacteria (defensins) or both gram- positive and gram-negative bacteria (cercropins). At- tacins and sarcotoxins are large polypeptides (>20 kDa) that are bacteriostatic rather than bacteriocidal. Many other species-specific inducible immune pro- teins are also known in insects. In addition to the inducible proteins, lysozymes and lectins are also im- portant in the immune response against invading or- ganisms. Lysozymes (=14 kDa) may be secreted by the fat body (Gillepsie et al. 1997) or the hemocytes (Cheng et al. 1979) following bacterial challenge. Ly- sozymes lyse gram-positive bacteria by hydrolyzing the peptidoglycan layer of their cell walls. Lectins (hemagglutinins), which are much larger proteins (•==70-1,500 kDa) (Gillespie et al. 1997) recognize nonself molecules and bind sugars, such as sialic acid. This enables hemocytes to phagocytize bacteria. Lec- tins also facilitate opsonization and cytolysis (Grub- hofferetal. 1991). Much less is known regarding the defense mecha- nisms that ticks use to prevent bacterial infection. According to Zhioua et al. (1996), hemocytes "play a dominant role in the tick immune response." As in insects, phagocytosis is accomplished by plasmato- cytes (Binnington and Obenchain 1981, Kuhn and Haug 1994), whereas encapsulation of larger organ- isms is done by granulocytes (Eggenberger et al. 1990). Podboronov (1991) reported an increase in phagocytosis within 2 h after introduction of bacteria into ticks, although this claim was not supported with numerical data. In the same report, he also reported evidence of lysozymes from different species of ticks, ranging in molecular weight from 13.8 to 15 kDa, but did not indicate whether the enzyme was an intracel- lular component of the hemocytes or free in the he- molymph plasma. Lectins similar to those reported from insects have been reported from ticks (Grub- hoffer et al. 1991). No evidence of defensin or cer- cropin-like peptides in ticks has been reported. A better understanding of tick antimicrobial defense is Downloaded from https://academic.oup.com/jme/article/35/4/458/860453 by guest on 09 January 2023