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In vivo effect of a natural Aedes aegypti defensin on Brugia pahangi development

Insects synthesize a range of potent peptides and proteins as part of their antimicrobial defence reaction (Cociancich et al., 1994). Insect defensins comprise one family of these inducible molecules. These are small cationic peptides with six disulphidelinked cysteine residues (Lambert et al., 1989), primarily active against Gram-positive bacteria. Insect defensins have been characterized from the mosquito, Aedes aegypti (L.) (Chalk etal., 1994,1995; Lowenberger et al., 1995). One defensin purified from the Refm strain of Ae.aegypti, homozygous for Red Eye and filaria susceptibility (Macdonald & Sheppard, 1965), also appears to be induced in response to Brugiapahangi (Buckley & Edeson) filaria infection. Ham et al. (1994) hypothesized that, if this peptide has a defence role against filaria, it may be localized on the worm surface. Here we present a preliminary report on the in vivo activity of purified Ae.aegypti defensin B, against B.pahangi. Defensin B was induced in the Rejin strain of Ae.aegypti via intrathoracic inoculation of 0.5 pl of a suspension of Escherichia coli, strain D-3 1, in TC199 at an optical density of 0.4 measured spectrophotometrically at 600 nm. Purification was carried out using ultrafiltration and reverse-phase HPLC (Lee & Brey, 1994; Chalk et al., 1995). The biological activity retained in the eluted fractions was evaluated by assaying against living Micrococcus luteus using a zone inhibition plate assay (Smithies, unpublished). The quantity of eluted active peptide was determined using a microassay kit (Pierce, Rochford, Ill., U.S.A.). The level to be administered to infected mosquitoes was determined by comparing SDS PAGE protein staining intensities (Schagger & Von Jagow, 1987) of known levels of the purified peptide, with those of haemolymph from individual filaria-infected mosquitoes. This resulted in an estimate of induced defensin-like peptide per mosquito (containing -2 pl haemolymph) of approximately 200 ng (equivalent to 25 p~). Hence this quantity was used in the experiments with B.pahangi-infected mosquitoes. In vivo assay of defensin B activity against B.pahangi was carried out by membrane-feeding 34-day-old mosquitoes a