Journal of Invertebrate Pathology 94 (2007) 108–118 www.elsevier.com/locate/yjipa 0022-2011/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2006.09.004 Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) Michael Abt, David B. Rivers ¤ Department of Biology, Loyola College, 4501 North Charles Street, Baltimore, MD 21210, USA Received 6 June 2006; accepted 6 September 2006 Available online 19 October 2006 Abstract Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modiWed dot blot analysis approach in which venom samples were applied to nylon membranes and incu- bated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider’s insect medium con- taining 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the Xy host, Sarcophaga bullata, was exposed to tyrosine. Only Xy larval plasma induced an enzyme reaction with the Schneider’s insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylXuoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest eVect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS– PAGE separation of crude venom followed by in-gel staining using L-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the impor- tance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the eVects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less eVective in aVording protection. These observations collectively argue that wasp venom PO is unique from that of the Xy hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.  2006 Elsevier Inc. All rights reserved. Keywords: Phenoloxidases; Immunosuppression; Wasp toxins; Hemocytes; Host manipulation; Cytotoxicity 1. Introduction Parasitic wasps produce secretions that possess a wealth of biologically active compounds that function- ally aid in conditioning the host for the beneWt of devel- oping wasp progeny. Ectoparasitic species appear to rely predominantly on venoms derived from the mother (Dey- rup and Matthews, 2003; Nakamatsu and Tanaka, 2003; Quicke, 1997; Rivers et al., 2005), although in some cases, regulatory factors are released from salivary glands of larvae (Doury et al., 1997; Hartzer et al., 2005; Richards and Edwards, 2000). Regardless of the source of ectopar- asitoid secretions, all seem to contribute either alone or in combination in the manipulation (e.g., alter behavior, change physiology, delay or halt development, suppress immunoresponses, and/or elicit death) of susceptible host insects (Jervis and Copeland, 1996; Quicke, 1997). Identi- Wcation of components in ectoparasitoid venoms that are responsible for host conditioning remains elusive, and has limited our understanding of the modes of action of * Corresponding author. Fax: +1 410 617 5682. E-mail address: drivers@loyola.edu (D.B. Rivers).