Insect Biochemistry and Molecular Biology 32 (2002) 559–566 www.elsevier.com/locate/ibmb Isolation of a protein lethal to the endoparasitoid Cotesia kariyai from entomopoxvirus-infected larvae of Mythimna separata Shohei Okuno * , Madoka Nakai, Tsuyoshi Hiraoka, Yasuhisa Kunimi Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Received 31 May 2001; received in revised form 27 August 2001; accepted 30 August 2001 Abstract Virion-free plasma from entomopoxvirus (MyseEPV)-infected larvae of the armyworm, Mythimna separata, contains a factor that adversely affects the survival of the gregarious braconid endoparasitoid, Cotesia kariyai. Heating or proteinase K treatment eliminates the toxic effect of virion-free plasma on the parasitoid, suggesting that the lethal factor is a protein. We purified the Protein Lethal to C. kariyai larvae (PLCK) from the virion-free plasma of MyseEPV-infected M. separata larvae by a three-step procedure using gel filtration and cation-exchange chromatography. Toxic activity was measured using an in vitro-cultured parasitoid bioassay. Parasitoid larvae cultured in IPL-41 medium (Weiss et al., In vitro 17 (1981), 495) containing 4.7 μg/ml purified PLCK shrank and died within 3 days. The molecular weight of PLCK was estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to be about 28 000, under both reducing and non-reducing conditions, indicating that in its native form the protein is a single 28-kDa polypeptide. Western blot analysis indicated that the lethal protein is not present in the hemolymph of uninfected host larvae, but is induced in the hemolymph by infection with MyseEPV. Western blot analysis also indicated that the proteins of virions and occlusion bodies of MyseEPV are not serologically related to PLCK.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Mythimna separata; Cotesia kariyai; Entomopoxvirus; Toxic protein; Host–parasitoid–virus interaction; In vitro culture 1. Introduction Endoparasitic wasps have evolved a variety of stra- tegies for evading host immune responses. Those that are not encapsulated by the circulating hemocytes of their common host usually complete development success- fully (Quicke, 1997). However, several authors have reported that immature parasitoids often perish if their hosts are infected with a virus. If the host insect dies prematurely due to viral infection, the parasitoids are unable to complete development and die (Brooks, 1993; Nakai et al., 1997; Nakai and Kunimi, 1998). Alterna- tively, some baculoviruses and an entomopoxvirus induce the infected host to produce a substance that adversely affects the development and survival of the * Corresponding author. (c/o Prof. Y. Kunimi). Fax: +81-423-67- 5696. E-mail address: sokuno@cc.tuat.ac.jp (S. Okuno). 0965-1748/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII:S0965-1748(01)00135-7 parasitoids (Brooks, 1993; Kyei-Poku and Kunimi, 1998; Kunimi et al., 1999). The gregarious braconid endoparasitoid, Cotesia kari- yai, is the predominant larval parasitoid of Mythimna separata (Sato et al., 1983). The parasitoids develop in the host for 10–12 days and then emerge from the host as third instar larvae. However, when female C. kariyai parasitize entomopoxvirus (MyseEPV)-infected M. separata larvae, the parasitoid progeny never emerge from the host, dying within it (Kyei-Poku and Kunimi, 1998). Virion-free plasma from MyseEPV-infected host larvae is lethal to parasitoid larvae when injected intrah- emocoelically into the host larvae, suggesting that the hemolymph of the MyseEPV-infected host contains factor(s) causing parasitoid death (Kyei-Poku and Kun- imi, 1998). In previous studies, we showed that C. kariyai embryos and larvae die when cultured in vitro with virion-free plasma from MyseEPV-infected M. separata larvae (Okuno et al., 2000). Since the parasitoids could not have been killed by the immune reaction of the host,