Effects of Simultaneous Expression of Two Sodium Channel Toxin Genes on the Properties of Baculoviruses as Biopesticides Grigori G. Prikhod’ko,* Holly J. R. Popham,* Thomas J. Felcetto,† Dan A. Ostlind,† Vivien A. Warren,† McHardy M. Smith,† Victor M. Garsky,‡ Jeffrey W. Warmke,† Charles J. Cohen,† and Lois K. Miller* ,1 *Departments of Entomology and Genetics, The University of Georgia, Athens, Georgia 30602; †Merck Research Laboratories, Rahway, New Jersey 07065; and ‡West Point, Pennsylvania 19486 Received August 7, 1997; accepted December 1, 1997 Previously, we have described the properties of re- combinant baculoviruses expressing three chimeric genes, mag4, sat2, and ssh1, that encode secretable insect selective sodium channel toxins, -Aga-IV from the spider Agelenopsis aperta, As II from the sea anemone Anemonia sulcata, and Sh I from the sea anemone Stichadactyla helianthus, respectively. We now show that -Aga-IV and As II act at distinct sites on voltage-sensitive sodium channels of insects and synergistically promote channel opening. We also show that these toxins have synergistic insecticidal activity against the blowfly Lucilia sericata and the fall army- worm Spodoptera frugiperda. To determine if toxin synergy also occurs in the context of virus replication, we inserted the chimeric toxin genes into nonessential sites of the Autographa californica nuclear polyhedro- sis virus (AcMNPV) genome under the control of either a modified promoter, P synXIV , or an insect derived pro- moter, P hsp70 . Comparative analyses showed that vi- ruses expressing toxin genes under the control of the P hsp70 promoter were more effective as biopesticides than under the control of P synXIV . Two toxins, -Aga-IV and As II, exerted the most potent effects in S. frugi- perda and Trichoplusia ni larvae, respectively. A virus simultaneously expressing two P hsp70 -promoted toxin genes, mag4 and sat2, exhibited properties similar to the two viruses expressing each of the toxin genes individually except that larval feeding time (FT 50 ) was reduced an additional 10%, indicating a small advan- tage to coproducing synergistic toxins.  1998 Academic Press INTRODUCTION Baculoviruses have long been considered potentially useful microbial agents for insect pest control (Stein- haus, 1949; Ignoffo, 1968). Members of this virus family exhibit a high degree of host specificity and thus provide a means of controlling pest species with mini- mum disruption of the natural balance of beneficial predators and parasitoids in the field (Gro ¨ner, 1986; Huber, 1986). However, industrial interest in pursuing development of baculoviruses as commercially avail- able pesticide products was low, owing in part to the slow rate at which baculoviruses kill their hosts com- pared to chemical pesticides. Indeed, under some cir- cumstances, baculovirus infection can actually increase the feeding damage incurred by the pest population immediately following biopesticide application (O’Reilly and Miller, 1991). Thus, there has been an interest in finding means of improving the rate at which baculovi- ruses stop the feeding of and/or kill their insect hosts (Maeda et al., 1991; McCutchen et al., 1991; O’Reilly and Miller, 1991; Stewart et al., 1991; Tomalski and Miller, 1991, 1992; reviewed by Black et al., 1997). Incorporation of an insect-selective neurotoxin gene into the genome of the baculovirus Autographa califor- nica nuclear polyhedrosis virus (AcMNPV) can dramati- cally reduce the time required for the virus to kill or otherwise debilitate its insect host and thereby makes the virus more attractive for development as a biopesti- cide (Black et al., 1997). The speed with which a toxin-expressing recombinant can reduce the time re- quired to kill or paralyze its insect host depends on the nature of the toxin, the type of promoter used to drive toxin gene transcription, and the insect species (Lu et al., 1996; Prikhod’ko et al., 1996). The hsp70 promoter of Drosophila melanogaster Meigen, P hsp70 , was found to be a particularly effective promoter for expression of the straw itch mite toxin gene, tox34, compared to the strong, very late, viral promoter, P synXIV (Lu et al., 1996). We have previously described the effects of express- ing three P synXIV -promoted toxin genes, mag4, sat2, and ssh1, that encode secretable and potent neurotoxins, μ-Aga-IV from the spider Agelenopsis aperta Gertsch, 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (706)542-2279. E-mail: miller@bscr.cc.uga.edu. BIOLOGICAL CONTROL 12, 66–78 (1998) ARTICLE NO. BC970605 66 1049-9644/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.