Infectivity, Speed of Kill, and Productivity of a Baculovirus Expressing the Itch Mite Toxin Txp-1 in Second and Fourth Instar Larvae of Trichoplusia ni John P. Burden, Rosemary S. Hails, John D. Windass,* Marie-Marthe Suner,* and Jennifer S. Cory 1 Ecology and Biocontrol Group, NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, United Kingdom; and *Zeneca Agrochemicals, Jealott’s Hill Research Station, Bracknell, Berkshire RG42 6ET, United Kingdom Received July 14, 1999; accepted December 14, 1999 A cDNA clone of the gene coding for the paralytic neurotoxin (tox34) from the female straw itch mite, Pyemotes tritici, was created by RT-PCR and in- serted into the genome of the Autographa califor- nica nucleopolyhedrovirus (AcMNPV) under the control of the AcMNPV p10 promoter. This recombi- nant virus, AcTOX34.4, caused a rigid paralysis in infected larvae. The infectivity of AcTOX34.4 was compared to the wild-type parent strain, AcMNPV- C6, in second and fourth instar larvae of the cabbage looper, Trichoplusia ni. There were no significant differences in LD 50 values between the recombinant virus and its wild-type parent strain but, as ex- pected, the LD 50 was lower for second instar larvae. The mean time to death and yield of occlusion bodies were measured in second and fourth instar T. ni larvae at a high (100% mortality) and low (<50% mortality) doses of the virus. The mean time to death of recombinant infected larvae was reduced by 50 – 60% compared to larvae infected with the wild-type strain, depending on virus dose and instar, with these larvae becoming paralysed after approxi- mately 60 h and dying 10–20 h later. This is among the fastest speeds of kill recorded for recombinant baculoviruses. Fourth instar larvae were found to succumb to the recombinant virus more quickly than the second instar larvae. The increase in the speed of kill of the recombinant virus was accompa- nied by a large reduction of approximately 95% in the yield of progeny virus. The yield of virus showed a highly significant relationship with time to death, but this relationship was complex and varied be- tween the different viruses, concentrations, and in- stars. The yield per unit weight of the larvae was found to be constant at a low virus dose and in- creased over time at a high virus dose, irrespective of instar and virus. It is predicted that these changes in the performance of the recombinant virus would act toward reducing its fitness, leading to it being outcompeted by the wild type in field situations. © 2000 Academic Press Key Words: Autographa californica; Pyemotes tritici; Trichoplusia ni; tox34; AcMNPV; recombinant baculo- virus; genetically modified organism; biopesticide. INTRODUCTION The relatively slow speed of kill of natural baculovi- rus insecticides, in comparison with synthetic chemical insecticides, has restricted their widespread use, par- ticularly in agriculture (Black et al., 1997). However, the recent development of faster acting genetically modified baculoviruses has provided the opportunity to improve this situation (Maeda, 1995; Black et al., 1997). Insertion of essential host regulatory genes into the baculovirus genome, such as those expressing ju- venile hormone esterase (Bonning et al., 1997), diuretic hormone (Maeda, 1989), and prothoracicotropic hor- mone (O’Reilly et al., 1995), has had variable results, often resulting in no or only limited improvement in speed of action. Overall, these modifications have been far less effective than the insertion of genes that code for insect-selective toxins (Tomalski and Miller, 1991; DuPont, 1996; Hughes et al., 1997). Although numerous recombinants have been devel- oped, mainly based on Autographa californica nucle- opolyhedrovirus (AcMNPV), expressing a variety of toxins, detailed studies on their effects in insect hosts are lacking. While limited comparative bioassays mea- suring speed of kill are necessary as an initial screen for successful recombinants, it is important to ascer- tain whether any alterations in the behaviour of the pathogen are affected by the age of the host, whether genetic modification alters other parameters, such as productivity, and how these different parameters in- teract. The only genetically modified baculoviruses 1 To whom correspondence should be addressed. Fax: (0)1865 281696. E-mail: jsc@wpo.nerc.ac.uk. Journal of Invertebrate Pathology 75, 226 –236 (2000) doi:10.1006/jipa.1999.4921, available online at http://www.idealibrary.com on 226 0022-2011/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.