Molecular Ecology (2003) 12, 493–504 © 2003 Blackwell Publishing Ltd Blackwell Science, Ltd Impact of oilseed rape expressing the insecticidal cysteine protease inhibitor oryzacystatin on the beneficial predator Harmonia axyridis (multicoloured Asian ladybeetle) N. FERRY,* R. J. M. RAEMAEKERS,* M. E. N. MAJERUS,† L. JOUANIN,‡ G. PORT,* J. A. GATEHOUSE § and A. M. R. GATEHOUSE * *School of Biology, University of Newcastle Upon Tyne, Newcastle, NE1 7RU, UK, †Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB3 3EH, UK, ‡Laboratoire de Biologie Cellulaire, INRA, Route de Saint Cyr, 78026 Verailles Cedex, France, § Department of Biological Sciences, University of Durham, Durham, DH1 3LE, UK Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on bene- ficial predators through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) on the predatory ladybird Harmonia axyridis were investigated using diamondback moth Plutella xylostella as the pest species. As expected, oilseed rape expressing OC-1 had no effects on either development or survival of the pest, which utilizes serine digestive proteases. Immunoassays confirmed accumulation of the transgene product in pest larval tissues at levels of up to 3 ng per gut. Characterization of proteolytic digestive enzymes of H. axyridis demonstrated that larvae and adults utilize cysteine and aspartic proteases; the former activity was completely inhibited by oryzacystatin in vitro. However, when H. axyridis larvae consumed prey reared on OC-1 expressing plants over their entire life cycle, no significant effects upon survival or overall development were observed. The inhibitor initially stimulated development, with a shortening of the developmental period of the second instar by 27% (P < 0.0001) accompanied by a 36% increase in weight of second instar larvae (P = 0.007). OC-1 had no detrimental effects on reproductive fitness of adult H. axyridis. Interestingly there was a significant increase in consumption of OC-1 dosed prey. The results show that prey reared on transgenic plants expressing a protein which inhibited ladybird digestive enzymes in vitro had no effects in vivo; the ladybird was able to up-regulate digestive proteases in response to the inhibitor. Keywords: beneficial predators, Harmonia axyridis, oryzacystatin (OC-1), Plutella xylostella, trans- genic oilseed rape, tritrophic interactions Received 5 July 2002; revision received 14 October 2002; accepted 14 October 2002 Introduction Genetic engineering of crops for enhanced levels of resistance to insect pests has the potential to offer large benefits to global agriculture, not least in a significant reduction in pesticide application. Insect-resistant transgenic crops expressing Bacillus thuringiensis ( Bt ) δ -endotoxins have been commercially grown for many years with minimal impact on nontarget organisms (Dogan et al . 1996; Riggin- Bucci & Gould 1997; Lozzia et al . 1998; Riddick & Barbosa 1998; Lozzia 1999; Riddick et al . 2000, 1998; Hellmich et al . 2001; Saxena & Stotzky 2001). However, field durability is predicted to be limited by the development of resistance to Bt toxins in pest populations (Gould 1998; Tabashnik et al . 2000; Carriere & Tabashnik 2001). Transgenic plants expressing other insect resistance genes, including plant-derived protease inhibitors, are currently being developed ( Jouanin et al . 1998; Gatehouse & Gatehouse 1999). Protease inhibitors are divided into four classes, inhibiting serine, cysteine, metallo- or aspartic proteases. They bind specifically to target gut proteases, and thus inhibit the ability of the insect to digest protein (Gatehouse & Gatehouse 1999). The potential of protease inhibitors to Correspondence: A. M. R. Gatehouse. Fax: 0191 2225228; E-mail: A.M.R.Gatehouse@ncl.ac.uk