Transgenic Research 12: 351–361, 2003. 351 © 2003 Kluwer Academic Publishers. Printed in the Netherlands. Tritrophic choice experiments with Bt plants, the diamondback moth (Plutella xylostella) and the parasitoid Cotesia plutellae Tanja H. Schuler1,∗, Roel P.J. Potting3, Ian Denholm1, Suzanne J. Clark2, Alison J. Clark1, C. Neal Stewart4 & Guy M. Poppy5 1Division of Plant and Invertebrate Ecology; 2Biomathematics Unit, IACR-Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK 3Laboratory of Entomology, Wageningen University, 6700 EH Wageningen, The Netherlands 4Department of Plant Sciences and Landscape Systems, The University of Tennessee, Knoxville, TN 37901, USA 5Division of Biodiversity and Ecology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK Received 3 August 2002; revised 24 January 2003; accepted 26 January 2003 Key words: Bacillus thuringiensis, biosafety, Brassica napus, non-target arthropods, transgenic plants, tritrophic interactions Abstract Parasitoids are important natural enemies of many pest species and are used extensively in biological and integrated control programmes. Crop plants transformed to express toxin genes derived from Bacillus thuringiensis (Bt) provide high levels of resistance to certain pest species, which is likely to have consequent effects on parasitoids specialising on such pests. A better understanding of the interaction between transgenic plants, pests and parasitoids is important to limit disruption of biological control and to provide background knowledge essential for implement- ing measures for the conservation of parasitoid populations. It is also essential for investigations into the potential role of parasitoids in delaying the build-up of Bt-resistant pest populations. The diamondback moth (Plutella xylostella), a major pest of brassica crops, is normally highly susceptible to a range of Bt toxins. However, extensive use of microbial Bt sprays has led to the selection of resistance to Bt toxins in P. xylostella. Cotesia plutellae is an important endoparasitoid of P. xylostella larvae. Although unable to survive in Bt-susceptible P. xylostella larvae on highly resistant Bt oilseed rape plants due to premature host mortality, C. plutellae is able to complete its larval development in Bt-resistant P. xylostella larvae. Experiments of parasitoid flight and foraging behaviour presented in this paper showed that adult C. plutellae females do not distinguish between Bt and wildtype oilseed rape plants, and are more attracted to Bt plants damaged by Bt-resistant hosts than by susceptible hosts. This stronger attraction to Bt plants damaged by resistant hosts was due to more extensive feeding damage. Population scale experiments with mixtures of Bt and wildtype plants demonstrated that the parasitoid is as effective in controlling Bt-resistant P. xylostella larvae on Bt plants as on wildtype plants. In these experiments equal or higher numbers of parasitoid adults emerged per transgenic as per wildtype plant. The implications for integrated pest management and the evolution of resistance to Bt in P. xylostella are discussed. Introduction concentrated on small-scale laboratory experiments or on large-scale field trials. Small-scale laboratory Studies of the effect of insect-resistant transgenic studies are often designed to represent worst-case plants on non-target insects have so far predominantly scenarios since they primarily aim to determine the inherent susceptibility of a non-target insect to the ∗ Author for correspondence transgene product (e.g., Hilbeck et al., 1998; Birch E-mail: [email protected] et al., 1999; Losey et al., 1999; Zwahlen et al., 2000). 352 However, this type of experiment only represents a pest present the other end of the spectrum (Obrycki first step towards assessing the side effects of trans- et al., 2001). genic plants on non-target organisms. Results of such A range of brassica crops have been engineered studies usually cannot be used directly to forecast ef- to express Bt toxin genes to provide resistance to the fects on non-target populations in the field, where diamondback moth (Plutella xylostella, Lepidoptera, many other factors, such as insect behaviour, variation Plutellidae) and other lepidopteran pests (Li et al., in the level of exposure, competition and other mor- 1995; Metz et al., 1995a,b; Stewart et al., 1996; Jin tality factors, come into play (Poppy, 2000; Schuler et al., 1999, 2000; Zhao et al., 2000; Kuvshinov et al., et al., 2000). Large-scale field studies represent the 2001). P. xylostella is a major pest of brassicas and most realistic scenario but patchy and fluctuating in- highly susceptible to a number of Bt toxins. However, sect populations combined with limited replication this pest has developed resistance to a wide range of make it difficult to detect small effects on non-target insecticides including, in some areas, to microbial Bt organisms at this scale. sprays (Talekar & Shelton, 1993; Tabashnik et al., There is therefore a need for an intermediate scale 1997). of experimentation, which incorporates more realism One of the main natural enemies of P. xylostella is than small-scale studies but at the same time allows for Cotesia plutellae (Hymenoptera, Braconidae) (Talekar appropriate replication and control over insect popula- & Shelton, 1993), a solitary larval endoparasitoid that tions. Some recent studies have already demonstrated is generally reported to be host-specific to P. xylostella the potential of this approach. Behavioural choice (Verkerk & Wright, 1996), although there are a few tests with maize expressing the Cry1Ab toxin of records on other lepidopterous hosts (e.g., Kaneko, Bacillus thuringiensis (Bt) and larvae of the preda- 1993; Lipa et al., 1993). Parasitic insects such as C. tory lacewing Chrysoperla carnea demonstrated that plutellae complete their larval development in a single the predator preferentially feeds on aphids rather than host insect, eventually leading to the host’s death on lepidopteran larvae (the targets of the Bt toxin) (Godfray, 1994). Their fate is therefore strongly linked (Meier & Hilbeck, 2001). This preference will re- with the fate of the host, at an individual as well as a duce the exposure of C. carnea to Cry1Ab toxin since population level, and both direct and indirect effects of aphids do not ingest the toxin when feeding on Bt transgenic plants on parasitoids have to be considered. maize (Raps et al., 2001). Population-scale labora- Bt oilseed rape caused 100% mortality of suscepti- tory experiments with the aphid Myzus persicae and ble P. xylostella larvae and small-scale no-choice ex- the aphid parasitoid Diaeretiella rapae showed that periments have shown that adult C. plutellae females D. rapae was as effective in controlling this non-target oviposited into such susceptible hosts but premature pest on Bt and proteinase inhibitor oilseed rape plants host mortality on Bt plants prevented the parasitoid as on untransformed plants (Schuler et al., 2001). larvae from successfully completing their develop- Glasshouse trials with potatoes expressing snowdrop ment. In contrast, C. plutellae developed normally in lectin (GNA), the moth Lacanobia oleracea and the larvae of a Bt-resistant P. xylostella strain (Schuler parasitoid Eulophus pennicornis indicated that the et al., 1999; Schuler et al., unpublished data). partially resistant GNA potatoes can augment biocon- The present study introduced choice into experi- trol of L. oleracea by E. pennicornis (Belletal., ments investigating the interactions between Bt plants 2001). and C. plutellae, thereby incorporating behaviour of Arthropod natural enemies differ widely in their the non-target organism. Behaviour plays a major role ecology and specialisation (Jervis & Kidd, 1996), and in the success of parasitoids. For example, a female the indirect effects of transgenic plants on their pop- not only faces the formidable task of locating a ‘hid- ulations will differ accordingly. Some are specialists den host’ in a complex environment but also has to that predominantly develop on one host species (e.g., decide whether a host is suitable for the development many larval endoparasitoids) while other natural en- of her offspring (Turlings et al., 1990; Vet & Dicke, emies attack a wider range of hosts (e.g., many egg 1992). Parasitoids use chemical stimuli from their parasitoids) or prey (most predatory arthropods). Pop- herbivorous hosts or the host’s food plant to locate ulations of generalist predators are least likely to be hosts. Although host-derived stimuli are reliable indi- negatively affected by the absence of the target pest in cators of host presence, they are, with the exception of a Bt plant crop (as they will be able to find other prey) host pheromones, difficult to detect at long distance while natural enemies specific to larvae of the target and are mostly used as contact stimuli. In contrast, 353 plant-derived stimuli are released in large amounts, from every batch of plants was tested in a bioassay are generally more easily detectable at a long distance with susceptible third instar P. xylostella larvae to de- and can be plant species and genotype-specific (Vet tect any decrease in the level of plant resistance and et al., 1991; Vet & Dicke, 1992). Many parasitoids, the plants always caused 100% mortality. including C. plutellae, discriminate in-flight between uninfested and host-infested plants (Potting et al., Insects 1999). Transgenic plants may differ