New Zealand Institute for Crop and Food Research Ltd, Private Bag 92169, Auckland, New Zealand b

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1 Biological Control 40 (2007) Impacts of Bacillus thuringiensis toxins on parasitoids (Hymenoptera: Braconidae) of Spodoptera litura and Helicoverpa armigera (Lepidoptera: Noctuidae) G.P. Walker a,, P.J. Cameron b, F.M. MacDonald a, V.V. Madhusudhan c, A.R. Wallace d a New Zealand Institute for Crop and Food Research Ltd, Private Bag 92169, Auckland, New Zealand b 20 Westminster Road, Mt. Eden, Auckland 1024, New Zealand c BioDiscovery NZ Ltd, 24 Balfour Road, Parnell, Auckland, New Zealand d New Zealand Institute for Crop and Food Research Ltd, Private Bag 4704, Christchurch, New Zealand Received 31 May 2006; accepted 15 September 2006 Available online 26 September 2006 Abstract Spodoptera litura (F.) and Helicoverpa armigera (Hübner) are potential non-target pests in Bacillus thuringiensis (Bt)-transformed potato and brassica crops that are currently being investigated as candidates for Weld release in New Zealand. The comparative susceptibility of these pests and two of their larval parasitoids to Bt toxins was examined using diets amended with commercial preparations of Bt sprays and Cry1Ac toxin preparations. Using concentrations that allowed survival of some of the parasitoids and their hosts, development and survival characteristics were compared at two rates of Dipel 2X, six rates of Cry1Ac, and two reduced-nutrition diets. For all these treatments, larval development of both parasitoids, Cotesia kazak (Telenga) and Meteorus pulchricornis (Wesmael), was delayed only when there was a signiwcant impact on the host Lepidoptera. Survival of M. pulchricornis was unavected even when the survival of S. litura larvae was mildly, but signiwcantly, reduced. In H. armigera, Bt diets that caused severely reduced survival of the host were accompanied by reductions in survival of M. pulchricornis. Similar or increased levels of survival of C. kazak were noted in H. armigera that were fed less toxic Bt-amended diets. Poor survival of parasitoids on reduced-nutrition diets suggested that reduced nutrition had a comparatively greater evect on parasitoid survival than Cry1Ac toxins. Overall, when Bt was ingested at concentrations that had minor evects on development or survival of host larvae, there was no impact on either parasitoid species. Further experiments with speciwc Bt cultivars proposed for release are required to conwrm that in any surviving larvae, the impacts of Bt on these parasitoids are likely to be fewer than the impacts on their non-target hosts Elsevier Inc. All rights reserved. Keywords: Bacillus thuringiensis; Non-target impacts; Lepidoptera; Spodoptera litura; Helicoverpa armigera; Parasitoids; Cotesia kazak; Meteorus pulchricornis 1. Introduction * Corresponding author. Fax: address: walkerg@crop.cri.nz (G.P. Walker). The development of Bacillus thuringiensis Berliner (Bt) transgenic crops provides prospects for widespread control of lepidopteran pests on Weld crops, but it is also likely to have impacts on non-target Lepidoptera feeding on these crops, and evects on the species that interact with Lepidoptera (Glare and O Callaghan, 2000). Environmental assessments of Bt crops have included studies of non-target insects, including evaluations of the impacts of these crops on non-target pests and natural enemies (Schuler et al., 2001). Prospective insect resistant Bt crops in New Zealand include potatoes (Davidson et al., 2002) and vegetable brassicas (Christey et al., 2006). Target pests in these crops are the potato tuber moth, Phthorimaea operculella (Zeller) on potatoes, and the diamondback moth, Plutella xylostella (L.) on vegetable brassicas. The interactions of P. operculella and its parasitoid Apanteles subandinus Blanchard /$ - see front matter 2006 Elsevier Inc. All rights reserved. doi: /j.biocontrol

2 G.P. Walker et al. / Biological Control 40 (2007) (Davidson et al., 2006), and P. xylostella and its parasitoids (Chilcutt and Tabashnik, 1997; Schuler et al., 1999) in Bttransgenic crops have been investigated previously. Two polyphagous Lepidopteran species: tomato fruitworm, Helicoverpa armigera (Hübner) and tropical armyworm, Spodoptera litura (F) are occasional pests on potatoes and vegetable brassicas in New Zealand, but they are not usually the subject of control operations and can thus be classiwed as non-target pests that may be avected by the deployment of Bt-transgenic crops. Helicoverpa armigera and S. litura are attacked by several larval parasitoids, some of which provide signiwcant control in other crops. For example, the braconid Cotesia kazak (Telenga), which is speciwc to H. armigera, causes 60 80% mortality of small larvae in processing tomatoes and signiwcantly reduces damage in this crop (Walker and Cameron, 1989). This parasitoid also attacks H. armigera in a number of other crops including brassicas (Walker et al., 2005) and reduction of its evectiveness by transgenic Bt is likely to be unfavorable in horticultural environments. A second braconid, Meteorus pulchricornis (Wesmael), similar to C. kazak in being a solitary endoparasitoid species, attacks both H. armigera and S. litura but is widely polyphagous (Berry and Walker, 2004). These two Lepidoptera and their parasitoids are appropriate model systems for examining the possible direct and indirect impacts of Bt crops on non-target Lepidoptera and their parasitoids in New Zealand. Lepidopteran parasitoids may be exposed directly to Bttransgenic toxins when adult parasitoids feed on Bt plant material or when larval parasitoids feed within hosts that have ingested toxins, either as single Cry toxins from Bt plants or as Cry toxin mixtures from Bt spray residues. Direct exposure of adults is unlikely as nectar does not contain Bt toxins (Groot and Dicke, 2003). Parasitoids of Lepidoptera are unlikely to be directly avected because Bt toxins bind to receptors in midgut epithelium of host larvae and lose their toxicity to natural enemies. However, immature parasitoids may be inxuenced indirectly through lethal or sublethal impacts on the health and development of hosts (Bernal et al., 2002). Lepidopteran-active Cry proteins are generally considered to lack direct toxicity to predators and parasitoids (Glare and O Callaghan, 2000). For example, lack of direct evects of Bt toxins on natural enemies has been established by direct feeding and choice experiments for adult parasitoids including Cotesia plutellae (Kurdjumov) (Chilcutt and Tabashnik, 1999; Schuler et al., 1999). Assessment of indirect impacts that may inxuence immature parasitoids through multi-trophic food chains is more complex. Previous studies of the impacts of Bt plants on non-target parasitoids have used commercial Bt insecticides on leaf disks (Monnerat and Bordat, 1998; Chilcutt and Tabashnik, 1999), Bt toxins in artiwcial diet (Hafez et al., 1995; Salama et al., 1996), laboratory studies with Bt-transformed plants (Schuler et al., 1999; Bernal et al., 2002; Baur and Boethel, 2003), and population studies on Bt plants (Schuler et al., 2001). Field studies surveying the relative abundance of natural enemies in Bt crops have emphasized predators rather than parasitoids (Reed et al., 2001; Conner et al., 2003). The objective of this study was to examine the potential direct evects of Bt toxins on larval parasitoids within their hosts, and the indirect host-mediated evects on parasitoids using the Lepidoptera-parasitoid systems described above. The methods were based on the use of concentrations of Bt toxin that would allow survival of suycient hosts and parasitoids to compare both their survival and development. For this we used laboratory assays with host Lepidoptera that were reared on artiwcial diets, to which commercial Bt preparations or Cry1Ac toxins were added. SigniWcant survival of hosts may occur especially when control of target pests in Bt-transgenic crops is not complete, or when non-target pests are tolerant of transgenic cultivars (Bernal et al., 2002) or the Bt toxins in foliar-applied sprays. Overall, our study aimed to determine whether non-target species on transgenic plants could be potentially subjected to concentrations of Bt toxin that would diverentially favor the population dynamics of either Lepidopteran species or their parasitoids. The work also contributes to an overall ecological risk assessment for the potential release of transgenic potatoes into the environment as described by Conner et al. (2003). 2. Materials and methods 2.1. Insects Spodoptera litura and H. armigera were obtained from laboratory colonies maintained by HortResearch at the Mt Albert Research Centre, Auckland, New Zealand. The colony of S. litura was originally established from moths collected in Queensland, Australia, and H. armigera from larvae collected in Christchurch, New Zealand. Both host colonies were reared on artiwcial diet and the parasitoids M. pulchricornis and C. kazak reared using methods based on those of Singh et al. (1982). In our experience, C. kazak is monophagous (Cameron et al., 1989) and in this study was always reared on H. armigera. To standardize hosts, the polyphagous M. pulchricornis was also reared on H. armigera. Rearing methods for M. pulchricornis were simpliwed because the strain in New Zealand is thelytokous, with no males recorded (Berry and Walker, 2004). Both parasitoid species were held in clear 3-L ventilated plastic jars (30 40 parasitoids per cage) with honey smears and 10% sugar water solution supplied for sustenance. Parasitoid cocoons were held at 20 C for adult eclosion and new adults were maintained at this temperature for an initial period of 3 4 days for mating and feeding. When not in use for experiments, parasitoids were held at 14 C except for twice-weekly feeding periods of 2 3 h at room temperature Diets A lima bean/wheat germ based diet (hereinafter referred to as the standard diet and used as a control), the recipe for

3 144 G.P. Walker et al. / Biological Control 40 (2007) Table 1 ArtiWcial diet used in bioassays with Helicoverpa armigera and Spodoptera litura and larval parasitoids Ingredient (unit) Standard Low nutrition 1/8th 1/6th Lima bean powder (g) Wheatgerm (g) Agar (g) Brewers yeast (g) Water (ml) Linseed oil (ml) Wheatgerm oil (ml) Vanderzant vitamin mix (g) Ascorbic acid (g) Methyl 4-hydroxybenzoate (g) Sorbic acid (g) Penicillin (mg) Streptomycin (mg) Prochloraz (mg) which is given in Table 1, was used in all experiments. Lownutrition diets contained the same ingredients as the standard diet but had proportionately smaller absolute amounts of key ingredients (Table 1). The standard diet was prepared by autoclaving a mixture of water and dry ingredients: lima bean powder (beans from various sources), wheat germ (Goodman Fielder, Auckland, NZ), agar (Danisco NZ Ltd, Auckland), Brewers yeast (Healtheries, Auckland, NZ), sorbic acid (Bronson and Jacobs, Auckland NZ), and methyl 4- hydroxybenzoate (Sigma Aldrich, Steinheim, Germany). Oils (Healtheries), vitamins [Vanderzant vitamin mix (Bio- Serv, Frenchtown, NJ, USA) and ascorbic acid (Lovetts, Auckland, NZ)], antibiotics (Sigma Aldrich) and the fungicide prochloraz (Octave 50 W, Schering AG, Berlin, Germany) were all added after the mixture had cooled to 65 C. Diets containing toxin were prepared in exactly the same way except that the toxin was added with the oils and vitamins to obtain the desired concentration Toxins Diets were incorporated either with Dipel 2X wettable powder containing 32 kiu of potency per mg Bt subsp. kurstaki (H-3a, 3b HD1) or especially prepared Cry1Ac toxin. The Cry1Ac protein used in all the experiments was prepared from Bacillus thuringiensis strain HD73, a strain known to produce only one endotoxin, Cry1Ac (Höfte and Whiteley, 1989). Activated Cry1Ac toxin was prepared from spore-crystal suspensions of HD73 as follows. Sevenday-old sporulated colonies (as conwrmed by stained slide mounts) were scraped into 11 ml of sterile deionized water. The resulting suspension was centrifuged and the supernatant discarded. The pellet was re-suspended in 11 ml of solubilization buver (0.1 M cyclohexylamino-1-propanesulfonic acid, ph 11.0 (CAPS), 20 mm dithiotheitol, 1 mg/ml trypsin) and allowed to solubilize overnight. The mixture was centrifuged, desalted in G-25 columns using 10 mm CAPS buver, ph 8.0, and stored frozen until required. The concentration of the crystal protein in toxin preparations Table 2 Concentrations of Bacillus thuringiensis toxins and low nutrition diets that were fed to the lepidopteran hosts Spodoptera litura and Helicoverpa armigera, with the parasitoids Meteorus pulchricornis and Cotesia kazak present or absent Treatment S. litura H. armigera M. pulchricornis M. pulchricornis C. kazak Dipel 2X 0.1 Weld rate a 0.1 Weld rate Dipel 2X Weld rate field rate Cry1Ac 276μg/ml 3.68μg/ml 0.3 μg/ml Cry1Ac 1.30μg/ml 0.15 μg/ml Cry1Ac 0.65 μg/ml Low nutrition 1/8 1/6 a Field rate is 50 g/100 l water. was estimated using the BCA reagent (Pierce Chemical Company, Rockford, IL, USA) as described in their protocol # Toxins activated using these procedures are widely accepted to be similar to those used in transgenic plants expressing Bt toxins (Hilbeck et al., 1998). For experiments with Dipel 2X (Abbott Laboratories, North Chicago, Illinois,USA) the rate used was 5 g/100 L or 50 g/100 L (the latter being the Weld rate). For experiments with Cry1Ac toxin, preliminary trials (V.V. Madhusudhan, unpublished data) were conducted to determine sub-lethal concentrations that would ensure survival of suycient numbers of host larvae for parasitization. Toxin in aqueous solution was incorporated into diet and dispensed into wells of a microplate using a 1 ml disposable syringe (100μl/ml diet per well). Neonate caterpillars of either S. litura or H. armigera were added individually to the wells and sealed in with Mylar Wlm ventilated with pin holes. Mortality was recorded at 7 days. Based on the dose-responses in these experiments; the initial Cry1Ac toxin concentration in the diet for S. litura was set at 276 μg/ml, whereas the diet for H. armigera contained 3.68 μg/ml. The concentration of toxin used against H. armigera was then reduced to 1.3 and 0.65 μg/ml. It was then reduced again to 0.30 and 0.15 μg/ml for an experiment with C. kazak because the previous experiment with M. pulchricornis on H. armigera suggested that lower concentrations would give better host and parasitoid survival, enabling more informative measurement of evects on C. kazak (Table 2) Low nutrition diets Two low nutrition diets containing key ingredients either at 1/8th or 1/6th of standard amounts were also used. These diets were prepared such that the concentrations of agar, vitamins and preservatives were kept constant while those of other key ingredients such as lima bean powder, wheat germ powder, yeast, and the oils were diluted (Table 1). These concentrations were chosen based on results of preliminary trials (V.V. Madhusudhan, unpublished data) that showed that caterpillars developing on these diets were of sizes similar to those exposed to sub-lethal concentrations of Bt chosen above. For these experiments, the bioassay methodology was exactly as described above, except that low nutrition diets without toxin were used.

4 G.P. Walker et al. / Biological Control 40 (2007) Laboratory bioassays and experimental design A series of laboratory bioassays was conducted, initially with a Bt foliar insecticide (Dipel 2X) and then with Cry1Ac toxin. Concentrations (listed in Section 2.3) were varied in an attempt to provide intoxicated host larvae that would support parasitoids and allow estimates of the development and survival of both trophic levels in each experiment. In later experiments, a low-nutrition diet treatment was added (Table 2). Actual concentrations used for each host parasitoid combination tested are given in the corresponding section of the results. For each experiment, neonate H. armigera or S. litura larvae were placed individually onto a 2 cc cube of diet in clear, sterile 60 ml containers with small holes in the lids for ventilation. H. armigera is highly cannibalistic so larvae of both species were reared singly to provide consistency. A layer of tissue underneath the lid absorbed excess moisture and prevented escape of neonate larvae. The diet was monitored to ensure that the larvae retained an adequate amount of quality food, and new diet was added as necessary. Vermiculite was added just before pupation to assist formation of moth pupae or parasitoid cocoons. All experiments were done at 19 2 C and a light:dark cycle of 14:10 h. Eight separate experiments were conducted, over an 11- month period. To ensure that variations in the populations of the hosts or parasitoids, or small changes in the timing of the use of individual cohorts in the experiments could not inxuence the evects of the experimental treatments all experiments had internal controls, half the larvae being fed the standard diet (Table 1). Comparisons of the controls across experiments conwrmed that consistent results were obtained. For each diet treatment, half the host larvae were reserved for parasitism at the 2nd instar by the parasitoids C. kazak or M. pulchricornis. This provided four treatments of n D larvae for each parasitized or non-parasitized and standard or amended diet combination. Thus, experiments with one diet required larvae. When larval host development was delayed by the diet treatment, parasitism was delayed by up to 2 weeks until larvae reached the 2nd instar. All female parasitoids had prior experience of oviposition through exposure to H. armigera larvae before the experiment. Parasitoids were held mainly at 14 C and used within 14 days of emergence. For oviposition by parasitoids, each host larva was overed singly on a Wne brush to a female parasitoid, within a parasitoid cage containing female parasitoids. After a single oviposition into the host was observed, the larva was returned to the diet. Larvae that were not successfully parasitized, as indicated by higher growth rates typical of normal larvae, were removed from this treatment. The remaining larvae were considered to be parasitized and provided the basis for calculating rates of successful development of adult parasitoids. Therefore, larvae that died before the 5th instar were assessed as parasitized and diverences in mortality between parasitized and non-parasitized treatments indicated interactions between parasitism and diet Data collected Non-parasitized host larvae and pupae, parasitized larvae, parasitoid cocoons, and emerged adult parasitoids were observed every 24 h at approximately the same time every day, normally between 1100 and 1300 h. Comparisons of development were based on weight of larvae (after days development), pupae, or cocoons, and in later experiments evects on adult parasitoid longevity were measured. Non-parasitized host larvae were weighed or measured when control larvae on the standard diet reached the 4th instar. Host pupae were weighed 2 3 days after pupation to allow for hardening of the pupa, and adult moths were weighed on the day of emergence. Parasitized larvae were monitored to note the time to formation of parasitoid cocoons, and these cocoons were weighed 2 days later to allow time for hardening to avoid damage. Those larvae in the parasitized treatment that were not successfully parasitized (as distinct from the non-parasitized treatment) were removed from the data. Emergence of adult parasitoids from cocoons was recorded, and the duration of the pupal stage in the cocoon was calculated. Adult longevity was also recorded in the experiments in which Cry1Ac-amended diets were being tested. For the Wrst of these, adult parasitoids were left after emergence in the larval rearing containers, with diet. However, longevity may have been inxuenced by divering levels of moisture in the diet in individual containers so, for later experiments, parasitoid cocoons were transferred to individual empty containers. Survivorship was calculated for each stage for hosts and parasitoids Statistical analysis Measures of survival and development were subject to analysis of variance using GenStat Release 6.2 (GenStat, 2002) and means for treated and control insects were compared with t-tests or estimates of least signiwcant diverences (LSDs). Standard residual plots were examined to check the validity of the assumptions for analysis of variance. Where variances divered signiwcantly, variables were compared with t-tests and approximate degrees of freedom applicable to such tests are quoted with results. For the Wnal set of experiments, with H. armigera and C. kazak, when four diets were compared simultaneously, many larvae failed to thrive on the three test diets, and the distributions of many measured characteristics were positively skewed and/or the variances of treated and control larvae varied signiwcantly. Accordingly, these variables were transformed where indicated (using the log transformation) for analysis. 3. Results 3.1. S. litura and M. pulchricornis There were three experiments with these species, using diets incorporating Dipel 2X at 0.1 and 1.0 times the Weld rate, and a diet with 276 μg/ml Cry1Ac (Table 3).

5 146 G.P. Walker et al. / Biological Control 40 (2007) Weld rate of Dipel 2X After 2 weeks on this diet, ANOVAs showed that S. litura larvae were 64 mg (31%) lighter (t D 4.6, n D 60, df D 58, P < 0.001), and pupae formed on average 1 day later (t D 4.4, n D 60, df D 58, P < 0.001) and were 50 mg (10%) lighter (t D 4.6, n D 60, df D 58, P < 0.001) than those reared on the control diet. However, there was no signiwcant diverence in overall time to moth emergence. Mortality of larvae was not signiwcantly diverent between treated and control diets. Ninety-three percent of non-parasitized S. litura on the 0.1 Weld rate Dipel 2X diet became moths, indicating that the diet was generally suitable for survival of these larvae (Table 3). This diet had very little evect on the developmental attributes recorded for the M. pulchricornis parasitoids, and their survival from oviposition to adult emergence was not signiwcantly diverent between treated and control diets (Table 3) Field rate of Dipel 2X After 19 days on this diet, the mean larval weight of S. litura was only 12% of that of larvae on the control diet. Time to pupal formation was extended by 10 days (td14.0, nd84, dfd57, P<0.001), pupae were 9% lighter than those reared on the control diet (td3.4, nd84, dfd82, P<0.001), and time to moth emergence was increased from 45 to 56 days (td13.6, nd80, dfd50, P<0.001). Fewer S.litura fed on the Weld rate of Dipel 2X survived to adults (72%) compared with controls (88%) (χ 2 D4.00, dfd1, P<0.05) (Table 3). Meteorus pulchricornis parasitoids reared on S. litura fed the Weld rate Dipel 2X diet required a signiwcantly longer (t D 4.8, n D 46, dfd 42, P < 0.001) and more variable amount of time to develop to cocoons than those reared on the control diet. The treatment also led to a signiwcant increase (t D 4.5, n D 46, df D 40, P < 0.001) of 2 3 days in overall time to adult parasitoid emergence, but this diverence was due to the diverence in parasitoid larval duration. The rate of survival to adult parasitoid was similar to that of larvae on the control diet (Table 3). Only one emerged parasitoid larva (pre-pupa) and no pupae (cocoons) died in both treatments, so there was no evidence of the Weld rate Dipel 2X diet avecting the Wnal adult emergence of the parasitoid Cry1Ac (276 μg/ml) diet After 19 days, this diet had reduced larval weights of S. litura to 34% of the mean weight of larvae on the control diet (t D 10.2, n D 92, df D 67, P < 0.001). Time to pupal formation was signiwcantly extended (t D 10.4, n D 91, df D 61, P < 0.001) by a mean of 4.8 days. Pupae reared on the Cry1Ac diet were 9% lighter than those reared on the control diet (t D 3.9, n D 91, df D 89, P < 0.001), which is similar to the reduction that occurred on the 0.1 Weld rate Dipel 2X and Weld rate Dipel 2X diets. Time to S. litura moth emergence was increased by a mean of 4.2 days (t D 8.8, n D 87, df D 85, P < 0.001). Moth emergence rates divered signiwcantly (χ 2 D 4.33, df D 1, P < 0.05), being 94% for larvae reared on the control diet but reduced to 80% for larvae reared on the Cry1Ac diet (Table 3). Time to formation of M. pulchricornis cocoons was delayed by 1 to 2 days (t D 7.2, n D 79, df D 61, P <0.001) and more variable in S. litura larvae fed this Cry1Ac diet, but the evects on both mean duration and its standard deviation were not as large as those observed for the Weld rate Dipel 2X diet. The increase in time to emergence of the adult parasitoid due to the Cry1Ac diet was also not as large as for the Weld rate Dipel 2X diet, averaging 0.7 days (95% conwdence limits days), but was statistically signiwcant (t D 3.3, n D 78, df D 47, P < 0.01). Surprisingly, Table 3 Development and survival (mean SEM) of Spodoptera litura (non-parasitized) and of Meteorus pulchricornis on Spodoptera litura Dipel 2X 0.1 Weld rate Control Dipel 2X Weld rate Control Cry1Ac 276 μg/ml Control Non-parasitized i.e. S. litura hosts Larvae reared (n) Larval weight (mg) b *** *** *** Days to pupa *** *** *** Pupal weight (mg) *** *** *** Days to adult Ns *** *** Survival to adult (%) 93 Ns * * 94 Parasitized i.e. M. pulchricornis on S. litura Larvae parasitized (n) Larvae dead (n) Days to cocoon Ns *** *** Days to Adult Ns *** ** Days as Cocoon ** Ns *** Days adult life ** Survival to adult (%) 96 Ns Ns Ns 86 Hosts were fed diet with 0.1 and 1.0 Weld rate of Dipel 2X, Cry1Ac (276 μg/ml), or control diet. SigniWcant diverences between treatment and corresponding controls are shown by asterisks. a a DiVerences at P < 0.05 are denoted by, P <0.01 by, P < by, and P >0.05 by Ns. b Larvae were weighed when 14, 19 and 19 days old for experiments with 0.1 Weld rate Dipel 2X, Weld rate of Dipel 2X and Cry1Ac (276 μg/ml), respectively. Experiments were started in November 2002, March 2003 and May 2003, respectively.

6 G.P. Walker et al. / Biological Control 40 (2007) the Cry1Ac diet led to a reduction (t D 7.6, n D 78, df D 76, P < 0.001) of about 1 day in the duration of the cocoon stage of the parasitoid. Adult parasitoids (held in the original larval rearing containers) had adult lives that were 1.6 days shorter (t D 2.7, n D 78, df D 65, P < 0.01) when they were reared on S. litura on the Cry1Ac diet compared with adults reared from larvae on the control diet but parasitoid adult emergence was not signiwcantly avected by the Cry1Ac diet (Table 3) H. armigera and M. pulchricornis Development and survival of H. armigera larvae and parasitization by M. pulchricornis were compared in three experiments: an experiment with diet containing the Weld rate of Dipel 2X, a second experiment with diet containing Cry1Ac at 3.68 μg/ml, and a third experiment with two concentrations of Cry1Ac (1.3 and 0.65 μg/ml), and a 1/8 low nutrition diet (Table 4). All H. armigera larvae on the Weld rate Dipel 2X diet died, so this experiment was abandoned. Diet containing 3.68 μg/ml Cry1Ac also killed all non-parasitized larvae, although one parasitoid was reared from parasitized larvae, taking 48 days to reach adulthood. Very few parasitoids were reared from the 1.3 μg/ml Cry1Ac and 1/8 nutrition diets, so detailed analyses were completed only for the 0.65 μg/ml Cry1Ac and control diets. Results for the other treatments are summarized Wrst /8 nutrition diet This diet resulted in all non-parasitized H. armigera larvae dying before pupation. At 20 days, the 38 surviving larvae had a mean weight of only 5 mg compared with 402 mg for those on the control diet. Only one parasitized larva produced a M. pulchricornis adult, and all the developmental stages except cocoon duration were delayed compared with controls (Table 4) Cry1Ac (1.3 μg/ml) diet On this diet, only three non-parasitized larvae, with reduced weight and delayed developmental attributes compared with controls, survived to produce moths. Two parasitoids were reared, also with reduced weight and delayed developmental attributes compared with controls (Table 4) Cry1Ac (0.65 μg/ml) diet This diet severely reduced the larval and pupal weights of non-parasitized H. armigera larvae and almost doubled the time to pupation. Mean time to moth emergence was more variable and was increased from 50 to 74 days (t D 4.5, n D 39, df D 4, P < 0.01) and moths were lighter (t D 4.5, n D 39, df D 37, P < 0.001). Only 12.5% of these larvae were reared to adults compared with 85% on the control diet (χ 2 D 42.1, df D 1, P < 0.001) (Table 4). Ninety-seven percent of the parasitized larvae on the control diet produced adult M. pulchricornis parasitoids. From the 40 parasitized H. armigera larvae on the 0.65 μg/ ml Cry1Ac diet 15 M. pulchricornis cocoons formed. Ten of the cocoons produced adults, the diverence in survival between the two diets being signiwcant (χ 2 D 41.8, df D 1, P < 0.001). Also, the survival of the M. pulchricornis parasitoids reared in H. armigera on the Cry1Ac diet was twice the rate of survival of the host larvae alone reared on the same diet (Table 4), although this diverence was not statistically signiwcant. The mean time to M. pulchricornis cocoon formation was approximately doubled, their weight was halved and the mean time to emergence of adult parasitoids was increased from 24 to 38 days by this diet, mostly due to diverences in larval development. Table 4 Development and survival (mean SEM) of Helicoverpa armigera (non-parasitized) and of Meteorus pulchricornis on H. armigera Low nutrition 1/8 Cry1Ac 1.3 μg/ml Cry1Ac 0.65 μg/ml Control Non-parasitized i.e. H. armigera hosts Larvae reared (n) Larvae dead (n) Larval weight (mg) a *** Days to pupa ** Pupal weight (mg) * Days to adult ** Adult weight (mg) *** Survival to adult (%) *** 85 Parasitized i.e. M. pulchricornis on H. armigera Larvae parasitized (n) Larvae dead (n) Days to cocoon *** Cocoon weight (mg) *** Days to adult *** Days as cocoon Ns Days adult life ** Survival to adult (%) *** 97 Hosts were fed diets with Cry1Ac at 1.3 and 0.65 μg/ml, 1/8 low nutrition diet, or control diet. SigniWcant diverences between the 0.65 μg/ml Cry1Ac diet and control are shown by asterisks as for Table 3. a Larvae on control diet were weighed when 20 days old, others when 21 days old. This experiment was started in June 2003.

7 148 G.P. Walker et al. / Biological Control 40 (2007) H. armigera and C. kazak Development and survival of H. armigera larvae and parasitization by C. kazak were compared in two experiments, one using 0.1 Weld rate of Dipel 2X, and a later one using diets containing Cry1Ac at two rates and a 1/6 lownutrition diet (Table 5) Weld rate of Dipel 2X Two weeks after starting the experiment, weights of larvae on this diet were heavily suppressed, to only one tenth of that of larvae on the control diet (t D 22.0, n D 56, df D 29, P < 0.001). Other development attributes were also adversely avected (P < 0.001), but host survival to adult was not signiwcantly avected (Table 5). Although there was no signiwcant diverence between diets in time to formation of parasitoid cocoons a 1-day increase in duration of the cocoon stage of parasitoids in hosts on this diet led to a corresponding increase in time to emergence of adult parasitoids. The sex ratio of 2.43:1 (male:female) was similar for both treatments and signiwcantly diverent from 1:1 (P < 0.05). Two parasitoid larvae and six cocoons died; seven of these deaths were from hosts on the control diet, reducing parasitoid survival for the control below that of the 0.1 Weld rate Dipel 2X diet (χ 2 D6.3, dfd1, P<0.05) (Table 5) Cry1Ac (0.15 and 0.3 μg/ml) diets After 14 days, both the Cry1Ac diets reduced mean host larval weights to 6 7% of controls, similar to that for the 0.65 μg/ml Cry1Ac diet (Section 3.2). Mean time to pupation was greatly extended, and pupae weighed about 40% less than the controls. For both diets time to moth emergence was delayed, moths were lighter and survival to pupation was high. Most mortality occurred in the pupal stage (20 of 26 deaths for the combined Cry1Ac diets), so overall survival to the adult stage on these diets was reduced to 60 75%, much less than the 97% survival of the controls (χ 2 D 13.4, df D 1, P < 0.001) (Table 5). The mean time to formation of C. kazak cocoons was signiwcantly increased (LSD 0.05 D 1.8, df D 116, P <0.001) when Cry1Ac diets were fed to H. armigera hosts, and parasitoid cocoon weights were signiwcantly reduced by about 22% by both Cry1Ac diets (LSD 0.05 D 0.42, df D 112, P < 0.001). Time for parasitoid development to adult was also signiwcantly extended by the Cry1Ac diet treatments and all diverences were signiwcant (P < 0.05), but the life span and sex ratio (2.0:1 male: female) of adult parasitoids was not avected (P > 0.05). Mortality of parasitoids varied signiwcantly among treatments. Only 50% of the 40 hosts on the control diet produced adult C. kazak, although all larvae were parasitized and the non-parasitized controls had excellent survival. Larvae reared on the 0.15 μg/ml Cry1Ac diet produced 28 C. kazak adults from the 39 parasitized host larvae (72% survival), which was signiwcantly higher than the control (χ 2 D 3.93, df D 1, n D 79, P <0.05). By contrast, the success rate of larvae reared on the 0.3 μg/ ml Cry1Ac diet was similar to that of the controls, with 48% of the 40 hosts producing adult C. kazak. Table 5 Development and survival (mean SEM) of Helicoverpa armigera (non-parasitized) and of Cotesia kazak on H. armigera Dipel 2X 0.1 Weld rate Hosts were fed diets with 0.1 Weld rate of Dipel 2X, Cry1Ac at 0.15 and 0.3 μg/ml, 1/6 low nutrition diet, or control diet. For the Dipel 2X diet at 0.1 Weld rate diverences from the control are shown by asterisks, and for the other treatments diverences are shown by an LSD (5%), since these four diets were tested at the same time. a LSD (5%) is the average over possible pair wise comparisons in the second part of the table, quoted because survival rates varied among treatments as shown. b Larvae were weighed when 14 days old, for both experiments, which were started in November 2002 and September 2003, respectively. c Values in brackets are means and LSD (5%) of log transformed data, where this was used to cater for skewness and heteroscedasticity in the raw data. d 3, 2 and 15 of these larvae produced parasitoid pre-pupae that died, for the 0.15 μg/ml, 0.3 μg/ml and 1/6 nutrition diets, respectively. e DiVerence from control is signiwcant (P < 0.05). Control Cry1Ac 0.15 μg/ml Cry1Ac 0.3 μg/ml Low nutrition 1/6 Control Non-parasitized i.e. H. armigera Larvae reared (n) Larval weight (mg) b *** Days to pupa *** (1.7) c 45.5 (1.7) 84.0 (1.9) 24.5 (1.4) (0.02) Survival to pupa (%) Pupal weight (mg) *** Days to adult *** (1.8) c 66.2 (1.8) (2.0) 46.3 (1.7) (0.02) Adult weight (mg) Survival to adult (%) 97 Ns Parasitized i.e. C. kazak on H. armigera Larvae parasitized (n) Larvae dead (n) d 8 d 20 d 7 Days to cocoon Ns Cocoon weight (mg) Days to adult *** Days as cocoon ** Days adult life Survival to adult (%) 97 * e LSD (5%) a

8 G.P. Walker et al. / Biological Control 40 (2007) Since independent estimates were available for the mortality of H. armigera reared on each diet in the absence of parasitoids, it was possible to estimate the parasitoid survival rate adjusted for host mortality (or potential survival rate ), assuming that the proportion of parasitoids that died due to death of the host (but not caused by the parasitoid) was equal to the proportion of non-parasitized hosts that died. Although rearing rates for the two Cry1Ac diets were signiwcantly diverent (χ 2 D 4.84, df D 1, n D 79, P < 0.05), the two data sets were pooled because their results were similar for other characters. The resulting estimates of potential survival rate were: control, 51%; 1/6 nutrition diet, 29%; Cry1Ac diets combined, 88%. A similar calculation compared the proportions of host mortality due to each of the Cry1Ac diets, adjusted for corresponding host control mortality, with that of the parasitoids. For the 0.15 Cry1Ac diet, the survival rate of 72% was signiwcantly greater than the expected rate based on applying to the parasitoids the host s speciwc mortality rate due to the diet (χ 2 D 9.48, df D 1, n D 39, P < 0.01). On the 0.3 Cry1Ac diet (Table 5) and the 0.65 Cry1Ac diet (Table 4) parasitoid survival was also greater than expected when compared with the reductions in survival of the H. armigera host on these diets, but the diverences were not signiwcant (χ 2 D 0.53 in both cases, df D 1, n D 40, P > 0.05). Thus the Cry1Ac diets used in this assay appear to have signiwcantly increased the potential parasitoid survival rate, and the 0.15 Cry1Ac diet has increased the survival rate of C. kazak compared with that of the host on this diet /6 nutrition This low nutrition diet generally had a greater evect on H. armigera than the Cry1Ac diets. After 14 days, the 1/6 nutrition diet suppressed the larval growth rate of non-parasitized larvae to about 1% of the control, mean time to pupation was more than tripled, to 84 days (df D 131, P < 0.001), but surviving pupae on the 1/6 nutrition diet emerged more quickly (t D 6.2, df D 105, P <0.001). The pupae and the moths had mean weights less than half that of the controls, and time to moth emergence was more than doubled. Only 45% of the H. armigera larvae on the 1/6 nutrition diet emerged as moths, with most mortality occurring at the larval stage (16 of 21 deaths) (Table 5). Mean time to formation of C. kazak cocoons was almost tripled, to 31 days (LSD 0.05 D 1.8, df D 116, P <0.001) by the 1/6 nutrition diet, and cocoon weight was signiwcantly reduced by an amount similar to that caused by the Cry1Ac diets. The mean time for parasitoid development to adult was almost doubled, from 20 to 39 days (LSD 0.05 D 2.3, df D 68, P < 0.001), but the life span and sex ratio of adult parasitoids was not avected (P > 0.05). Twenty (53%) of the parasitoids died as larvae but 15 of these had emerged as C. kazak pre-pupae (pre-cocoons) before dying (Table 5). Parasitoid cocoons suvered even higher mortality (72%), so that only Wve C. kazak adults (13%) were reared from parasitized H. armigera larvae on the 1/6 nutrition diet, in spite of 97% of the host larvae being parasitized. 4. Discussion The diverent developmental and survival responses of S. litura and H. armigera to the range of Bt diets tested here are consistent with numerous studies showing that Spodoptera spp. are moderately tolerant of Bt toxins, whereas Helicoverpa spp. are moderately susceptible (Glare and O Callaghan, 2000; Adamczyk et al., 2001). These two Lepidoptera species, therefore, allowed comparisons between parasitoid hosts that had ingested widely divering levels of Bt toxins. This range should be investigated because longer exposure and increasing expression in future transgenic plants may lead to increased exposure of parasitoids to transgenic toxins (Groot and Dicke, 2003). Our experiments investigated diverential evects of a single Bt toxin (Cry1Ac), a mixture of Bt toxins (Dipel 2X), and nutritional dilutions on hosts and parasitoids at concentrations that imposed stress during their development but allowed survival of both these trophic levels. The combined results for Dipel 2X at Weld rate and Cry1Ac toxins indicated that Bt intoxication of S. litura host larvae produced only minor impacts on M. pulchricornis even when the development rates and survival of the host were signiwcantly reduced. Delays in parasitoid development occurred only when host development was signiwcantly delayed. For example, the developmental delays for parasitoids ranged from 3 to 11% compared with 9 25% for moths. This sub-lethal evect on M. pulchricornis is similar to that obtained for the development of other parasitoids, including Parallorhogas pyralophagus (Marsh) parasitizing a tropical stem borer Eoreuma loftini (Dyar) (Bernal et al., 2002), two parasitoids of Pseudoplusia includens Walker (Baur and Boethel, 2003), and two braconid parasitoids of potato tuber moth Phthorimaea operculella (Salama et al., 1996). Lethal evects showed similar trends. Survival of M. pulchricornis was unavected even when the survival of S. litura larvae was signiwcantly reduced (72 80%) by the Weld rate of Dipel 2X or Cry1Ac at 276 μg/ml. Several prior experiments have obtained similar results using diverent techniques. For example, parasitoids have been reared successfully from Bt-resistant populations of diamondback moth that survive feeding on microbial Bt (Chilcutt and Tabashnik, 1997) or Bt-transgenic rape (Schuler et al., 1999). These results contrast with records of reduced survival of parasitoids when susceptible hosts are fed Bt crops (Bernal et al., 2002; Baur and Boethel, 2003). In summary, our results indicate that the survival of M. pulchricornis in S. litura is unlikely to be avected by a wide range of Bt toxin levels. Rearing of H. armigera on diets containing 0.1 Weld rate of Dipel 2X or Cry1Ac concentrations of 0.65, 0.3 and 0.15 μg/ml all provided enough survivors for comparisons of the evects of Bt toxins on hosts and parasitoids. For all these treatments, delays in larval development of M. pulchricornis and C. kazak were associated with delays in host development, as observed in experiments with S. litura. For M. pulchricornis, the Cry1Ac diets caused

9 150 G.P. Walker et al. / Biological Control 40 (2007) delays of about 45% in the time to adult emergence for both non-parasitized hosts and parasitoids, whereas development of C. kazak was delayed by only 25% compared with 48% for hosts. This evect, together with reductions in cocoon weight associated with Cry1Ac treatments, is similar to previous observations for other parasitoids of Lepidoptera (Salama et al., 1996; Bernal et al., 2002; Baur and Boethel, 2003). Bt-amended diets appeared to produce varying evects for survival of the two parasitoids in H. armigera. For M. pulchricornis, the 0.65 μg/ml Cry1Ac diet reduced parasitoid survival to about 25%, but this impact was only half that recorded in non-parasitized hosts (Table 4). The relatively small sample sizes that were possible in our work mean that this result is not statistically signiwcant, but when taken in conjunction with other survival data reported here it is worth noting. For example, the survival of one to two parasitoids in three even more stressful diet treatments (Cry1Ac at 3.68 μg/ml and 1.3 μg/ml, and 1/8 nutrition diets) that killed most or all host larvae in nonparasitized treatments indicated that parasitoids may be less avected by Bt toxins than their hosts. Further, survival of C. kazak in H. armigera fed 0.15 μg/ml or 0.3 μg/ ml Cry1Ac and the 0.1 Weld rate of Dipel 2X produced similar or higher levels of parasitoid survival compared with controls. Also, the speciwc survival rate for C. kazak on the 0.15 μg/ml Cry1Ac diet (adjusted for control mortality) was signiwcantly higher than the corresponding rate for their H. armigera hosts. This appears to be possible because the parasitoids complete their larval development and are able to emerge and pupate before their sick host would have died if it were not parasitized, so long as the Bt toxin level is present at a level that does not cause death of the host at an early instar. Increased survival of parasitoids in larvae fed some Bt-amended diets compared with controls or hosts indicated that the toxins benewted parasitoids in some experiments. This phenomenon may be related to hormetic responses including increased survivorship caused by sub-lethal exposures to some pesticides (Morse, 1998). Although Morse (1998) points out that for conventional insecticides, many Weld rates are toxic to natural enemies, the dose responses of parasitoids to transgenic dosages of Bt toxins are largely unknown, so positive evects may occur. No similar observations of increased survival of parasitoids with continuous exposure to Bt-treated hosts have been located for other species. Reduced nutrition diets for H. armigera were added to the experiments to investigate hypotheses that evects of Bt treatments may be largely the result of poor host nutrition caused by the evects of Bt toxins on host gut membranes (Groot and Dicke, 2003). Where the reduced nutrition diets allowed signiwcant survival of host larvae (1/6 nutrition diet), poor survival of parasitoids suggested that reduced nutrition had a comparatively greater evect on parasitoid survival than Cry1Ac toxins. We noted the emergence, before the hosts died, of 15 pre-pupae (pre-cocoons) from host larvae that had been fed the 1/6 nutrition diet (Table 5). These pre-pupae also died, but their emergence parallels the ability of the parasitoids to develop successfully in sick hosts fed the Bt toxin. Comparisons of the evects of starvation or diet incorporation of Cry1Ac with the light brown apple moth Epiphyas postvittana (Walker) showed that only Cry1Ac caused cell lysis but both treatments caused similar weight loss compared with controls (Sutherland et al., 2003). However, after these treatments, larvae initially starved made lower weight gains than those fed Cry1Ac, which suggests that starvation would have greater evects on parasitoids than Bt toxins. Overall, our studies conwrm previous observations that there are no direct evects of Bt toxins on developing parasitoid larvae and provide additional information to support the hypothesis that the evects are indirect and mediated through the health of the host. Firstly, exposure of M. pulchricornis in S. litura to a wide range of concentrations of Bt toxins produced no change in survival of the parasitoid. By comparison, survival of the same parasitoid in H. armigera was severely reduced by Bt toxins at much lower rates than used for S. litura, but only at concentrations that avected the health of the host as indicated by measures of survival and development. Thus, at a concentration of 0.65 μg/ml Cry1Ac in H. armigera, the toxins signiwcantly reduced parasitoid survival, whereas at over 400 times this concentration (276 μg/ml) in S. litura, no evect was detected. In addition to studies of larval survival and development, full evaluation of the impacts of Bt toxins on parasitoids requires examination of other phases of the host parasitoid association, from habitat and host location, to oviposition preferences, and performance of the surviving adults. In Welds of Bt toxic plants, a reduction in host- Wnding cues may cause a depression of parasitoid activity although oviposition choice between intoxicated and normal hosts does not appear to be avected (Schuler et al., 1999). Our experiments detected some evects of larval parasitoid exposure to Bt toxins on the adult longevity of individuals that develop successfully to this stage, but we obtained no measures of their fecundity. Few studies have examined impacts of transgenic toxins on subsequent performance of adult parasitoids. Baur and Boethel (2003) reported signiwcantly fewer ova in Cotesia marginiventris (Cresson) developing inside P. includens larvae fed Bt cotton (NuCotn 33B) expressing Cry1A(c), while Bell et al. (1999) observed a positive inxuence of snowdrop lectin (GNA) on brood size of Eulophus pennicornis (Nees) reared on Laconobia oleracea L. feeding on GNA-expressing plants. To date, our results support hypotheses that Bt toxins act only indirectly on parasitoids, but these results require conwrmation using transgenic Bt toxins expressed at concentrations relevant to the target species. In addition, assessment of potential impacts of Bt-transgenic crops on the ecological aspects of host parasitoid interactions, including host location and synchrony with hosts, will require preliminary Weld trials.

10 G.P. Walker et al. / Biological Control 40 (2007) Acknowledgments We thank Anne Barrington and Vicky Bush for technical assistance with diet preparation. The laboratory cultures of Spodoptera litura and Helicoverpa armigera were supplied by HortResearch. This project was funded by the New Zealand Foundation for Research, Science and Technology. References Adamczyk, J.J., Hardee, D.D., Adams, L.C., Sumerford, D.V., Correlating diverences in larval expression and development of bollworm (Lepidoptera: Noctuidae) and fall armyworm (Lepidoptera: Noctuidae) to diverential expression of Cry1A(c) delta-endotoxin in various plant parts among commercial cultivars of transgenic Bacillus thuringiensis cotton. J. Econ. Entomol. 94, Baur, M.E., Boethel, D.J., EVect of Bt-cotton expressing Cry1A(c) on the survival and fecundity of two hymenopteran parasitoids (Braconidae, Encyrtidae) in the laboratory. Biol. 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