Impact of snowdrop lectin (Galanthus nivalis agglutinin; GNA) on adults of the green lacewing, Chrysoperla carnea

Journal of Insect Physiology 55 (2009) 136–143

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Impact of snowdrop lectin (Galanthus nivalis agglutinin; GNA) on adults of the green lacewing, Chrysoperla carnea Yunhe Li, Jo¨rg Romeis * Agroscope Reckenholz-Ta¨nikon Research Station ART, Reckenholzstr. 191, 8046 Zurich, Switzerland

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 February 2008 Received in revised form 26 September 2008 Accepted 30 October 2008

Based on the finding that Galanthus nivalis agglutinin (GNA) has direct negative effects on larvae of Chrysoperla carnea, laboratory experiments were conducted to investigate its toxicity to the adults. While the ingestion of GNA dissolved in an artificial diet did not affect adult longevity, there were concentration-dependent negative effects on the pre-oviposition period, daily fecundity and total fecundity (number of eggs laid). When GNA was ingested by larvae of C. carnea, it caused a significant extension of larval development time. Adults that had emerged from GNA-fed larvae did not differ from those that developed from control larvae in terms of adult fresh weight, pre-oviposition period and daily or total fecundity. However, fertility (proportion of hatching eggs) was significantly decreased in adults raised from GNA-treated larvae. Western blots revealed that GNA ingested by larvae of C. carnea was partly transferred to the adult stage and was subsequently excreted or digested within a few days. Our toxicity studies (Tier-1 tests) clearly established a hazard of GNA to adult C. carnea when administered to larvae or adults at high concentrations. Implications of these toxicity data for the non-target risk assessment of GNA-expressing transgenic crops are discussed. ß 2008 Elsevier Ltd. All rights reserved.

Keywords: Genetically modified plants Hazard assessment Non-target effects Risk assessment Tier-1 testing

1. Introduction Genes encoding plant-derived lectins have been introduced through genetic engineering into a number of crops with the aim of increasing the plant’s resistance to insect pests (Malone et al., 2008). The snowdrop lectin (Galanthus nivalis agglutinin, GNA), derived from the snowdrop lily (G. nivalis L., Amaryllidaceae; Van Damme et al., 1987), has received the most attention since it acts on sap-feeding insects, such as Aphididae, Cicadellidae and Delphacidae belonging to the Hemiptera (Powell et al., 1993, 1995, 1998; Rahbe´ et al., 1995; Sauvion et al., 1996). These pests are not affected by the well-known insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) which are expressed in the current commercialized insect-resistant transgenic crops (Shelton et al., 2002). In addition to effects on sap-feeding pests, the insecticidal effects of GNA have also been found to extend to the Lepidoptera (Fitches and Gatehouse, 1998; Se´tamou et al., 2002a, 2003), Coleoptera (Nutt et al., 1999) and Acari (McCafferty et al., 2008). Genes that encode GNA have been incorporated into a range of crops including potato (Gatehouse et al., 1996; Down et al., 1996), rice (Foissac et al., 2000; Nagadhara et al., 2003, 2004; Rao

* Corresponding author. Tel.: +41 44 377 72 99; fax: +41 44 377 72 01. E-mail address: [email protected] (J. Romeis). 0022-1910/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2008.10.015

et al., 1998; Wu et al., 2002), maize (Wang et al., 2005), tobacco (Hilder et al., 1995), wheat (Stoger et al., 1999), tomato (Wu et al., 2000) and sugarcane (Se´tamou et al., 2002a, b, 2003), conferring partial resistance to sap-feeding pests (Malone et al., 2008). In contrast to the specific Bt Cry proteins, lectins like GNA have a higher potential to cause unintended effects on non-target organisms due to their relatively broad range of activity (O’Callaghan et al., 2005; Malone et al., 2008). Such non-targets may include arthropod predators and parasitoids that are important for natural pest regulation (Romeis et al., 2008a). As the target insect pests are only partially controlled by GNA-expressing plants, the additional impact of antagonists such as predators and parasitoids is required for effective pest control. A number of studies have investigated the effects of GNAexpressing plants on hymenopteran parasitoids. Most studies in which the parasitoids have been provided with GNA-fed, sublethally affected aphids or lepidopteran larvae as hosts have reported negative effects on the parasitoids (Malone et al., 2008). However it remains unclear whether the observed effects were caused directly by the insecticidal protein or indirectly a consequence of effects on the host (Romeis et al., 2006). Clear direct toxic effects have only been established in studies where the parasitoids had been directly fed with high concentrations of GNA dissolved in a sucrose solution (Romeis et al., 2003; Bell et al., 2004) or contained in aphid honeydew (Hogervorst et al., 2009).

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In the case of arthropod predators, Birch et al. (1999) reported reduced fecundity, egg viability and a reduction in female longevity of Adalia bipunctata (Coleoptera: Coccinellidae) when preying on the aphid Myzus persicae (Hemiptera: Aphididae), which had fed on GNA-expressing potatoes. These results were, however, not confirmed in subsequent studies by Down et al. (2000, 2003) where a range of life-table parameters of A. bipunctata larvae and adults were not affected when feeding on aphids reared on GNA potatoes or on GNA-containing artificial diet. Few studies provide evidence for direct toxic effects of GNA on predatory insects. Bell et al. (2003) reported adverse effects on the predatory bug Podisus maculiventris (Heteroptera: Pentatomidae) when the insects were reared on larvae of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae) that had been artificially injected with GNA. Hogervorst et al. (2006) investigated direct effects of GNA on larvae of three species of important aphid predators, i.e. Chrysoperla carnea (Neuroptera: Chrysopidae), A. bipunctata and Coccinella septempunctata (Coleoptera: Coccinellidae). Longevity of the larvae of all three species was directly affected by GNA, when fed a sucrose solution containing GNA at a high concentration of 1% (weight per volume, w/v). In addition, the study revealed that gut enzymes from none of the three species were able to break down GNA. Nevertheless, when the ladybird larvae were transferred to a diet devoid of GNA, the GNA in their bodies decreased over time, probably due to excretion (Hogervorst et al., 2006). In contrast the GNA concentration remained high in C. carnea, and this was explained by the fact that the larvae lack a connection between the mid- and hindguts and are therefore not able to excrete the protein (Yazlovetsky, 2001). Based on these previous findings, the present study addressed the following three questions: (i) Does GNA have a direct toxic effect on adult C. carnea? (ii) Does consumption of GNA by larvae of C. carnea have subsequent effects on the fitness of adults? (iii) What is the fate of GNA after ingestion by larvae of C. carnea? The data from these Tier-1 toxicity (hazard) studies are discussed in respect to the potential risk that GNA-expressing transgenic plants would pose to C. carnea (Romeis et al., 2008b). 2. Materials and methods 2.1. Reagents Lyophilized GNA was obtained from Els J.M. van Damme (Ghent University, Belgium), which was isolated from snowdrop bulbs as described by Van Damme et al. (1987). All the GNA used in this study was from the same batch for which biological activity was confirmed by an agglutination test using rabbit erythrocytes (Van Damme et al., 1987). GNA primary antibodies were provided by Prof. J.A. Gatehouse (University of Durham, UK). Bradford reagent and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories GmbH (Mu¨nchen, Germany). Sucrose was purchased from Merck (Darmstadt, Germany). All other chemicals were from Sigma Chemical Company (Buchs, Switzerland) and were of analytical class unless otherwise stated. 2.2. Insects Eggs of C. carnea were collected from a permanent laboratory colony (for details on the rearing see Romeis et al., 2004) and kept in a climatic chamber at 22  1 8C, 75  5% RH and 16:8 L:D until they hatched. When adults of C. carnea were used in the experiments, the larvae were reared on eggs of Ephestia kuehniella (Lepidoptera: Pyralidae) provided by Biotop (Valbonne, France). After the larvae had reached the second instar, they were kept individually in plastic tubes

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(5.5 cm high, 1.5 cm diameter) and supplied ad libitum with eggs of E. kuehniella until pupation. 2.3. Adult feeding bioassay Pairs of newly emerged adults of C. carnea were individually confined to plastic cylinders (6.0 cm diameter, 8.5 cm high) ventilated by a gauze window (about 4 cm diameter) in the lid. The gauze also served as an oviposition substrate. At the bottom of each plastic container, there was a hole (about 1 cm diameter) through which the insects could obtain water from a wetted cotton roll which connected with water in a reservoir. The insects were provided ad libitum with water and an artificial diet (sucrose: brewer’s yeast: water at 7:4:4) commonly used for rearing adult C. carnea (Hagen and Tassan, 1966, 1970; Jones et al., 1977) containing GNA at one of the following concentrations: 0%, 0.01%, 0.1% or 1% (w/v). The diets and cages were replaced every 3 days. The experiment was conducted in a controlled climatic chamber (22  1 8C, 75  5% RH, 16:8 L:D) and lasted for about six months until the last insect died. Insects were checked daily and the following parameters were recorded: survival, pre-oviposition period (mean number of days from adult emergence to the first oviposition), daily fecundity (number of eggs laid per female per day), total fecundity (total number of eggs laid per female), and fertility (number of eggs hatching divided by number of eggs laid). Eggs laid by each female were counted daily and removed from the test container. To estimate egg fertility, eggs collected every 3rd–5th day from the day after the onset of oviposition until the female died were left on their oviposition substrates (gauze) and placed in separate containers together with E. kuehniella eggs. The provision of ad libitum prey ensured minimal cannibalism of hatching larvae. After 7 days, the larvae were counted and egg hatching rates were calculated. Since some adults escaped, were killed due to handling or never laid fertile eggs, some test units were excluded from further analyses. In total, data from 10–14 pairs of C. carnea were analyzed for each of the different treatments. Pair-wise comparisons were done for each of the GNA concentrations with the control. Bonferroni correction was applied for 3 pair-wise comparisons leading to an adjusted a = 0.017. Longevity and total fecundity data were analyzed using Student’s ttests. Repeated-measures ANOVA (RM-ANOVA) were performed using daily fecundity data. Data on the pre-oviposition period and fertility were compared using Mann–Whitney U-test, since these data were not normally distributed and variances were not homogenous. Percentage fertility data were transformed using the arcsine of the square root prior to the analysis. 2.4. Larval feeding bioassay 2.4.1. GNA-ingestion by larvae Freshly hatched C. carnea larvae (<12 h old) were placed individually in plexiglass containers (2 cm  2 cm  1.5 cm). Each insect received either two droplets of a pure 2 M sucrose solution or a sucrose solution containing GNA at a concentration of 1% (w/v) on the first 2 days of each larval instar. After each session of sucrose-feeding, C. carnea larvae were transferred to new containers and fed with E. kuehniella eggs ad libitum until their next moult. The concentration of 1% GNA was used since previous studies had shown significant adverse effects of GNA on different life-table parameters of C. carnea larvae at this concentration (Hogervorst et al., 2006; Lawo and Romeis, 2008). The experiment was carried out in a controlled climate chamber at 22  1 8C, 85  5% RH and 16:8 L:D until adult emergence. The larvae were checked twice per

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day (9 a.m. and 5 p.m.), and the following parameters were recorded: mortality, development time of larvae (i.e. days required to reach pupal stage) and pupation rate. Larval development was considered completed when the pre-pupal cocoon was formed. In order to confirm that larvae had ingested GNA, 10 larvae were collected from each treatment group 1 day before they were estimated (from preliminary experimentation) to begin pupating and stored at 80 8C for later GNA-detection by Western blot. Larvae that escaped or were killed due to handling during this experiment were discarded from statistical analyses. This resulted in a total of 106 insects in the GNA treatment and 103 insects in the control. Data on larval mortality and pupation rates were analyzed using Chi-square tests. Larval development time was analyzed using Mann–Whitney U-test. 2.4.2. Subsequent effects on adults Adults that had emerged within 12 h from pupae produced in the experiment above were sexed and weighed. These adults were then paired within 48 h after emergence, and each pair was confined to a plastic cylinder (6.0 cm diameter, 8.5 cm high) and provided with the same artificial diet as described above, but devoid of GNA (regardless of their diet during the larval stage). In total, 18 pairs derived from GNA-fed larvae and 17 pairs from the control larvae were tested. The experimental system and methods were similar to those described in Section 2.3. This experiment was terminated after 18 days. Insects were checked daily and the following parameters were recorded: survival, pre-oviposition period, daily and total fecundity and fertility. For the fertility investigation, eggs were collected from three particular days (at the beginning, middle and end of the oviposition period). Other parameters were recorded using the same methods as described in Section 2.3. Adults that escaped or were killed due to handling and those which never laid fertile eggs were discarded from statistical analyses. In total, data from 17 pairs of adults were analyzed for the GNA treatment and 14 pairs of adults for the control. Since preoviposition data were not normally distributed and variances were not homogenous they were analyzed using Mann–Whitney U-test. Daily fecundity was analyzed using RM-ANOVA. Data on all other parameters assessed were analyzed using Student’s t-test. Percentage fertility data were transformed using the arcsine of the square root prior to the analysis.

transferred to 0.2 mm nitrocellulose membranes (Schleicher & Schuell, BA83). The membranes were developed for immunoassay by Western blotting, using polyclonal antibodies raised against GNA as the primary antibody, and HRP-conjugated goat anti-rabbit IgG as the secondary antibody as described by Gatehouse et al. (1996). Specifically bound antibody was detected by enhanced chemiluminescence (ECL) according to the manufacturer’s instructions. 3. Results 3.1. Adult feeding bioassay GNA incorporated in artificial diet had no significant effect on the longevity of female C. carnea adults at the concentrations provided when compared to the control (Student’s t-test, adjusted a = 0.017; 0.01% GNA: t23 = 0.71, P = 0.486; 0.1%: t22 = 0.11, P = 0.910; 1%: t26 = 0.60, P = 0.553) (Fig. 1A). Similarly, male C. carnea lived on average between 169 and 188 days and also no differences between any GNA treatment and control was observed (P > 0.05). However, the pre-oviposition periods of female adult C. carnea fed with 0.1% or 1% GNA were significantly reduced compared with the control (Mann–Whitney U-test, adjusted a = 0.017; 0.01%: U = 60.5, P = 0.524; 0.1%: U = 25.0, P = 0.013; 1%: U = 33.0, P = 0.005) and total fecundity was significantly reduced in those fed with 1% GNA (Student’s t-test, adjusted a = 0.017; 0.01%: t22 = 0.78, P = 0.444; 0.1%: t21 = 1.85, P = 0.079; 1%: t25 = 3.83, P < 0.001) (Fig. 2B and C). While insects fed control diet laid an average of 1797.6 eggs during their lifetime, this was reduced to a total of 1035.4 eggs in the 1% GNA treatment. Similarly, a pair-wise RM-ANOVA revealed that only adult females fed with 1% GNA had significantly reduced daily fecundity, compared with the control (0.01%: F1,21 = 0.73, P = 0.404; 0.1%: F1,19 = 2.12, P = 0.162; 1%: F1,20 = 11.31, P = 0.003) (Fig. 2). Day  treatment interactions were also significant (F103,2060 = 1.58, P < 0.001) for the daily fecundity data. Fertility was significantly reduced for eggs laid by adults exposed to 0.1% GNA when compared to eggs from adults fed control diet. In contrast, egg fertility was not reduced when adults were fed with diet containing 0.01% or 1% GNA (Mann–Whitney U-tests, adjusted a = 0.017; 0.01%: U = 61.0 P = 0.381; 0.1%: U = 28.0, P = 0.014; 1%: U = 60.0, P = 0.081).

2.5. Fate of GNA after ingestion by C. carnea larvae 3.2. Larval feeding bioassay Freshly hatched C. carnea larvae (<12 h old) were fed on E. kuehniella eggs ad libitum individually in plexiglass containers (2 cm  2 cm  1.5 cm). Upon reaching their last instar, larvae were transferred to new containers and provided with two droplets of a pure 2 M sucrose solution or a sucrose solution containing 1% GNA (w/v) for 3 days to ensure that larvae contained GNA before pupation. Subsequently, larvae were fed E. kuehniella eggs until pupation. After emergence of adults, 10 females and 10 males were randomly collected from each treatment and immediately stored at 20 8C. The remaining insects were fed on artificial diet devoid of GNA. After 1, 2, 4, 8 and 16 days, samples of 10 females and 10 males each were taken in the same way. After all the samples had been collected, the frozen adults were thawed on ice, their wings removed using scissors, and homogenized in 0.4 ml PBS buffer per 10-adult sample. The resultant suspension was shaken at 4 8C for 15 min, centrifuged at 13,000  g for 5 min, and the supernatant collected. Total soluble protein was determined by Bradford assay according to the manufacturer’s instructions. Sample proteins were separated by SDS-PAGE (12.5%) (approximately 10 mg total soluble protein was loaded), then electrophoretically

3.2.1. Effect of GNA on larvae When larvae of C. carnea were provided with a sucrose solution containing 1% GNA or a pure sucrose solution, survival and pupation rates did not differ between the two treatments, while larval development time was significantly prolonged (by 3.7 days on average) in the GNA treatment (Table 1). Western blots indicated the presence of GNA in C. carnea larvae just prior to pupation (data not presented), which confirmed the ingestion of GNA during larval development. 3.2.2. Subsequent effect on adults The ingestion of GNA (at the high concentration of 1%) during the larval stage had no effect (P > 0.05) on the tested life-table parameters during the adult stage, i.e. male and female weights, pre-oviposition period, and total fecundity (Table 1). Similarly, daily fecundity remained unaffected (RM-ANOVA; F1,29 = 1.25, P = 0.27) (Fig. 3). The only significant difference that was detected between the two treatments was a reduced fertility for eggs produced by adults that had ingested GNA during the larval stage (Table 1).

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Fig. 1. Different life-table parameters of female Chrysoperla carnea, when fed artificial diet containing 0, 0.01, 0.1 and 1% snowdrop lectin (GNA) (w/v) (n = 10–14 pairs per treatment). Asterisks above bars indicate results which differ significantly from the control ((A and C) Student’s t-test; (B and D) Mann–Whitney U-test with Bonferroniadjusted a = 0.017).

3.3. Fate of GNA after ingestion by larvae To determine whether GNA was transferred to the adult stage after ingestion during the larval stage, and the endogenous stability of GNA in adult C. carnea, protein extracts from adults that had consumed GNA as larvae were analyzed at different time intervals after adult emergence. As shown in Fig. 4, GNA could be detected in adults by Western blotting. In the case of female C. carnea, the GNA protein present in their body tissues decreased over time, and could not be detected 4 days after emergence. However, GNA was detected for much longer in male insects and

was still confirmed after 8 days. Eventually the protein also disappeared from male bodies and was not detectable after 16 days. 4. Discussion 4.1. Direct effects of GNA on adult C. carnea Female C. carnea adults fed with control artificial diet lived for an average of 102.2 days, started laying eggs after about 3.6 days, and laid a total of 1797.6 eggs during their life time. When adults

Fig. 2. Mean daily fecundity (number of eggs laid per female per day) of Chrysoperla carnea adults fed with artificial diet containing snowdrop lectin (GNA) at 0, 0.01, 0.1 and 1% (w/v) (n = 10–14 pairs per treatment). Daily fecundity of insects fed 1% GNA diet was significantly decreased when compared to the control (repeated-measures ANOVA, P = 0.003).

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Table 1 Impact of snowdrop lectin (GNA) on larvae and adults of Chrysoperla carnea. Larvae were fed either with 2 M sucrose solution (control) or with 2 M sucrose solution containing 1% GNA (w/v) during the first 2 days of each larval stage and provided with E. kuehniella eggs during the remainder of each instar. Emerging adults were fed with standard artificial diet devoid of GNA for 18 days. Number of replicates is given in parentheses. Parameters

GNA

Control

Statistics

Larval survival (%) Larval development time (d  SE) Pupation rate (%) Male fresh weight (mg  SE) Female fresh weight (mg  SE) Pre-oviposition period (d  SE) Total number of eggs per female (SE) Fertility (% eggs hatched)

97.17 (106) 18.8  0.21 (95) 92.20 (103) 9.3  0.20 (30) 11.0  0.20 (24) 3.6  0.15 (17) 270.7  23.67 (17) 56.2  6.26 (17)

98.06 (103) 15.1  0.13 (96) 95.05 (101) 9.1  0.11 (32) 11.1  0.19 (28) 3.4  0.14 (14) 308.0  22.94 (14) 75.5  5.61 (14)

x2 = 0.18, P = 0.67a

a b c

U = 716.0, P < 0.001b x2 = 0.68, P = 0.41a t = 0.96, P = 0.34c t = 0.29, P = 0.77c U = 90.0, P = 0.25b t = 1.12, P = 0.27c t = 2.36, P = 0.03c

Chi-square test. Mann–Whitney U-test. Student’s t-test.

cidae), has been shown to be a subunit of ferritin, which suggests that the lectin may interfere with iron homeostasis. More recently, it has been shown that GNA binds to glycoproteins in the guts of larvae of predator species, i.e. C. carnea, A. bipunctata and C. septempunctata (Hogervorst et al., 2006). Additionally GNA has been reported to bind to the gut tissue of larvae of A. bipunctata (Down et al., 2000) and that of pupae and adults of the aphid parasitoid Aphelinus abdominalis (Hymenoptera: Aphelinidae) (Couty et al., 2001) when offered GNA-fed aphids as prey or hosts. It has also been suggested that passage of GNA into the haemolymph, internal organs and glands can make an important contribution to the toxicity, since the lectin may cause general systemic effects (Powell et al., 1998). Previous studies have shown that GNA can be transported across the midgut epithelial barrier into the haemolymph in N. lugens (Powell et al., 1998) and L. oleracea (Fitches et al., 2001). A recent study also indicated that GNA is occasionally present in the haemolymph of C. carnea and A. bipunctata larvae, which have fed on a sucrose solution containing 0.1% GNA (w/v) (Hogervorst et al., 2006).

were fed with artificial diet containing different concentrations of GNA, direct, negative, concentration-dependent effects were detected for the pre-oviposition period and for total and daily fecundity. Interestingly, C. carnea survival remained unaffected even at the highest GNA concentration (1%). A small but significant reduction in egg fertility was also detected, but only among adults fed with 0.1% GNA and not the higher dose. One possible explanation is that adults receiving this concentration of GNA had their fecundity reduced, but were still able to lay some GNAdamaged eggs which failed to hatch, whereas in the adults given the highest concentration of GNA such eggs were so damaged that they were not even laid (indicated by the reduced fecundity). Since the GNA-containing diet was changed every 3 days, GNA is known to be a very stable protein (Van Damme et al., 1987; Hogervorst et al., 2006) and the fresh weight of the diet decreased only by around 8% during the 3-day period (own observations), we believe that the insects were exposed to a relative constant concentration of insecticidal protein throughout the bioassay. Despite the fact that GNA has been found to cause direct toxic effects to a range of arthropods, the toxicity mechanism of GNA, and lectins in general, is not yet fully understood (Czapla, 1997; Harper et al., 1995; Hogervorst et al., 2006). Even though binding to glycoproteins in the insect’s midgut is considered a prerequisite for the toxicity of lectins (Czapla, 1997) it is not an absolute predictor of subsequent toxicity (Harper et al., 1995). It has been reported that GNA binds to midgut epithelial cells of sensitive insects and causes morphological changes which are thought to affect nutrient absorption (Powell et al., 1998). In addition, Du et al. (2000) demonstrated that one of the receptors for GNA in the gut of the rice brown planthopper, Nilaparvata lugens (Hemiptera: Delpha-

The longevity of C. carnea larvae was directly affected by GNA when they were fed a sucrose solution containing 1% GNA (Hogervorst et al., 2006). However, since a sucrose solution alone does not provide the nutrients essential for development of the predator to the next larval stage, it was not possible in that study to assess the consequent effects of GNA on the next life stages, such as pupae and adults. In the present study, the larvae of C. carnea were provided with a 2 M sucrose solution containing GNA or a pure

Fig. 3. Mean daily fecundity (number of eggs laid per day) of Chrysoperla carnea adults fed with artificial diet (sucrose:water:yeast = 7:4:4) for 18 days. As larvae, these adults had been fed with 2 M sucrose solution containing 1% GNA (w/v) or a pure sucrose solution (control) on the first 2 days of each larval stage, and then eggs of E. kuehniella for the remainder of each instar. Repeated measured ANOVA showed no statistical difference between GNA and control treatments (P = 0.27).

Fig. 4. Western blot of GNA-persistence study with Chrysoperla carnea adults that had been fed with GNA as larvae. Freshly emerged last instar larvae were provided a 2 M sucrose solution with 1% GNA (w/v) for 3 days, and subsequently offered eggs of E. kuehniella until pupation. Surviving adults were fed with an artificial diet. Samples of adults were taken 0, 1, 2, 4, 8, and 16 days after emergence. Protein extracts from samples comprising 10 pooled females or males were resolved by SDS-PAGE, electroblotted onto nitrocellulose and probed with anti-GNA antibodies. The first lane (GNA) contains 0.1 mg of GNA. The GNA bands appeared at a size of approximately 12.5 kDa.

4.2. Consequences of GNA-ingestion by C. carnea larvae for adults

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sucrose solution only on the first 2 days of each larval stage, then fed with E. kuehniella eggs for the rest of each instar, so that the larvae could successfully develop into adults. The ingestion of GNA had no negative effects on larval survival, pupation rate, and the fresh weights of newly emerged adults, while it caused a significant extension in larval development time. These results were consistent with the findings of a recent study by Lawo and Romeis (2008), except that a reduction in larval survival was also detected in their study. Once C. carnea adults had developed from the GNA-fed or control larvae, both groups received an artificial diet without GNA for 18 days to evaluate whether GNA-feeding during the larval stage causes detrimental effects on adult life-table parameters. No significant differences attributable to GNA were found in the pre-oviposition period or the daily and total fecundity of these adults. Surprisingly the fertility of females that had developed from GNA-fed larvae was reduced by almost 20% when compared to control insects. In addition, it was found that in the GNA treatment many newly hatched larvae died immediately after emergence while they had not yet fully escaped from the egg shell. This was rarely observed in the control treatment. It thus appears that the detrimental effect of the high (1%) dose of GNA on the C. carnea larvae was partly transferred to the adult stage. The reasons for this are unclear but likely a consequence of the damage caused during the larval stage and not a direct effect of the GNA on the adults. Western blot studies of whole insects indicated that the GNA rapidly disappeared in the bodies of adult lacewings after emergence (see below). We can, however, not rule out the possibility that low amounts of protein crossed the gut boundary into the haemolymph which were below our detection limit but still sufficient to cause the observed detrimental effects. However, we found in this and a previous study (Li et al., 2008) that egg fertility was not affected when adults were directly fed with artificial diet containing 1% GNA for 28 days. 4.3. Fate of GNA after ingestion by C. carnea larvae The finding that GNA ingested during the larval stage is transferred to the subsequent adult stage of a holometabolous insect such as C. carnea might be explained by two facts. First, larvae of C. carnea lack a connection between the mid- and hindgut (Yazlovetsky, 2001) and are therefore not able to excrete the ingested GNA. Second, the digestive enzymes of C. carnea larvae cannot digest GNA (Hogervorst et al., 2006) which is known to be a particularly stable protein since it also remains unaltered after passing through the gut system of lepidopteran larvae (Gatehouse et al., 1995; Fitches and Gatehouse, 1998). In an earlier study, Couty et al. (2001) also detected GNA in the pupae and adults of A. abdominalis that had developed in GNA-fed aphids. The transfer of exogenously derived larval toxicants to pupal and adult stages has been reported from a number of holometabolous insect species (Bowers, 1992; Dettner et al., 1997). It may reflect a degree of adaptation to cope with the ingested toxicants and provide the insects with chemicals that protect them from attack by their antagonists (Brown, 1984; Schaffner et al., 1994). It would be interesting to conduct further studies to determine whether GNA is also transferred from the larval to the adult stage in other holometabolous insect species. After emergence of C. carnea adults, the amount of GNA present in their body tissues decreased over time. GNA disappeared in females within 4 days, but could still be detected in males 8 days after emergence. This difference between the sexes is probably due to the fact that females replace their gut contents shortly after emergence due to immediate food-ingestion, while males consume comparably less food so that their gut contents are replaced

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more slowly (Li & Romeis, unpublished data). Based on these observations the disappearance of the GNA from the adult bodies was more likely due to excretion rather than digestion. 4.4. Ecological implications Earlier studies by our group (Hogervorst et al., 2006; Lawo and Romeis, 2008) have shown that feeding on high concentrations of GNA causes direct detrimental effects on C. carnea larvae. The present study clearly established a hazard of GNA to adult C. carnea at a high concentration (0.1–1%, w/v). Interestingly, adult C. carnea were not only affected when they directly consumed GNA but also as a consequence of GNA-feeding during the larval stage. These findings imply that to fully assess the risk of GNA-expressing plants, non-target studies may need to cover the whole life cycle of the test organism. However, to establish whether GNA-expressing transgenic plants would pose a risk to C. carnea under field conditions one would have to consider the level at which these predators may be exposed to this insecticidal protein (Romeis et al., 2008b). Since GNA-expressing plants that provide sufficient target pest control are not available yet, a detailed exposure assessment is impossible as it is unknown at which level the insecticidal protein would be present in different plant tissues. A number of studies have shown, however, that a GNA concentration of 0.1% in the phloem sap is required to achieve a detectable impact for example on aphids (Down et al., 1996; Sauvion et al., 1996; Couty et al., 2001). Adult C. carnea are known to feed on honeydew, sugar-rich excretions produced by sap-feeding Sternorrhynchae (Principi and Canard, 1984; Hogervorst et al., 2007). The insecticidal proteins expressed by transgenic crops targeting sap-feeding pests, such as GNA-transgenic plants, may be transferred to honeydew excreted by the phloem feeders (Romeis et al., 2008a; Hogervorst et al., 2009). For example, GNA has been found in the honeydew produced by peach potato aphids (M. persicae) feeding on GNA-transgenic tobacco plants (Shi et al., 1994). Powell et al. (1998) showed that the GNA concentration in the honeydew is close to that in the food source for the rice brown planthopper, N. lugens, when feeding on artificial diet containing GNA. However, the amount of GNA in Rhopalosiphum padi (Hemiptera: Aphididae) honeydew was only about 10–40% of the concentrations contained in the artificial diet (Hogervorst et al., 2009). Moreover, a recent study with a honeydewfeeding aphid parasitoid suggests that honeydew-feeders might also indirectly be affected by the fact that the composition of honeydew is altered because the sap-feeders are themselves sub-lethally affected by GNA (Hogervorst et al., 2009). Besides honeydew, adult C. carnea are known to feed on pollen (Sundby, 1967; Sheldon and MacLeod, 1971; Rousset, 1984; Villenave et al., 2005; Li et al., 2008). Thus, pollen feeding may be another important route of exposure when the GNA is expressed in this plant part which has not been reported so far. In a recent study, however, Lehrman (2007) reported for a genetically modified oilseed rape expressing the pea lectin gene under control of an anther specific promoter that pollen can contain close to 0.1% lectin (w/w). For regulatory risk assessment, hazard studies are generally initiated with toxicity tests at elevated exposure doses, typically 10 times the expected environmental concentration (Romeis et al., 2008b). Given the amount of lectin detected in honeydew and pollen in the studies mentioned above, the toxin doses applied in this study are high but not unrealistic doses for testing. In order to fully understand the ecological relevance of the toxicity impact of GNA on adult C. carnea, further higher tier studies should be conducted under more realistic exposure conditions in case GNAexpressing plants with potential for commercial application have become available.

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