Environmental risk assessment for the small tortoiseshell Aglais urticae and a stacked Bt-maize with combined resistances against Lepidoptera and Chrysomelidae in central European agrarian landscapes

The cultivation of Lepidoptera‐resistant Bt‐maize may affect nontarget butterflies. We assessed the risk posed by event MON89034 × MON88017 (expressing Cry1A.105 and Cry2Ab2 against corn borers) to nontarget Lepidoptera. Using the small tortoiseshell Aglais urticae, a butterfly species common in central Europe, as a test organism we (i) assessed the toxicity of Bt‐maize pollen on butterfly larvae; (ii) measured pollen deposition on leaves of the host plant Urtica dioica; (iii) mapped the occurrence and distribution of host plants and larvae in two arable landscapes in Germany during maize anthesis; and (iv) described the temporal occurrence of a 1‐year population of A. urticae. (i) Larvae‐fed 200 Bt‐maize pollen grains/cm² had a reduced feeding activity. Significant differences in developmental time existed at pollen densities of 300 Bt‐maize pollen grains/cm² and in survival at 400 grains/cm². (ii) The highest pollen amount found was 212 grains/cm² at the field margin. Mean densities were much lower. (iii) In one region, over 50% of A. urticae nests were located within 5 m of a maize field, while in the other, all nests were found in more than 25 m distance to a maize field. (iv) The percentage of larvae developing during maize anthesis was 19% in the study area. The amount of pollen from maize MON89034 × MON88017 found on host plants is unlikely to adversely affect a significant proportion of larvae of A. urticae. This paper concludes that the risk of event MON89034 × MON88017 to populations of this species is negligible.     

Biological Activity of Cry1Ab Toxin Expressed by Bt Maize Following Ingestion by Herbivorous Arthropods and Exposure of the Predator Chrysoperla carnea

A major concern regarding the deployment of insect resistant transgenic plants is their potential impact on non-target organisms, in particular on beneficial arthropods such as predators. To assess the risks that transgenic plants pose to predators, various experimental testing systems can be used. When using tritrophic studies, it is important to verify the actual exposure of the predator, i.e., the presence of biologically active toxin in the herbivorous arthropod (prey). We therefore investigated the uptake of Cry1Ab toxin by larvae of the green lacewing (Chrysoperla carnea (Stephens); Neuroptera: Chrysopidae) after consuming two Bt maize-fed herbivores (Tetranychus urticae Koch; Acarina: Tetranychidae and Spodoptera littoralis (Boisduval); Lepidoptera: Noctuidae) by means of an immunological test (ELISA) and the activity of the Cry1Ab toxin following ingestion by the herbivores. Moreover, we compared the activity of Cry1Ab toxin produced by Bt maize to that of purified toxin obtained from transformed Escherichia coli, which is recommended to be used in toxicity studies. The activity of the toxin was assessed by performing feeding bioassays with larvae of the European corn borer (Ostrinia nubilalis (Hübner); Lepidoptera: Crambidae), the target pest of Cry1Ab expressing maize. ELISA confirmed the ingestion of Bt toxin by C. carnea larvae when fed with either of the two prey species and feeding bioassays using the target pest showed that the biological activity of the Cry1Ab toxin is maintained after ingestion by both herbivore species. These findings are discussed in the context of previous risk assessment studies with C. carnea. The purified Cry1Ab protein was more toxic to O. nubilalis compared to the plant-derived Cry1Ab toxin when applied at equal concentrations according to ELISA measurements. Possible reasons for these findings are discussed.     

A synthesis of laboratory and field studies on the effects of transgenic Bacillus thuringiensis (Bt) maize on non‐target Lepidoptera

One of the major applications of transgenic crops in agriculture are the so‐called Bacillus thuringiensis Berliner (Bt) plants, in particular Bt maizes, which produce insecticidal Cry proteins that target specific orders, such as the Lepidoptera or Coleoptera. We reviewed publications that reported on the direct toxic effects of Bt‐maize and/or Cry proteins of current Bt‐maize events on larvae of non‐target butterflies and moths (Lepidoptera). In total, 20 peer‐reviewed publications were identified, of which 16 papers contributed laboratory‐based data and seven field‐based data. An adverse effect on caterpillars was recorded in 52% of all laboratory‐based and in 21% of all field‐based observations. The variables most often studied and having the highest occurrence of effects were larval survival, body mass, and developmental time. Parameters of the adult stage were under‐represented in the studies. Overall, 11 lepidopteran species were tested. The majority of the studies originated from the USA, with the Monarch butterfly being the most studied, whereas other species and other parts of the world were widely neglected. Laboratory experiments were often run under unrealistic conditions from an ecological point of view. Although the papers we reviewed indicated a potential hazard for Lepidoptera that are exposed to and feed on lepidopteran‐specific Bt‐maize pollen, a general conclusion on the level of risk for butterflies and moths cannot as yet be drawn. A comprehensive risk characterization would require thorough hazard identification, exposure assessment, and impact assessment. However, our review showed that even the basic level of hazard characterization is as yet incomplete. Reasons for this are the still‐limited numbers of publications and concurrent lack of knowledge, the restriction of data to only a few species, the over‐representation of North American species, and the identified limitations of both laboratory and field experiments. The findings of this review suggest that more realistic, ecologically meaningful, and detailed experiments and analyses are crucial to improve the present assessment of Bt‐maize cultivation effects on Lepidoptera.     

Does Cry1Ac Bt‐toxin impair development of worker larvae of Africanized honey bee?

The western honey bee (Apis mellifera L.) is a widespread pollinator species. The present study aimed to test if Africanized honey bee larvae are negatively affected by the ingestion of diet contaminated with the Bacillus thunringiensis toxin Cry1Ac, which is expressed in GM cotton plants. The toxin activity was confirmed in bioassays with the velvetbean caterpillar (Anticarsia gemmatalis), a soybean pest species susceptible to Cry1Ac. The honey bee larvae were subjected to ingestion of either pure larval diet (control), diluted larval diet (diluted control) or larval diet diluted in a Cry1Ac solution at a concentration compatible with the maximum possible field exposure. Although diluted diet slightly increased larval mortality, Cry1Ac ingestion did not affect survival, developmental time, and neither adult body mass nor size, indicating that GM plants are unlikely to significantly impair the development of honey bee larvae. The larval‐rearing system reported here was suitable to assess the lethal and sub‐lethal effects of GM expressed toxins against honey bee larvae.     

Rapid selection and characterization of Cry1F resistance in a Brazilian strain of fall armyworm

Transgenic maize (Zea mays L., Poaceae) event TC1507, producing the Cry1F protein of Bacillus thuringiensis Berliner, has been used for management of the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), in Brazil since 2009. A strain of S. frugiperda, obtained from field collections of larvae in TC1507 maize in Minas Gerais state in 2010, was selected in the laboratory for resistance to Cry1F using leaves of TC1507 maize in two selection regimes. Continuous exposure of larvae to Cry1F was more effective than exposure for 6, 8, and 10 days in the selection of resistant S. frugiperda individuals. With only four generations of laboratory selection, a strain with high levels of resistance to Cry1F was obtained, as indicated by the survival of insects reared on leaves of TC1507 maize plants and by the more than 300‐fold resistance level measured in bioassays with the purified Cry1F protein. Importantly, reciprocal crosses between control and the Cry1F‐selected strains revealed that the resistance is autosomal and incompletely recessive, and the response obtained in the backcross of the F₁ generation with the resistant strain was consistent with simple monogenic inheritance. Additionally, there were no apparent fitness costs associated with resistance either for survival or larval growth on non‐Bt maize leaves. Our findings provide experimental evidence for rapid evolution of Cry1F resistance in S. frugiperda in the laboratory and further reinforce the potential of this species to evolve field resistance to the TC1507 maize as previously reported. The resistant strain isolated in this study provides an opportunity to estimate the resistance allele frequency in the field and to determine the biochemical and molecular basis of the resistance, which should provide further information to assist in the resistance management of S. frugiperda on transgenic maize producing B. thuringiensis proteins.     

Preference of Bt‐resistant and susceptible Busseola fusca moths and larvae for Bt and non‐Bt maize

The sustainability of genetically engineered insecticidal Bacillus thuringiensis Berliner (Bt) maize, Zea mays L. (Poaceae), is threatened by the evolution of resistance by target pest species. Several Lepidoptera species have evolved resistance to Cry proteins expressed by Bt maize over the last decade, including the African maize stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae). The insect resistance management (IRM) strategy (i.e., the high‐dose/refuge strategy) deployed to delay resistance evolution is grounded on certain assumptions about the biology and ecology of a pest species, for example, the interactions between the insect pest and crop plants. Should these assumptions be violated, the evolution of resistance within pest populations will be rapid. This study evaluated the assumption that B. fusca adults and larvae select and colonize maize plants at random, and do not show any preference for either Bt or non‐Bt maize. Gravid female B. fusca moths of a resistant and susceptible population were subjected to two‐choice oviposition preference tests using stems of Bt and non‐Bt maize plants. Both the number of egg batches as well as the total number of eggs laid on each stem were recorded. The feeding preference of Bt‐resistant and susceptible neonate B. fusca larvae were evaluated in choice test bioassays with whorl leaf samples of specific maize cultivars. Although no differential oviposition preference was observed for either resistant or susceptible female moths, leaf damage ratings indicated that neonate larvae were able to detect Bt toxins and that they displayed feeding avoidance behaviour on Bt maize leaf samples.     

Effect of Cry1F maize on the behavior of susceptible and resistant Spodoptera frugiperda and Ostrinia nubilalis

Understanding the behavior of pests targeted with Bacillus thuringiensis Berliner (Bt) crops is important to define resistance management strategies. Particularly the study of larval movement between plants is important to determine the feasibility of refuge configurations. Exposure to Bt maize, Zea mays L. (Poaceae), has been suggested to increase larval movement in lepidopteran species but few studies have examined the potential for resistance to interact with behavioral responses to Bt toxins. Choice and no‐choice experiments were conducted with Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae) and Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) to determine whether Cry1F resistance influences neonate movement. Leaf discs of Cry1F maize and the corresponding isoline were used to characterize behavioral responses. In both experiments, the location (on or off of plant tissues) and mortality of susceptible and Cry1F resistant neonates was recorded for 5 days, but the analysis of larvae location was performed until 7 h. Our results indicated no strong difference between resistant and susceptible phenotypes in S. frugiperda and O. nubilalis, although a small percentage of susceptible neonates in both species abandoned maize tissue expressing Cry1F. However, significant behavioral differences were observed between species. Ostrinia nubilalis exhibited increased movement between leaf discs, whereas S. frugiperda selected plant tissue within the first 30 min and remained on the chosen plant regardless of the presence of Cry1F. Spodoptera frugiperda reduced larval movement may have implications to refuge configuration. This study represents the first step toward understanding the effects of Cry1F resistance on Lepidoptera larval behavior. Information regarding behavioral differences between species could aid in developing better and more flexible resistance management strategies.     

Bt maize pollen exposure and impact on the garden spider, Araneus diadematus

Concerns have been raised that Bt maize pollen may have adverse effects on non‐target organisms; consequently, there is a general call for Bt maize risk assessment evaluating lethal and sublethal side effects. Spiders play an important economic and ecological role as pest predators in various crops, including maize. Web‐building spiders, especially, may be exposed to the Cry1Ab toxin of Bt maize by the ingestion of pollen via ‘recycling’ of pollen‐dusted webs and intentional pollen feeding. In this study, the potential Bt maize pollen exposure of orb‐web spiders was quantified in maize fields and adjacent field margins, and laboratory experiments were conducted to evaluate the possible effects of Bt maize pollen consumption on juvenile garden spiders, Araneus diadematus (Clerck) (Araneae: Araneidae). In maize fields and neighbouring field margins, web‐building spiders were exposed to high amounts of Bt maize pollen. However, a laboratory bioassay showed no effects of Bt maize pollen on weight increase, survival, moult frequency, reaction time, and various web variables of A. diadematus. A pyrethroid insecticide (Baythroid) application affected weight increase, survival, and reaction time of spiders negatively. In conclusion, the insecticide tested showed adverse effects on the garden spider, whereas the consumption of Bt maize pollen did not. This study is the first one on Bt maize effects on orb‐web spiders, and additional research is recommended in order to account for further spider species, relative fitness parameters, prey‐mediated effects, and possible long‐term chronic consequences of Bt exposure.     

Survival of Helicoverpa armigera larvae on and Bt toxin expression in various parts of transgenic Bt cotton (Bollgard II) plants

There is no conclusive evidence that Helicoverpa spp. (Lepidoptera: Noctuidae) in Australia have evolved significant levels of resistance to Bollgard II^® cotton (which expresses two Bt toxin genes, cry1Ac and cry2Ab). However, there is evidence of surviving larvae on Bollgard II cotton in the field. The distribution and survival of early‐instar Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae were examined on whole Bollgard II and non‐Bt cotton plants in greenhouse bioassays. The expression of Cry toxins in various parts of Bollgard II plants was compared to the survival of larvae in those locations. Only 1% of larvae survived after 6 days on greenhouse‐grown Bollgard II plants compared to 31% on non‐Bt cotton plants. Overall, and across all time intervals, more larvae survived on reproductive parts (squares, flowers, and bolls) than on vegetative parts (leaves, stems, and petioles) on Bollgard II plants. The concentration of Cry1Ac toxin did not differ between plant structures, whereas Cry2Ab toxin differed significantly, but there was no relationship between the level of expression and the location of larvae. This study provides no evidence that lower expression of Cry toxins in the reproductive parts of plants explains the survival of H. armigera larvae on Bollgard II cotton.     

The effect of Bt maize on Sesamia calamistis in South Africa

Bt maize, Zea mays L. (Poaceae) expressing Cry 1Ab insecticidal proteins was introduced for control of Busseola fusca (Fuller) (Lepidoptera: Noctuidae) and Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) in South Africa after its development for control of crambid borers in North America. In the light of the reportedly lower toxicity of Bt maize to certain Noctuidae borers, the effect of Bt maize was evaluated on Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae). The characteristic larval behaviour of S. calamistis may result in reduced exposure to Bt toxin and subsequent high levels of survival. Larvae do not feed on plant whorls like other borer species but penetrate stems directly from behind leaf sheaths where eggs are laid. Greenhouse and laboratory bioassays were done with three Bt maize hybrids and their iso‐hybrids. ‘Whole plant methods’ were used and potted plants artificially infested with eggs or larvae and survival recorded over time. Larval survival was also determined on different plant parts (whorls, stems, tillers, and ears) over time. Bt maize was shown to be highly toxic to S. calamistis. No larvae survived longer than 12–18 days on Bt maize plants in any of the experiments. Adults did not differentiate between Bt and non‐Bt plants in oviposition choice experiments. Sesamia calamistis is polyphagous and occurs in mixed populations with other borer species with which it shares many parasitoid species in Africa. The ecological impact of local extinction of S. calamistis caused by this highly effective transgenic event is therefore not expected to be great.     

Selection for Cry1F resistance in the European corn borer and cross-resistance to other Cry toxins

Evolution of resistance by insect pests is the greatest threat to the continued success of Bacillus thuringiensis (Bt) toxins used in insecticide formulations or expressed by transgenic crop plants such as Cry1F‐expressing maize [(Zea mays L.) (Poaceae)]. A strain of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), obtained from field collections throughout the central US Corn Belt in 1996 was selected in the laboratory for resistance to Cry1F by exposure to the toxin incorporated into artificial diet. The selected strain developed more than 3000‐fold resistance to Cry1F after 35 generations of selection and readily consumed Cry1F expressing maize tissue; yet, it was as susceptible to Cry1Ab and Cry9C as the unselected control strain. Only a low level of cross‐resistance (seven‐fold) to Cry1Ac was observed. These lacks of cross‐resistance between Cry1F and Cry1Ab suggest that maize hybrids expressing these two toxins are likely to be compatible for resistance management of O. nubilalis.     

Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance

We evaluated the insecticidal toxicity of Cry1Aa, Cry1Ab and Cry1Ac toxins against neonate larvae of sugarcane shoot borer Chilo infuscatellus Snellen (Lepidoptera: Crambidae) in vitro on diet surface. With the lowest LC₅₀ value, Cry1Ab emerged as the most effective among the three toxins. Sugarcane cultivars Co 86032 and CoJ 64 were transformed with cry1Ab gene driven by maize ubiquitin promoter through particle bombardment and Agrobacterium-mediated transformation systems. Gene pyramiding was also attempted by retransforming sugarcane plants carrying bovine pancreatic trypsin inhibitor (aprotinin) gene, with cry1Ab. Southern analysis confirmed multiple integration of the transgene in case of particle bombardment and single site integration in Agrobacterium-mediated transformants. The expression of cry1Ab was demonstrated through Western analysis and the toxin was quantified using ELISA. The amount of Cry1Ab protein in different events varied from 0.007 to 1.73% of the total soluble leaf protein; the events transformed by Agrobacterium method showed significantly higher values. In in vivo bioassay with neonate larvae of shoot borer, transgenics produced considerably lower percentage of deadhearts despite suffering feeding damage by the borer compared with the untransformed control plants. Expressed Cry1Ab content was negatively related to deadheart damage. Aprotinin-expressing sugarcane pyramided with cry1Ab also showed reduction in damage. The potential of producing sugarcane transgenics with cry1Ab and aprotinin genes resistant to early shoot borer was discussed in the light of the results obtained.     

An evaluation of methods for assessing the impacts of Bt‐maize MON810 cultivation and pyrethroid insecticide use on Auchenorrhyncha (planthoppers and leafhoppers)

1 Auchenorrhyncha (Planthoppers and Leafhoppers) are not only pests of many crops, but they are also nontarget organisms with respect to Bt‐protein expressing genetically modified plants. As herbivorous arthropods, planthoppers and leafhoppers ingest Cry proteins depending on their feeding behaviour. Consequently, they are directly exposed to these entomotoxic proteins and can also serve as a source of Cry protein exposure to predatory arthropods. Therefore, it is reasonable to use Auchenorrhyncha in the risk assessment of genetically modified crops. 2 During a 2‐year field study, we evaluated four different methods in terms of their feasibility to assess the impacts of plant‐incorporated protectants from Bt‐maize and of insecticide use on this group of arthropods. Visual assessment of plants, sweep netting, yellow traps and custom made sticky traps were utilized in field plots of Bt‐maize MON810, untreated near‐isogenic maize and insecticide‐treated near‐isogenic maize and were compared with respect to their capability to reflect the diversity and abundance of Auchenorrhyncha species. 3 Zyginidia scutellaris (Herrich‐Schäffer) (Cicadomorpha: Cicadellidae) represented more than 94% of all captured individuals in both years. The analysis of Z. scutellaris data showed no consistent differences between Bt‐maize MON810 and the untreated near isogenic hybrid, demonstrating no negative impact of MON810 on this species. Insecticide treatment, on the other hand, was not equivalent to the isogenic maize in terms of Z. scutellaris densities. Based on the collected data and on practical considerations, we recommend the combined use of transect‐wise sweep netting and sticky traps for the sampling of Auchenorrhyncha in maize.     

Expression of Bacillus thuringiensis Cry1Ac protein in cotton plants, acquisition by pests and predators: a tritrophic analysis

1. Studies have shown that Cry proteins of the bacterium Bacillus thuringiensis expressed in transgenic plants can be acquired by nontarget herbivores and predators. A series of studies under field and controlled conditions was conducted to investigate the extent to which Cry1Ac protein from Bt transgenic cotton reaches the third trophic level and to measure the amount of protein that herbivores can acquire and expose to predators. 2. Levels of Cry1Ac in Bt cotton leaves decreased over the season. Among herbivores (four species), Cry1Ac was detected in lepidopteran larvae and the amount varied between species. Among predators (seven species), Cry1Ac was detected in Podisus maculiventris and Chrysoperla rufilabris. 3. In the greenhouse, only 14% of the Cry1Ac detected in the prey (Spodoptera exigua larvae) was subsequently found in the predator P. maculiventris. Detection of Cry1Ac protein in Orius insidiosus, Geocoris punctipes and Nabis roseipennis was probably limited by the amount of prey consumed that had fed on Bt cotton. 4. Purified Cry1Ac was acquired by the small predatory bug G. punctipes but at much higher concentration than found in plants or in lepidopteran larvae. 5. Bt protein was shown to move through prey to the third trophic level. Predatory heteropterans acquired Cry1Ac from prey fed Bt cotton, but acquisition was dependent on the concentration of Cry1Ac conveyed by the prey and the amount of prey consumed. The type and availability of prey capable of acquiring the protein, coupled with the generalist feeding behaviour of the most common predators in the cotton ecosystem, probably constrain the flow of Cry1Ac through trophic levels.     

Response of Danaus plexippus to pollen of two new Bt corn events via laboratory bioassay

Laboratory bioassays were conducted to evaluate the response of first instar larvae of the monarch butterfly, Danaus plexippus L. (Lepidoptera: Danaidae), a non‐target species, to pollen from corn, Zea mays L. (Commelinales: Poaceae), from two new corn hybrids genetically modified to express different types of insecticidal proteins derived from the bacterium Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt). One hybrid expresses both Cry1Ab and Cry2Ab2 proteins (MON 810 × MON 84006), active against lepidopteran pests, and the other expresses Cry3Bb1 protein (MON 863), targeted against coleopteran pests. First instar larvae were placed on milkweed leaves (Asclepias syriaca L.) (Gentianales: Asclepiadaceae) dusted with doses of either Bt pollen or its nonexpressing (isoline) pollen counterpart ranging from 50 to 3200 grains cm^?2 of milkweed leaves, or no pollen at all. Larvae were exposed to pollen for 4 days, then moved to pollen‐free leaves and observed for another 6 days. Survival was observed after 2, 4, and 10 days. Weight gain was estimated after 4 and 10 days, leaf consumption after 2 and 4 days, and larval development after 10 days. Exposure to pollen of the Cry1Ab/Cry2Ab2‐Bt expressing hybrid reduced larval survival approximately 7.5–23.5% at the dose ranges tested relative to a no pollen control. Larval weight gain and consumption were reduced for larvae exposed to pollen of this hybrid and a small minority of larvae (3.1%) never developed past the third instar after 10 days of observation. Exposure to pollen of the Cry3Bb1‐Bt expressing hybrid had no negative effects on larval mortality, weight gain, consumption, or development relative to the consumption of Bt‐free corn pollen. The relevance of these findings to the risk that these Bt corn hybrids pose to monarch populations is discussed.     

Can the growing of transgenic maize threaten protected Lepidoptera in Europe?

We evaluated whether protected European butterflies can potentially be at risk if transgenic maize is extensively grown in Central Europe. We explored potential consequences of both insect resistant (IR) and herbicide resistant (HR) transgenic maize. IR maize can produce pollen that is toxic to lepidopteran larvae, and this puts butterfly species at possible risk if the presence of young larvae coincides with maize flowering, during which large quantities of maize pollen can be deposited on vegetation. By considering the timing of maize flowering in Europe and the phenology of the protected Lepidoptera species, we found that 31 species had at least one generation where 50% of the larval stage overlapped with maize flowering, and 69 species for which first instar larvae were present during maize pollen shedding. HR maize allows high concentration herbicide treatments on fields without seasonal limitation, which can drastically reduce weed densities. In cases where such weed species are host plants for protected butterflies, reduced host plant/food availability can result, causing population decreases. By using published information, we first identified the important weed species in major maize-growing European countries. Subsequently, we checked whether the host plants of protected Lepidoptera included species that are common maize weeds. We identified 140 protected species having food plants that are common weeds in one or more of the major European maize-growing countries. If HR maize is grown in Europe, there is a potential hazard that their food plants will seriously decline, causing a subsequent decline of these protected species.     

Effects of Bt maize on Frankliniella tenuicornis and exposure of thrips predators to prey-mediated Bt toxin

As a part of a risk assessment procedure, the impact of Bt maize expressing Cry1Ab toxin on the thrips Frankliniella tenuicornis (Uzel) (Thysanoptera: Thripidae) was investigated, and the potential risks for predators feeding on thrips on Bt maize were evaluated. The effects of Bt maize on F. tenuicornis were assessed by measuring life‐table parameters when reared on Bt and non‐Bt maize. The content of Cry1Ab toxin in different stages of F. tenuicornis reared on Bt maize and the persistence of the toxin in adults where determined in order to evaluate the possible exposure of predators when feeding on thrips. In addition, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) was used as a model predator to assess how the behaviour of prey and predator may influence the exposure of a natural enemy to the Bt toxin. Life‐table parameter results showed that F. tenuicornis was not affected when it was reared on Bt maize. This indicates that the potential for prey quality‐mediated effects on predators is low. Bt content was highest in thrips larvae and adults, and negligible in the non‐feeding prepupal and pupal stages. The persistence of the Cry1Ab toxin in adult F. tenuicornis was short, resulting in a decrease of 97% within the first 24 h. Predation success by young C. carnea larvae varied among the thrips stages, indicating that exposure of predators to Bt toxin can additionally depend on the prey stage. When combining the current knowledge of the susceptibility of major thrips predators with our findings showing no potential for prey quality‐mediated effects, relatively low toxin content in thrips as well as short persistence, it can be concluded that the risks for predators when feeding on thrips in or next to Bt maize fields are negligible.     

Bio‐safety evaluation of Cry1Ac,Cry2Ab,Cry1Ca,Cry1F and Vip3Aa on Harmonia axyridis larvae

Dietary exposure studies are initial steps in environmental risk assessments of genetically engineered plants on non‐target organisms. These studies are conducted in the laboratory where surrogate species are exposed to purified and biologically active insecticidal compounds at higher concentrations than those expected to occur in transgenic crops foliage. Thus, dietary exposure (early tier) tests provide robust data needed to make general conclusions about the susceptibility of the surrogate species to the test substance. For this, we developed suitable artificial diet and used it to establish a dietary exposure test for assessing the toxicity of midgut‐active insecticidal compounds to the larvae of the Asian ladybird beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Using boric acid as a model compound, we validated the bioassay established for H. axyridis larvae. An artificial diet containing boric acid which negatively affected survival, development and adult weights was offered to larvae and indicated that the bioassay was able to detect toxic effects of insecticidal substances incorporated in diets. Using this dietary exposure test, environmental risk assessment of Cry1Ac, Cry2Ab, Cry1Ca, Cry1F and the non‐Cry protein Vip3Aa was evaluated by analysing pupation rates, adult emergence rates, 7‐day larval weights, and freshly emerged male and female weights among the toxin treatments and a pure artificial diet. These life‐table parameters did not vary among artificial diets containing 200 μg/g Bt proteins or pure artificial diet. In contrast, boric acid adversely affected all life‐table parameters. Thus on these bases, we concluded H. axyridis larvae are not sensitive to these Bt proteins expressed in genetically engineered crops.     

Potential impact of genetically modified Lepidoptera‐resistant Brassica napus in biodiversity hotspots: Sicily as a theoretical model

The general increase of the cultivation and trade of Bt transgenic plants resistant to Lepidoptera pests raises concerns regarding the conservation of animal and plant biodiversity. Demand for biofuels has increased the cultivation and importation of oilseed rape (Brassica napus L.), including transgenic lines. In environmental risk assessments (ERAs) for its potential future cultivation as well as for food and feed uses, the impact on wild Brassicaeae relatives and on non‐target Lepidoptera should be assessed. Here we consider the potential exposure of butterflies as results of possible cultivation or naturalization of spilled seed in Sicily (Italy). Diurnal Lepidoptera, which are pollinators, can be exposed directly to the insecticidal proteins as larvae (mainly of Pieridae) through the host and through the pollen that can deposit on other host plants. Adults can be exposed via pollen and nectar. The flight periods of butterflies were recorded, and they were found to overlap for about 90% of the flowering period of B. napus for the majority of the species. In addition, B. napus has a high potential to hybridise with endemic taxa belonging to the B. oleracea group. This could lead to an exposure of non‐target Lepidoptera if introgression of the Bt gene into a wild population happens. A rank of the risk for butterflies and wild relatives of oilseed rape is given. We conclude that, in environmental risk assessments, attention should be paid to plant–insect interaction especially in a biodiversity hotspot such as Sicily.     

Target and non‐target effects of a spider venom toxin produced in transgenic cotton and tobacco plants

The peptide ω‐Hexatoxin‐Hv1a (Hvt) is one of the most studied spider toxins. Its insecticidal potential has been reported against species belonging to the arthropod orders Lepidoptera, Diptera and Orthoptera. The gene encoding Hvt has been transformed into cotton and tobacco to protect the plants from damage by lepidopteran pests. This study evaluated the expression of the ω‐HXTX‐Hv1a gene in transgenic plants, and the toxicity of plant‐expressed and purified Hvt on target lepidopteran insects and on several non‐target species. Transgenic Bollgard II cotton plants, which produce Cry1Ac and Cry2Ab2 and purified Cry2Ab2 protein were included in the study as comparators. LC₉₅ values of purified Hvt against Spodoptera littoralis and Heliothis virescens were 28.31 and 27.57 μg/ml of artificial diet, respectively. Larval mortality was 100% on Hvt‐transgenic tobacco plants but not on Hvt‐transgenic cotton, probably because of the significantly lower toxin expression level in the transgenic cotton line. Non‐target studies were conducted with larvae of the predators Chrysoperla carnea and Coccinella septempunctata, adults of the aphid parasitoid Aphidius colemani, and adult workers of the honey bee, Apis mellifera. Even at 40 μg/ml, Hvt did not adversely affect the four non‐target species. Purified Cry2Ab2 at 10 μg/ml also did not adversely affect any of the non‐target species. Our results show that Hvt might be useful for developing insecticidal plant varieties to control pest Lepidoptera.