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Acquisition of Cry1Ac Protein by Non-Target Arthropods in Bt Soybean Fields

  • Huilin Yu,

    Affiliation State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

  • Jörg Romeis,

    Affiliations State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, Institute for Sustainability Sciences ISS, Agroscope, Zurich, Switzerland

  • Yunhe Li,

    Affiliation State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

  • Xiangju Li,

    Affiliation State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

  • Kongming Wu

    kmwu@ippcaas.cn

    Affiliation State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

Abstract

Soybean tissue and arthropods were collected in Bt soybean fields in China at different times during the growing season to investigate the exposure of arthropods to the plant-produced Cry1Ac toxin and the transmission of the toxin within the food web. Samples from 52 arthropod species/taxa belonging to 42 families in 10 orders were analysed for their Cry1Ac content using enzyme-linked immunosorbent assay (ELISA). Among the 22 species/taxa for which three samples were analysed, toxin concentration was highest in the grasshopper Atractomorpha sinensis and represented about 50% of the concentration in soybean leaves. Other species/taxa did not contain detectable toxin or contained a concentration that was between 1 and 10% of that detected in leaves. These Cry1Ac-positive arthropods included a number of mesophyll-feeding Hemiptera, a cicadellid, a curculionid beetle and, among the predators, a thomisid spider and an unidentified predatory bug belonging to the Anthocoridae. Within an arthropod species/taxon, the Cry1Ac content sometimes varied between life stages (nymphs/larvae vs. adults) and sampling dates (before, during, and after flowering). Our study is the first to provide information on Cry1Ac-expression levels in soybean plants and Cry1Ac concentrations in non-target arthropods in Chinese soybean fields. The data will be useful for assessing the risk of non-target arthropod exposure to Cry1Ac in soybean.

Introduction

Genetically modified (GM) crops expressing cry genes from Bacillus thuringiensis (Bt) are widely used to control major insect pests and are an important component of integrated pest management (IPM) systems [1][4]. The damage caused by lepidopteran pests greatly reduces soybean yield and quality [5]. Recently, Monsanto Company has developed an insect-resistant transgenic soybean cultivar called MON87701. This soybean line expresses the cry1Ac gene and exhibits excellent efficacy against some lepidopteran pests [6], [7].

Before a new GM plant is grown in the field, its potential for harming valuable non-target organisms (NTO) is determined as part of an environmental risk assessment [8][11]. Risk is a function of hazard (here: toxicity of the insecticidal compound) and the likelihood that this hazard will be realized (here: likelihood of exposure to hazardous concentrations of the insecticidal compound) [8], [9]. Knowledge about the NTOs most likely to be exposed to the insecticidal compound enables researchers to determine which species should be the focus of risk assessment [12][14].

Herbivores are directly exposed to Bt proteins when feeding on transgenic plant tissues [15]. The quantity of Bt protein ingested can differ widely among herbivore species. This variation reflects the time and site of toxin expression in the plant, the feeding ecology of the herbivores, and the amount of plant material that is ingested [15][17]. For example, the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), has been reported to contain high concentrations of Cry proteins when fed Bt maize or Bt cotton; the concentrations were equal to or even higher than the levels in the plant tissues [18][20]. In contrast, larvae of Lepidoptera like Helicoverpa amigera (Noctuidae) or Spodoptera littoralis (Noctuidae) contain Cry protein levels that are one or two orders of magnitude lower than those in Bt plant tissues [21], [22]. Interestingly, sucking pests such as aphids and planthoppers were reported to contain no or only trace amounts of Cry proteins after feeding on Bt crops [23], [24].

Predators are exposed to Bt toxins mainly by consuming herbivores that have ingested the toxin [15]. Omnivorous species can also acquire Cry proteins by directly feeding on pollen or other Bt plant tissue. This has, for example, been reported for various species of Heteroptera, such as Orius spp. (Anthocoridae), adult Chrysoperla spp. (Neuroptera: Chrysopidae), and species of ladybird beetles (Coleoptera: Coccinellidae) [25][29]. Tri-trophic laboratory studies have revealed that various species of predators belonging to different arthropod orders contain Cry proteins after feeding on prey reared on Bt-transgenic maize, rice, or cotton plants [15], [19], [27], [30][40]. In general, the concentrations of Bt proteins were significantly diluted when transferred to higher trophic levels, and there has been no indication of toxin accumulation, which is also consistent with field studies with different Bt plants [30], [40], [41][45].

The objective of our study was to characterize the level at which different arthropod species are exposed to the Cry protein when foraging in Bt soybean fields. The larger goal was to identify non-target species that are most likely at risk as a consequence of such exposure. These data will provide baseline information for further non-target risk assessment of transgenic Bt soybean.

Materials and Methods

Ethics statement

No specific permits were required for the described field studies. The soybean fields from which the arthropods used in this study were originally collected were owned by the authors' institute (Institute of Plant Protection, Chinese Academy of Agricultural Sciences, CAAS). These field studies did not involve endangered or protected species.

Plants

Experiments were conducted with transgenic soybean MON87701, which produces the Cry1Ac protein (Bt), and the corresponding non-transformed near-isoline A5547. Soybean seeds were supplied by Monsanto Company (St. Louis, MO, USA). In 2010, the two soybean lines were sown at the Agriculture Experiment Station of the Chinese Academy of Agriculture Sciences (CAAS) located at Langfang, Hebei Province. Soybeans were managed according to the common growing practices in the region but without pesticide application. Bt soybean and control soybean were grown in separate fields at a distance of about 500 m. Each field was divided into four 180-m2 plots (length×width: 15 m×12 m). Plots were isolated by belts of maize plants (length×width: 5 m×12 m).

Bt protein content in transgenic soybean plants

Different soybean plant tissues were collected at different growth stages in 2010: before anthesis (V6–8 and V11–12; 1 to 25 August); during anthesis (R1 and R3; 26 August to 10 September); and after anthesis (R5, R6, and R7; 15 September to 10 November) [46], [47]. Only leaves were collected before anthesis; leaves and flowers were collected during anthesis; and leaves and pods were collected after anthesis. At each sampling date, 30 leaves or 50 flowers or 20 pods were collected from 10 randomly selected soybean plants from each of the four soybean plots as one sample, resulting in a total 44 samples [leaves: 4 replications (plots)×7 growth stages, resulting in 28 samples; flowers: 4 replications (plots)×1 growth stage (anthesis), resulting in 4 samples; pods: 4 replications (plots)×3 growth stages (R5, R6, R7), resulting in 12 samples] for each Bt soybean and control soybean. The leaves were the third fully expanded trifoliate leaves, and the flowers were taken from the upper part of the plants. All samples were kept at −80°C for later ELISA analyses.

Cry protein content in arthropod species collected from Bt soybean plots

Arthropods were collected from soybean plots using a sweep net between 5 August and 30 October 2010 when the soybean plants were at the V6 to R7 growth stage. In addition, leaves covered with aphids were cut, put into a plastic bag, and transported to the laboratory; soybean aphids were collected from the underside of the leaves using a camel-hair brush and amicroscope. Immediately after they were collected in the field, all other arthropods were placed individually in 2- or 5-ml centrifuge tubes and were kept in a cooling box to reduce metabolism and to reduce excretion of Bt protein. Once transferred to the laboratory, the arthropods were immediately stored in a −80°C freezer. Since the experimental plots were not large enough, and the arthropods were not evenly distributed, for some species, we could not collect enough individuals for ELISA measure in some plots, while in other plots excess individuals were collected. In addition, ELISA measures showed that Bt concentrations in samples collected from different field plots were not significantly different. Therefore, we pooled arthropod individuals of the same species collected from the different plots and subsequently divided them in equal sub-samples for the ELISA analyses. The arthropod species that were analysed are listed in Tables 1 and 2 and in Table S1. Most species names were verified using the Catalogue of Life (www.catalogueoflife.org) and Fauna Europaea (www.faunaeur.org). Species names that were not included in the databases were confirmed by experts from China Agriculture University and Northwest A&F University.

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Table 1. Cry1Ac concentrations in arthropods collected in Bt soybean before, during, and after anthesis in 2010.

https://doi.org/10.1371/journal.pone.0103973.t001

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Table 2. Detection of Cry1Ac in arthropods collected from Bt soybean plots at different growth stages for which only one or two sub-samples were analysed (by ELISA).

https://doi.org/10.1371/journal.pone.0103973.t002

ELISA measurement

The concentrations of Cry1Ac in fresh soybean leaves and insect samples were measured by double-sandwich ELISA using the Cry1Ac detection kit from Envirologix Inc. (Portland, ME, USA). Before measurement, the collected arthropods were identified to species and then washed in deionized water to remove any Bt protein from their outer surface before lyophilization. For small arthropods, several or many individuals were pooled as a sample. For larger arthropods (e.g., the grasshopper Atractomorpha sinensis; Orthoptera: Acrididae), one individual was analyzed per sample. Thus, the dry weight of the arthropod samples ranged from 1.6 to 100 mg. Table 1 and Table S1 provide information about the number of individuals that were pooled per sample. Whenever possible, arthropods were split into three samples that were analysed separately. Arthropod samples were homogenized in phosphate-buffered saline with 0.05%Tween-20 (PBST). The ratio of lyophilized sample (dry weight, DW) to extraction buffer was 20 mg∶1 ml. After PBST was added to the samples in a centrifuge tube, the samples were fully ground with a Tissuelyser II mill from QIAGEN (Germany) (frequency: 28/s, 4 min). After centrifugation at 12000× g and appropriate dilution of the supernatants, ELISA was performed according to the manufacturer's instructions. The measured OD values were calibrated to a range of concentrations of purified Cry1Ac protein purchased from Envirotest-China (agent for Envirologix Inc., Portland, ME, USA; www.envirotest-china.com). The protoxin from B. thuringiensis had been expressed as single-gene products in Escherichia coli at Case Western Reserve University (USA). The protoxin inclusion bodies were then dissolved and trypsinized, and then isolated and purified by ion exchange HPLC; the pure fractions were then desalted and lyophilized. The purity was about 94–96% (Marianne P. Carey, Case Western Reserve University, personal communication). The toxin was considered not detectable if the concentration was lower than three-fold concentrations of blank optical density (about 0.02 µg/g DW).

Samples from various species belonging to the different arthropod orders addressed in the present study that were collected in control soybean plots were also analysed by ELISA to test for any cross-reaction of arthropod proteins with the ELISA. No such cross-reaction was apparent.

Statistical analyses

For comparison of Cry protein concentrations in soybean tissue at different growth stages, one-way ANOVAs were carried out followed by Tukey's HSD test using SPSS 13.0.

Results

Bt protein content in transgenic soybean plants

Concentrations of Cry1Ac differed significantly among different soybean plant tissues collected at different growth stages (one-way ANOVA, F10,65 = 314.80, P<0.0001) (Fig. 1). The Cry1Ac content in leaves ranged from 25.50 to 37.50 µg/g DW. Before anthesis, the leaves collected from Bt soybean plants at V6–8 and V11–12 had similar Cry1Ac contents (P = 0.70). During anthesis, Cry1Ac levels in leaves significantly declined, i.e., levels were significantly lower at R1 and R3 than at V6–8 and V11–12. After anthesis, the Cry1Ac content rebounded, reaching a level at R5 (37.50 µg/g DW) that was similar to the levels before anthesis. Subsequently, the concentration in leaves declined significantly, but the levels at R6 and R7 remained higher than the levels measured during anthesis. The Cry1Ac concentration was significantly lower in flowers than in any of the leaf samples but was significantly higher than in pod samples (P<0.0001). Cry1Ac contents were similar in pods at R5 and R6 but declined significantly at R7 (P<0.0001). No Cry1Ac was found in control soybean tissues.

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Figure 1. Cry1Ac toxin concentrations (µg/g dry weight, mean+SE) in plant tissues of Bt soybean from the field.

Samples were taken before (I), during (II) and after anthesis (III) (n = 6). Bars with different letters are significantly different at P<0.05.

https://doi.org/10.1371/journal.pone.0103973.g001

Cry1Ac toxin content in arthropods represented by three samples

In total, we collected and analysed different life stages of more than 50 arthropod species/taxa belonging to 42 families in 10 orders (Tables 1, 2, Table S1). ELISA results for species for which three sub-samples were analysed are presented in Table 1.

Among the species/taxa for which three samples were analysed, a total of 17 were positive for Cry1Ac, i.e., the protein levels were higher than the detection limit (Table 1). For all positive samples, the amounts of Cry1Ac were lower than the amounts measured in soybean leaf tissue (based on a mean level of 32 µg/g DW). By far the highest concentration was detected in adults of the herbivorous grasshopper A. sinensis, which contained up to 16.24 µg Cry1Ac/g DW, representing about half of the concentration detected in soybean leaves. For other samples, the species contained between 1 and 10% of the amount of Cry1Ac in soybean leaves, and these species included adult Misumenopos tricuspidatus (Araneae: Thomisidae), adults of unidentified Anthocoridae (Hemiptera), adults of Deraeocoris punctulatus and nymphs of Lygus spp. (both Hemiptera: Miridae), nymphs of Dolycoris baccarum and Halyomorpha halys (both Hemiptera: Pentatomidae), and nymphs of Cicadella viridis (Homoptera: Cicadellidae).

For some species for which different life stages were collected, the quantity of Cry1Ac significantly differed between adults and larvae/nymphs. This was the case for three species of herbivores, including Lygus spp. and C. viridis, in which Cry1Ac concentrations were generally higher in the nymphs than in adults, and A. sinensis, in which levels were higher in adults than in nymphs (Table 1). In the case of Trigonotylus ruficornis (Hemiptera: Miridae), Cry1Ac levels in adults and nymphs were always below the detection limit. For predators, two species contained higher concentrations in the larvae than in adults, and these were Propylea japonica (Coleoptera: Coccinellidae) and Chrysoperla spp. In the case of Misumenopos tricuspidatus (Araneae: Thomisidae), levels did not differ between adults and juveniles (Table 1).

For some species, Cry1Ac concentrations differed among the three sampling periods, i.e., before, during, and after anthesis (Table 1). In the case of adults and nymphs of the herbivore Lygus spp. and adults of the predator D. punctulatus, Cry1Ac concentrations were highest before anthesis. In contrast, Cry1Ac concentrations in nymphs of D. baccarum were highest after anthesis. For adults of Paraluperodes suturalis nigrobilineatus (Coleoptera: Chrysomelide), Cry1Ac levels were similar before, during, and after anthesis.

Cry1Ac was not detected in any arthropod sample from control soybean plots.

Cry1Ac toxin content in arthropods represented by one or two samples

Results for species for which only one or two sub-samples were analysed are summarized in Table 2. Rather than presenting mean values for Cry1Ac concentration, Table 2 divides the species into those which were positive or negative for Cry1Ac. Additional details are provided in Table S1. Among species for which analyses were replicated fewer than three times, 12 species/taxa were positive for Cry1Ac (Table 2, Table S1). A significant amount of Cry1Ac (about 13% of that detected in soybean leaves) was detected in unidentified spiders belonging to the Thomisidae. In addition, relatively high amounts of Cry1Ac (1 to 10% of that in soybean leaves) were detected in adults of Sympiezomias velatus (Coleoptera: Curculionidae), adults and nymphs of Riptortus pedestris (Hemiptera: Alydidae), larvae of Spilosoma niveus (Lepidoptera: Arctiidae), and adults of an unidentified species of Zygoptera (Odonata). Nineteen species/taxa contained Cry1Ac levels below the detection limit (Table 2, Table S1).

Discussion

Throughout the growing season, Cry1Ac protein concentrations in Bt soybeans were higher in leaves than in other tissues. The concentrations reached a maximum of 37.50 µg/g DW, which is approximately equivalent to 13.4 µg/g fresh weight (FW) [7]. Thus, the Cry1Ac concentrations in Bt soybean leaves were higher than the Cry1A concentrations reported from leaves of field-grown Bt cotton (0.7 µg/g FW), Bt rice (8 µg/g FW), and Bt maize (4 µg/g FW) [48][50]. The Cry1Ac concentration in soybean leaves declined significantly during anthesis and then gradually rebounded. A similar expression pattern has been reported for Bt cotton [48]. It is well established that the Cry protein concentration in Bt-transgenic crops varies with plant variety, plant age, and environmental conditions including temperature, relative humidity, light, and soil properties [51][57]. It might thus be useful to study the expression levels and patterns for other Bt soybean varieties in other geographical regions where the plants will be grown. The Cry protein concentrations reported also vary with the detection method, including the extraction procedure and the ELISA kit [58][60]. Because we used the same methods to analyse all of our samples, the values reported within our study are comparable.

For the assessment of the exposure of non-target species to Cry1Ac toxin in Bt soybean fields, it is important to determine which arthropod species are likely to be exposed to the toxin under field conditions and at what level. We thus collected a total of 52 species or taxa belonging to 42 families in 10 different arthropod orders from Bt soybean fields and measured their Cry1Ac content.

Among herbivores, no toxin was detected in the soybean aphid Aphis glycines (Hemiptera: Aphididae), a species that feeds exclusively on phloem sap. This result agrees with previous reports that phloem feeders ingest little or no Cry protein when feeding on Bt plant tissues [23], [61], [62]. In another sap-sucking herbivore, the leafhopper C. viridis, significant amounts of Cry1Ac were found in the nymphal stages but not in the adults. Previous studies in Bt maize and Bt cotton fields also reported a low level of Cry proteins in different species of Cicadellidae [43], [44], [63]. Mesophyll-feeding bugs (Hemiptera) belonging to the Miridae (i.e., Lygus spp.) and Pentatomidae (D. baccarum and H. halys) contained measurable concentrations of Cry1Ac (between 1 and 10% of the concentration measured in soybean leaves). In contrast, levels detected in Rhopalus maculates (Hemiptera: Rhopalidae) were less than 15% of that in leaves. In the case of Lygus spp., nymphs contained much higher (5- to 23-fold) concentrations than adults. A similar difference between life stages has been reported for Lygus rugulipennis (Hemiptera: Miridae) collected in Cry3Bb1-expressing Bt maize [44]. An artificial diet study with Lygus hesperus (Hemiptera: Miridae) revealed that only a small portion of the ingested Cry1Ac toxin was absorbed into the hemolymph while most was excreted in a still biologically active form [64]. Field investigations revealed that the abundances of Lygus spp., C. viridis, and D. Baccarum were similar in Bt soybean and control soybean (Yu et al., unpublished data). Although these hemipteran pests ingested the Cry1Ac toxin, they did not appear to be adversely affected [65].

In our study, a number of leaf-feeding herbivores contained considerable amounts of Cry1Ac. By far the highest concentration was detected in adults of the grasshopper A. sinensis (levels reached 50% of that in soybean leaves), while their nymphs contained amounts of Cry1Ac that were 75- to 8100-times lower. The large difference between the life stages could be explained by the fact that adults ingest significantly more food than nymphs [66], [67]and that adults are apparently inefficient at digesting or excreting the ingested Cry1Ac protein. In contrast, adults of the leaf beetle P. suturalis nigrobilineatus (Coleoptera: Chrysomelidae) contained low Cry1Ac toxin concentrations (less than 1% of that in soybean leaves), and the concentrations did not differ among soybean growth stages. This despite the fact that adults of this species are reported to feed on soybean plants [68].The low Cry1Ac concentration detected is surprising given that, in previous studies, adults of Oulema species in Bt maize fields contained among the highest concentrations found in the sampled arthropods [43], [44]. In the case of Lepidoptera that were analysed in the current study, Cry1Ac was detected at low levels (up to 2% of the concentration in soybean leaves) in larvae, while no toxin was found in the adult stages, regardless of species.

Besides herbivores, a number of common predatory species were collected and analysed in the current study. We found detectable amounts of Cry1Ac in adults and larvae of Chrysoperla spp., the larvae of P. japonica, the adults and nymphs of Geocoris pallidipennis (Hemiptera: Lygaeidae), the adults and nymphs of Orius spp., and the nymphs of Nabis stenoferus (Hemiptera: Nabidae). The level was highest in G. pallidipennis at 0.3 µg/g DW, which was 100-fold lower than the concentration in Bt soybean leaves. Overall, our data are similar to those reported for generalist predators in Bt cotton, Bt rice, and Bt maize [41][45], [69]. The level of exposure of predatory species to plant-expressed Bt Cry proteins is highly variable and can differ depending on the prey consumed, on the time of the last consumption, and on whether the predators have directly consumed plant tissue such as pollen [15]. The predators Geocoris spp. and Nabis spp., for example, also directly feed on green leaf tissue [70], [71]. Throughout the soybean season, higher levels of Cry1Ac toxin were detected in larvae than in adults of Chrysoperla spp. and P. japonica. A likely explanation for the difference observed in the case of Chrysoperla spp. is the difference in the food consumed. While Chrysoperla spp. larvae are predaceous, adults consume pollen, nectar, and aphid honeydew [72]. For P. japonica, the situation is less clear because adults and larvae have a similar feeding habit, i.e., both feed on aphids and other small arthropods [73] and also utilize plant pollen and nectar as supplemental food sources [74]. It is thus unclear what caused the difference in Cry1Ac concentration in the two life stages of P. japonica. Interestingly, similar results were reported from a field study with Cry1Ab-expressing Bt maize in that field-collected larvae of the spider mite predator Stethorus punctillum (Coleoptera: Coccinellidae) contained about three-times more Cry protein than adults [43]. Even under controlled laboratory conditions where S. punctillumwas fed ad libitum with Bt maize-reared T. urticae, the larvae contained significantly more Cry protein than the adults [20]. Anthocorids such as Orius spp. are known to feed on pollen in addition to prey. This is likely why Orius spp. collected in flowering Bt maize fields contained more Cry protein than specimens collected before or after anthesis [43]. In our study, however, Orius spp. and an unidentified Anthocorid species contained higher amounts of Cry1Ac when collected after soybean flowering than during flowering. This suggests that pollen feeding is not very important for these two species in soybean fields. The adults of Deraeocoris punctulatus (Hemiptera: Miridae) contained relatively high amounts of Cry1Ac toxin when collected before anthesis, but not during or after anthesis. This might be explained by the fact that D. punctulatus adults ingests Cry1Ac when they supplement their diet by feeding on soybean tissues [75]. Adults of the aphid predator Aphidoletes abietis (Diptera: Cecidomyiidae) are known to feed on nectar and honeydew [76] and are thus not exposed to the Cry protein.

Predatory spiders such as M. tricuspidata and E. graminicolum are true generalists and play an important role in controlling pests such as thrips, spider mites, leafhoppers, aphids, and lepidopteran larvae in Bt crop fields [77]. In addition to encountering Bt toxin when feeding on above-ground herbivores, spiders may also be exposed to Bt toxin when feeding on pollen and when feeding on soil-associated prey; the latter may have acquired the toxin as a consequence of root feeding or as a consequence of the exudation of toxin by roots and its subsequent passage through the detrital food web [78], [79]. Despite their contribution to biological control and despite the multitude of pathways through which spiders may be exposed to Bt toxins in agroecosystems, few studies have measured the uptake of Bt toxin by spiders and their exposure level in the field. In three previous studies with Bt maize or Bt cotton, field-collected spiders of different families (Linyphiidae, Araneidae, Tetragnathidae, and Theridiidae) were found to contain detectable concentrations of Cry toxin, and the amount of uptake of Cry toxin was associated with their prey spectrum [41], [44], [63]. Generally, the uptake of Bt toxin by spiders (Theridiidae and Lycosidae) is low because the toxin is diluted as it is transferred from the first trophic level (Bt crops) to the second (prey) and because rapid excretion and digestion likely prevent the toxin from accumulating in spider bodies [33], [40]. In our study, three species of spiders (M. tricuspidata, E. graminicolum, and an unidentified species of Thomisidae) collected from Bt soybean were found to contain Cry1Ac toxin, indicating that exposure pathways exist for these spiders in soybean fields. Although relatively high amounts of Cry1Ac (about 13% of the level in soybean leaves) were found in the unidentified species of Thomisidae, the data should be considered with caution because the analysis was not replicated. Lower levels of Cry1Ac (about 1% of that detected in soybean leaves) were found in adult M. tricuspidatus throughout the soybean season, which suggests that M. tricuspidatus in Bt soybean field is likely to consume prey that contain a similar Cry concentration throughout the season and that pollen consumption is not an important exposure pathway.

Conclusions

The current report provides the first data concerning the exposure of non-target arthropods to Cry proteins in Bt soybean fields. The Cry1Ac concentration detected in arthropods varied among arthropod species/taxa, between arthropod life stages, and among the growth stages of the soybean plants. The highest Cry1Ac concentration, which was about 50% of that in soybean leaves, was found in adults of a grasshopper species. Other herbivorous arthropods that were positive for Cry1Ac contained levels between 1 and 10% of that found in the plants; these included a cicadellid sap-feeder, and a number of hemipteran species that are known to feed on mesophyll tissue, and the adults of a curculionid beetle. Among the predators, the highest concentrations were detected in a thomisid spider and an unidentified species of Anthocoridae. For the remaining species/taxa, concentrations were <1% or even below the detection limits of the ELISA. Such information on the exposure of different arthropod groups to the plant-expressed Cry protein within complex food webs is required for non-target risk assessment. More specifically, such information enables researchers to focus on those species that are most likely to be at risk from the insecticidal compound in Bt crops [15], [23], [80], [81].

Supporting Information

Table S1.

Cry1Ac concentrations in arthropods collected in Bt soybean before, during, and after anthesis in 2010. ELISA results below the limit of detection (LOD) are indicated as ‘<’ with the corresponding LOD value.

https://doi.org/10.1371/journal.pone.0103973.s001

(DOCX)

Acknowledgments

We thank Yiping Li (Northwest A&F University, China) and Wenliang Li (China Agriculture University) for identifying species of arthropods; Xiaodan Nan (Northwest A&F University, China) and Jiao Wei (YangZhou University, China) for helping to collect the arthropods; and Huaiheng Wu (Institute of Plant Protection and soil Fertilizer, Hubei Academy of Agricultural Science, China) for providing ELISA technical assistance. Special thanks to Honghua Su (YangZhou University, China) for helping to research the literature and to Michael Meissle (Agroscope, Switzerland) for comments on an earlier draft of the manuscript. We also thank Monsanto Company for providing soybean seeds.

Author Contributions

Conceived and designed the experiments: HLY JR KMW XJL. Performed the experiments: HLY. Analyzed the data: HLY. Wrote the paper: HLY JR YHL.

References

  1. 1. Romeis J, Meissle M, Bigler F (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nat Biotechnol 24: 63–71.
  2. 2. Romeis J, Shelton AM, Kennedy GG (2008) Integration of Insect-Resistant Genetically Modified Crops within IPM Programs. Spring Science+Business Media B.V. 429 p.
  3. 3. Naranjo SE (2011) Impact of Bt transgenic cotton on integrated pest management. J Agric Food Chem 59: 5842–5851.
  4. 4. Lu YH, Wu KM, Jiang YY, Guo YY, Desneux N (2012) Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services. Nature 487: 362–365.
  5. 5. Rai PS, Seshu Reddy KV, Govindan R (1973) A list of insect pests of soybean in Karnataka state. Curr Res 2: 97–98.
  6. 6. Bernardi O, Malvestiti GS, Dourado PM, Oliveira WS, Martinelli S, et al. (2012) Assessment of the high-dose concept and level of control provided by MON87701×MON89788 soybean against Anticarsia gemmatalis and Pseudoplusia includes (Lepidoptera: Noctuidae) in Brazil. Pest Manag Sci 68: 1083–1091.
  7. 7. Yu HL, Li YH, Li XJ, Romeis J, Wu KM (2013) Expression of Cry1Ac in transgenic Bt soybean lines and their efficiency in controlling lepidopteran pests. Pest Manag Sci 69: 1326–1333.
  8. 8. Garcia-Alonso M, Jacobs E, Raybould A, Nickson TE, Sowig P, et al. (2006) A tiered system for assessing the risk of genetically modified plants to non-target organisms. Environ Biosafety Res 5: 57–65.
  9. 9. Romeis J, Bartsch D, Bigler F, Candolfi MP, Gielkens MMC, et al. (2008) Assessment of risk of insect-resistant transgenic crops to non-target arthropods. Nat Biotechnol 26: 203–208.
  10. 10. Romeis J, Hellmich RL, Candolfi MP, Carstens K, De Schrijver A, et al. (2011) Recommendations for the design of laboratory studies on non-target arthropods for risk assessment of genetically engineered plants. Transgenic Res 20: 1–22.
  11. 11. Wolt JD, Keese P, Raybould A, Fitzpatrick JW, Burachik M, et al. (2010) Problem formulation in the environmental risk assessment for genetically modified plants. Transgenic Res 19: 425–436.
  12. 12. Romeis J, Raybould A, Bigler F, Candolfi MP, Hellmich RL, et al. (2013) Deriving criteria to select arthropod species for laboratory tests to assess the ecological risks from cultivating arthropod-resistant transgenic crops. Chemosphere 90: 901–909.
  13. 13. Romeis J, Meissle M, Álvarez-Alfageme F, Bigler F, Bohan DA, et al. (2014) Potential use of an arthropod database to support the non-target risk assessment and monitoring of transgenic plants. Transgenic Res DOI:https://doi.org/10.1007/s11248-014-9791-2.
  14. 14. Carstens K, Cayabyab B, De Schrijver A, Gadaleta PG, Hellmich RL, et al. (2014) Surrogate species selection for assessing potential adverse environmental impacts of genetically engineered insect-resistant plants on non-target organisms. GM Crops and Food 5: 11–15.
  15. 15. Romeis J, Meissle M, Raybould A, Hellmich RL (2009) Impact of insect-resistant transgenic crops on above-ground non-target arthropods. In: Ferry N, Gatehouse AMR, editors. Environmental Impact of Genetically Modified Crops. CABI, Wallingford, UK. pp.165–198.
  16. 16. Dutton A, Romeis J, Bigler F (2003) Assessing the risks of insect resistant transgenic plants on entomophagous arthropods: Bt-maize expressing Cry1Ab as a case study. BioControl 48: 611–636.
  17. 17. Devos Y, De Schrijver AD, de Clercq P, Kiss J, Romeis J (2012) Bt-maize event MON88017 expressing Cry3Bb1 does not cause harm to non-target organism. Transgenic Res 21: 1191–1214.
  18. 18. Torres JB, Ruberson JR (2008) Interactions of Bacillus thuringiensis Cry1Ac toxin in genetically engineered cotton with predatory heteropterans. Transgenic Res 17: 345–354.
  19. 19. García M, Ortego F, Castañera P, Farinós GP (2010) Effects of exposure to the toxin Cry1Ab through Bt maize fed-prey on the performance and digestive physiology of the predatory rove beetle Atheta coriaria. Biol Control 55: 225–233.
  20. 20. Li YH, Romeis J (2010) Bt maize expressing Cry3Bb1 does not harm the spider mite, Tetranychus urticae, or its ladybird beetle predator, Stethorus punctillum. Biol Control 53: 337–344.
  21. 21. Obrist LB, Dutton A, Romeis J, Bigler F (2006) Biological activity of Cry1Ab toxin expressed by Bt maize following ingestion by herbivorous arthropods and exposure of the predator Chrysoperla carnea. BioControl 51: 31–48.
  22. 22. Lawo NC, Wäckers FL, Romeis J (2010) Characterizing indirect prey-quality mediated effects of a Bt crop on predatory larvae of the green lacewing, Chrysoperla carnea. J Insect Physiol 56: 1702–1710.
  23. 23. Romeis J, Meissle M (2011) Non-target risk assessment of Bt crops-Cry protein uptake by aphids. J Appl Entomol 135: 1–6.
  24. 24. Chen Y, Tian JC, Wang W, Fang Q, Akhtar ZR, et al. (2012) Bt rice expressing Cry1Ab does not stimulate an outbreak of its non-target herbivore, Nilaparvata lugens. Transgenic Res 21: 279–291.
  25. 25. Lundgren JG, Wiedenmann RN (2004) Nutritional suitability of corn pollen for the predator Coleomegilla maculata (Coleoptera: Coccinellidae). J Insect Physiol 50: 567–575.
  26. 26. Duan JJ, Teixeira D, Huesing JE, Jiang CJ (2008) Assessing the risk to non-target organisms from Bt corn resistant to corn rootworms (Coleoptera: Chrysomelidae): Tier-I testing with Orius insidiosus (Heteroptera: Anthocoridae). Environ Entomol 37: 838–844.
  27. 27. Li YH, Meissle M, Romeis J (2010) Use of maize pollen by adult Chrysoperla carnea (Neuroptera: Chrysopidae) and fate of Cry proteins in Bt-transgenic varieties. J Insect Physiol 56: 157–164.
  28. 28. Lumbierres B, Albajes R, Pons X (2012) Positive effect of Cry1Ab-expressing Bt maize on the development and reproduction of the predator Orius majusculus under laboratory conditions. Biol Control 63: 150–156.
  29. 29. Wang YY, Li YH, Romeis J, Chen XP, Zhang J, et al. (2012) Consumption of Bt rice pollen expressing Cry2Aa does not cause adverse effects on adult Chrysoperla sinica Tjeder (Neuroptera: Chrysopidae). Biol Control 61: 246–251.
  30. 30. Torres JB, Ruberson JR, Adang MJ (2006) Expression of Bacillus thuringiensis Cry1Ac protein in cotton plants, acquisition by pests and predators: a tritrophic analysis. Agric Forest Entomol 8: 191–202.
  31. 31. Álvarez-Alfageme F, Ferry N, Castañera P, Ortego F, Gatehouse AMR (2008) Prey mediated effects of Bt maize on fitness and digestive physiology of the red spider mite predator Stethorus punctillum Weise (Coleoptera: Coccinellidae). Transgenic Res 17: 943–954.
  32. 32. Álvarez-Alfageme F, Ortego F, Castañera P (2009) Bt maize fed-prey mediated effect on fitness and digestive physiology of the ground predator Poecilus cupreus L. (Coleoptera: Carabidae). J Insect Physiol 55: 144–150.
  33. 33. Chen M, Ye GY, Liu ZC, Fang Q, Hu C, et al. (2009) Analysis of Cry1Ab toxin bioaccumulation in a food chain of Bt rice, an herbivore and a predator. Exotoxicology 18: 230–238.
  34. 34. Li YH, Romeis J, Wang P, Peng YF, Shelton AM (2011) A comprehensive assessment of the effects of Bt cotton on Coleomegilla maculata demonstrates no detrimental effects by Cry1Ac and Cry2Ab. PLoS ONE 6: e22185 DOI:https://doi.org/10.1371/journal.pone.0022185.
  35. 35. Stephens EJ, Losey JE, Allee LL, DiTommaso A, Bodner C, et al. (2012) The impact of Cry3Bb1 Bt-maize on two guilds of beneficial beetles. Agric Ecosyst Environ 156: 72–81.
  36. 36. Tian JC, Chen Y, Li ZL, Li K, Chen M, et al. (2012) Transgenic Cry1Ab rice does not impact ecological fitness and predation of a generalist spider. PLoS ONE 7: e35164 DOI:https://doi.org/10.1371/journal.pone.0035164.
  37. 37. Tian JC, Collins HL, Romeis J, Naranjo SE, Hellmich RL, et al. (2012) Using field-evolved resistance to Cry1F maize in a lepidopteran pest to demonstrate no adverse effects of Cry1F on one of its major predators. Transgenic Res 21: 1303–1310.
  38. 38. Tian JC, Wang XP, Long LP, Romeis J, Naranjo SE, et al. (2013) Bt crops producing Cry1Ac, Cry2Ab and Cry1F do not harm the green lacewing, Chrysoperla rufilabris. PLoS ONE 8: e60125 DOI:https://doi.org/10.1371/journal.pone.0060125.
  39. 39. Tian JC, Long LP, Wang XP, Naranjo SE, Romeis J, et al. (2014) Using resistant prey demonstrates that Bt plants producing Cry1Ac, Cry2Ab and Cry1F have no negative effects on Geocoris punctipes and Orius insidiosus. Environ Entomol 43: 242–251.
  40. 40. Meissle M, Romeis J (2012) No accumulation of Bt protein in Phylloneta impressa (Araneae: Theridiidae) and prey arthropods in Bt maize. Environ Entomol 41: 1037–1042.
  41. 41. Harwood JD, Wallin WG, Obrycki JJ (2005) Uptake of Bt endotoxins by nontarget herbivores and higher order arthropod predators: molecular evidence from a transgenic corn agroecosystem. Mol Ecol 14: 2815–2823.
  42. 42. Harwood JD, Samson RA, Obrycki JJ (2007) Temporal detection of Cry1Ab-endotoxins by coccinellid predators in fields of Bacillus thuringiensis corn. Bull Entomol Res 97: 643–648.
  43. 43. Obrist LB, Dutton A, Albajes R, Bigler F (2006) Exposure of arthropod predators to Cry1Ab toxin in Bt maize fields. Ecol Entomol 31: 143–154.
  44. 44. Meissle M, Romeis J (2009) The web-building spider Theridion impressum (Araneae: Theridiidae) is not adversely affected by Bt maize resistant to corn rootworms. Plant Biotechnol J 7: 645–656.
  45. 45. Zhang QL, Li YH, Hua HX, Yang CJ, Wu KM, et al. (2013) Exposure degree of important non-target arthropods to Cry2Aa in Bt rice field. Chin J Appl Ecol 24: 1647–1651 (In Chinese).
  46. 46. McWilliams DA, Berglund DR, Endres GJ (1999) Soybean Growth and Management Quick Guide. North Dakota State University and University of Minnesota.
  47. 47. Chen XZ, Li X, Yang JY, Xie H, Han FT (2003) The relationship analyses on the bearing term structure and agronomic characters of the summer seeding soybean. Chin Agric Sci Bull 19: 64–68 (in Chinese).
  48. 48. Wan P, Zhang YJ, Wu KM, Huang MS (2005) Seasonal expression profiles of insecticidal protein and control efficacy against Helicoverpa armigera for Bt cotton in the Yangtze River valley of China. J Econ Entomol 98: 195–201.
  49. 49. Nguyen HT, Jehle JA (2007) Quantitative analysis of the seasonal and tissue-specific expression of Cry1Ab in transgenic maize Mon810. J Plant Dis Protect 114: 82–87.
  50. 50. Zhang YJ, Li YH, Zhang Y, Chen Y, Wu KM, et al. (2011) Seasonal expression of Bt proteins in transgenic rice lines and the resistance against Asiatic Rice Borer Chilo suppressalis (Walker). Environ Entomol 40: 1323–1330.
  51. 51. Kranthi KR, Naidu S, Dhawad CS, Tatwawadi A, Mate K, et al. (2005) Temporal and intra-plant variability of Cry1Ac expression in Bt-cotton and its influence on the survival of the cotton bollworm, Helicoverpa armigera (Hübner) (Noctuidae: Lepidoptera). Curr Sci 89: 291–298.
  52. 52. Rochester IJ (2006) Effect of genotype, edaphic, environmental conditions and agronomic practices on Cry1Ac protein expression in transgenic cotton. J Cotton Sci 10: 252–262.
  53. 53. Addison SJ, Rogers DJ (2010) Potential impact of differential production of the Cry2Ab and Cry1Ac proteins in transgenic cotton in response to cold stress. J Econ Entomol 103: 1206–1215.
  54. 54. Chen FJ, Wu G, Ge F, Parajulee MN (2011) Relationships between exogenous-toxin quantity and increased biomass of transgenic Bt crops under elevated carbon dioxide. Ecotoxicol Environ Safe 74: 1074–1080.
  55. 55. Ranjithkumar L, Patil BV, Vijaykumar NG (2011) Impact of irrigation and fertilizer levels on Cry1Ac protein content in Bt cotton. Res J Agr Sci 2: 33–35.
  56. 56. Chen Y, Wen YJ, Chen Y, Cothren JT, Zhang X, et al. (2012) Effects of extreme air temperature and humidity on the insecticidal expression level of Bt cotton. J Integr Agric 11: 1836–1844.
  57. 57. Hagenbucher S, Olson DM, Ruberson JR, Wäckers FL, Romeis J (2013) Resistance mechanisms against arthropod herbivores in cotton and their interactions with natural enemies. Crit Rev Plant Sci 32: 458–482.
  58. 58. Crespo ALB, Spencer TA, Nekl E, Pusztai-Carey M, Moar WJ, et al. (2008) Comparison and validation of methods to quantify Cy1Ab toxin from Bacillus thuringiensis for standardization of insect bioassays. Appl Environ Microbiol 74: 130–135.
  59. 59. Nguyen HT, Hunfeld H, Meissle M, Miethling-Graff R, Pagel-Wieder S, et al. (2008) Round robin quantitation of Cry3Bb1 using the qualitative PathoScreen ELISA. IOBC/WPRS Bull 33: 59–66.
  60. 60. Székács A, Weiss G, Quist D, Takács E, Darvas B, et al. (2012) Inter-laboratory comparison of Cry1Ab toxin quantification in MON810 maize by enzyme-immunoassay. Food Agri Immunol 23: 99–121.
  61. 61. Burgio G, Dinelli G, Marotti I, Zurla M, Bosi S, et al. (2011) Bt-toxin uptake by the non-target herbivore, Myzus persicae (Hemiptera: Aphididae), feeding on transgenic oilseed rape in laboratory conditions. Bull Entomol Res 101: 241–247.
  62. 62. Zhao Y, Ma Y, Niu L, Ma W, Mannakkaraa A, et al. (2013) Bt cotton expressing Cry1Ac/Cry2Ab or Cry1Ac/epsps does not harm the predator Propylaea japonica through its prey Aphis gossypii. Agric Ecosyst Environ 179: 163–167.
  63. 63. Dhillon MK, Sharma HC (2013) Comparative studies on the effects of Bt-transgenic and non-transgenic cotton on arthropod diversity, seedcotton yield and bollworms control. J Environ Biol 34: 67–73.
  64. 64. Brandt SL, Coudron TA, Habibi J, Brown GR, Ilagan OM, et al. (2004) Interation of two Bacillus thuringiensis delta-endotoxins with the digestive system of Lygus hesperus. Curr Microbiol 48: 1–9.
  65. 65. Chougule NP, Bonning BC (2012) Toxins for transgenic resistance to Hemipteran pests. Toxins 4: 405–429.
  66. 66. Yang FA, Huang YZ, Wang YL (1996) Biological characteristics of Atractomorpha sinensis. Entomol Knowledge 33: 278 (In Chinese).
  67. 67. Han FY, Ren AJ, Jin JB (1999) The relationship between climatic factors and mating and laying in Atractomopha sinensis I. Bol. J Shanxi Univ 22: 270–273 (In Chinese).
  68. 68. Sun X, An MX, Zhao KJ, Liu J (2012) The occurrence and integrated control of two striped leaf beetle Paraluperodes suturalis nigrobilineatus in Heilongjiang province. Mordernizing Agr 3: 4–5 (In Chinese).
  69. 69. Zhang GF, Wan FH, Guo JY, Hou ML (2004) Expression of Bt toxin in transgenic Bt cotton and its transmission through pests Helicoverpa armigera and Aphis gossypii to natural enemy Propylaea japonica in cotton plots. Acta Entomol Sin 3: 334–341.
  70. 70. Ridgway RL, Jones SL (1968) Plant feeding by Geocoris pallens and Nabis americoferus. Ann Entomol Soc Am 63: 232–233.
  71. 71. Stoner A (1970) Plant feeding by a predaceous insect, Geocoris punctipes. J Econ Entomol 63: 1911–1915.
  72. 72. McEwen P, New TR, Whittington AE (2001) Lacewings in the crop Environment. Cambridge University Press, Cambridge. 547p.
  73. 73. Song HY, Wu LY, Chen GF, Wang ZC, Song QM (1988) Biological characters of lady-beetle, Propylaea japonica (Thunberg). Nat Enemies Insects 10: 22–33 (In Chinese).
  74. 74. Li KS, Chen XD, Wang HZ (1992) New discovery of feeding habitats of some ladybirds. Shanxi Forest Sci Technol 2: 84–86 (In Chinese).
  75. 75. Lu CZ (1999) Study on the biology and predaceous role of Deraeocorls Punctulaus to cotton aphid. J TalimUniv Agri Reclamation 11: 13–16 (In Chinese).
  76. 76. Kuo-Sell HL (1987) Some bionomics of the predacious aphid midge, Aphidoletes aphidimyza (Rond.) (Diptera: Cecidomyiidae), and the possibility of using the rose grainaphid, Metopolophium dirhodum (Wlk.), as an alternative prey in an open rearing unit in greenhouses. In: Cavalloro R, editor. Integrated and biological control in protected crops. Proceedings of a Meeting of the EC Experts' group. Balkema, Rotterdam, pp. 151–156.
  77. 77. Wu KM, Guo YY (2005) the evolution of cotton pest management practices in China. Annu Rev Entomol 50: 31–52.
  78. 78. Peterson JA, Lundgren JG, Harwood JD (2011) Interactions of transgenic Bacillus thuringiensis insecticidal crops with spiders (Araneae). J Arachnol 39: 1–21.
  79. 79. Meissle M (2013) Side effects of Bacillus thuringiensis toxins on spiders. In: Nentwig W, editor. Spider Ecophysiology. Springer-Verlag, Berlin Heidelberg, Germany. pp. 429–440.
  80. 80. Raybould A (2007) Environmental risk assessment of genetically modified crops: General principles and risks to non-target organisms. BioAssay 2: 8 Available at http://www.seb.org.br/bioassay.
  81. 81. Li YH, Peng YF, Hallerman EM, Wu KM (2014) Biosafety management and commercial use of genetically modified crops in China. Plant Cell Rep 33: 565–573.