parasitoid, *Diaeretiella rapae*. *Biol Control* (2004) **31**(2):133–7. doi:10.1016/j. biocontrol.2004.03.010

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Mullin, Fine, Reynolds and Frazier. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.  There are a number of reviews on the health consequences of exposure to EDCs including a comprehensive report by the World Health Organization and the United Nations Environment Programme (1). Recently, De Falco et al. (2) addressed the impact of EDCs on male reproductive system, with special reference to the effects of bisphenol A (BPA), alkylphenols, and phthalates. Jeng (3) also reviewed the epidemiological data on the adverse effects of EDCs on male reproduction and experimental studies that could shed light on mechanisms (disruption of steroidogenesis, oxidative stress, and epigenetic changes) through which EDCs could impair male reproductive health. Both articles are essentially narrative reviews of the abundant and highly controversial literature on the health consequences of exposures to EDCs.

A key feature that distinguishes a narrative review from a systematic review is that the former review does not include a comprehensive and meticulous search of all potentially relevant articles on specified sources, and does not use explicit and reproducible criteria to selected articles for review (4). Compared to systematic reviews, narrative reviews of the literature are more likely to error and bias in the selection of relevant studies (4, 5). Moreover, if research designs, methods, and study characteristics do not undergo a critical appraisal, summary, and conclusions of literature reviews are even more prone to bias.

De Falco et al. were unable to convey to readers an unbiased review of the empirical evidence suggesting that environmental exposures to EDCs might affect male reproduction. The authors, for instance, did not disclose the conflicting evidence on the enlargement of prostate after developmental exposure to BPA. In the mid-1990s, a set of studies by vom Saal and coworkers showed that prenatal exposure to β-estradiol (EST), diethylstilbestrol (DES), or BPA led to enlarged ventral prostate in adult mice (6, 7). The observation that enlargement of prostate resulted from prenatal exposures to low doses of estrogenic compounds (e.g., supra-physiological levels of EST), and exhibited non-monotonic dose–response relationships, fueled considerable debate over the adverse health consequences of environmental exposure to EDCs. Several studies, however, failed to reproduce these findings not only with BPA but also with EST and DES (8–10). Although reproducibility is one hallmark of experimental sciences, the foregoing discrepancy between studies by different authors has remained unexplained (11).

#### *Edited by:*

*Robin Mesnage, King's College London, UK*

*Reviewed by: Hui Hu, University of Florida, USA*

*\*Correspondence: Francisco José Roma Paumgartten<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Environmental Health, a section of the journal Frontiers in Public Health*

> *Received: 02 May 2015 Accepted: 09 June 2015 Published: 22 June 2015*

#### *Citation:*

*Paumgartten FJR (2015) Commentary: "Estrogenic and anti-androgenic endocrine disrupting chemicals and their impact on the male reproductive system". Front. Public Health 3:165. doi: 10.3389/fpubh.2015.00165*

Furthermore, authors' statements that "*. . .*over 50 years, the global average sperm count dropped by half*. . .*" and that "studies of the last decade strongly support that male reproductive health has been deteriorating*. . .*" were unaccompanied by any reference to the conflicting evidence on this matter (2). The widespread notion that semen quality has decreased over the past decades stands on some retrospective studies [Ref. (12, 13), and others]. Nonetheless, resultsfrom a number of other studies (not cited by the authors) are inconsistent with this hypothesis.Most studies showing downward trends in sperm counts included samples coming from different populations and places that do not necessarily allow a valid comparison over time. For instance, a re-analysis of US data used by Carlsen et al. (12) found no decline in sperm counts when data from New York were excluded from the regression analysis (14). Therefore, apparent time trend toward lower concentrations reported by Carlsen et al. (12) resulted, in fact, from geographic variations in sperm counts (14, 15). Moreover, a longitudinal study of sperm concentrations for Danish military draftees (5000 men), collected annually for 15 years (1996–2010), found no indication that semen quality has changed during the monitoring period (16). Although several studies precipitated by reports on "downward temporal trends in sperm counts" refuted its existence, the "sperm crisis" notion is still a highly controversial issue in the literature (17–20). Temporal trends to increasing birth prevalence of male reproductive tract defects such hypospadias and cryptorchidism described by some authors are far from being a consistent finding among studies (21, 22).

#### **References**

The "endocrine disruptor hypothesis," a landmark of which was the Wingspread Conference Statement in 1990s (23), fits like a glove to the beliefs of the public that pesticides and other manmade chemicals in the environment are undermining human health and fertility. Two seminal books by Rachel Carlson (Silent Spring, 1962) and Theo Colborn (Our Stolen Future, 1996) boosted considerably these concerns on the deleterious effects of environmental chemicals on human fertility and health. Notwithstanding the fact that ED hypothesis is instigating, the notion that "male reproductive health has been deteriorating," as asserted by De Falco et al. (2) and others, lacks an unequivocal demonstration by soundly designed epidemiology studies. It is of note that, even if a temporal trend toward a worse male reproductive health had been demonstrated consistently, it would still be missing to prove that there is a causal link between EDCs, identified as such in experimental tests, and the incidence of the adverse health outcome in the human population. A step forward to identifying relevant research gaps and to unveiling the real impact of EDCs on male fertility and reproductive health would be to conduct less and less narrative and potentially biased reviews and more and more good quality systematic reviews of the literature on the topic. Finally, we highlight that a critical appraisal of the quality of original studies is required for both a good quality narrative and a good quality systematic review. If unbiased, good quality narrative reviews can also be helpful. Systematic reviews, however, are a more reliable approach to avoid bias in the selection of studies.

23. Krimsky S. Environmental endocrine hypothesis and public policy. In: Kroll-Smith S, Brown P, Gunter VJ, editors. *Illness and the Environment*. New York, NY: University Press (2000). p. 95–107.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Paumgartten. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Lény Teyssier1,2,3, Julie Colussi1 , Stéphanie Delemasure4 , Johanna Chluba1 , David Wendehenne2,3, Olivier Lamotte2,3\* and Jean-Louis Connat1 \**

*1Univ. Bourgogne Franche-Comté, LNC UMR866, Dijon, France, 2UMR1347 Agroécologie, AgroSup Dijon, INRA, Univ. Bourgogne Franche-Comté, Dijon, France, 3ERL CNRS 6003, Dijon, France, 4Cohiro Biotechnology, Faculté de médecine, Dijon, France*

Plant defense stimulators (PDSs) rely on the activation of plant innate immunity in order

#### *Edited by:*

*Robin Mesnage, King's College London, UK*

#### *Reviewed by:*

*Leticia Moreno-Fierros, Universidad Nacional Autónoma de México, Mexico Md. Asaduzzaman, University of Dhaka, Bangladesh*

#### *\*Correspondence:*

*Olivier Lamotte<EMAIL_ADDRESS>Jean-Louis Connat<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Environmental Health, a section of the journal Frontiers in Public Health*

*Received: 19 July 2016 Accepted: 21 March 2017 Published: 24 April 2017*

#### *Citation:*

*Teyssier L, Colussi J, Delemasure S, Chluba J, Wendehenne D, Lamotte O and Connat J-L (2017) Inflammatory Effects of the Plant Protection Product Stifenia (FEN560) on Vertebrates. Front. Public Health 5:74. doi: 10.3389/fpubh.2017.00074*

to protect crops against various pests. These molecules are thought to be a safer alternative to classical plant protection products. Given that innate immune systems share common features in plants and vertebrates, PDS can potentially cross-react with innate immunity of non-target organisms. To test this hypothesis, we studied effects of the commercial PDS Stifenia (FEN560), which is composed of crushed fenugreek seeds. We tested various concentrations of Stifenia (0.03–1 mg mL−1 ) on human peripheral blood mononuclear cells and checked, 20 h later, cell metabolic activity (MA) using XTT assay, cell death by flow cytometry analysis, and IL-1β inflammatory cytokine released in the culture medium using ELISA. Stifenia induced a general decrease of the cell MA, which was concomitant with a dose-dependent release of IL-1β. Our results highlight the activation of human immune cells. The inflammatory effect of Stifenia was partially inhibited by pan-caspase inhibitor. Accordingly, Stifenia induced the release of p20 caspase-1 fragment into the culture medium suggesting the involvement of the NLRP3 inflammasome. Furthermore, we observed that Stifenia can induce cell death. We also tested the effect of Stifenia on Zebrafish larvae. After 24 h of exposure, Stifenia induced a dose-dependent IL-1β and TNFα gene expression. The human-cell-based approach developed in this work revealed a high sensitivity concerning inflammatory properties of a plant protection product. These tests could be routinely used to screen the potential adverse effects of this type of compounds. Finally, our results suggest a potential danger of using extensively certain PDS for crop protection.

Keywords: peripheral blood mononuclear cells, zebrafish, IL-1**β**, pesticides, plant defense stimulator, fenugreek

### INTRODUCTION

In the context of pesticides reduction, alternative strategies to protect crops have emerged, including use of transgenic crops, resistant hybrids, or integrated pest management methods. Among these, stimulation of the plant immune system with various molecules is promising. Plant defense stimulators (PDSs), plant defense inducers, or elicitors define a class of compounds of diverse origins, which can induce disease resistance-related mechanisms by mimicking a pathogen attack or a danger state, resulting in reduced levels of plant infection. They comprise a range of purified or mixture-based natural or synthetic compounds that have been shown to protect plants efficiently (1, 2). To use a new molecule for crop protection in France, an authorization is needed according to the European Union (EC No. 1107/2009) and French regulations. Various toxicological and ecotoxicological tests are required for the production of an initial draft assessment report by EU-designated rapporteur member state (RMN) (3)*.* Stifenia (FEN560), which is exclusively composed of grounded fenugreek seeds (*Trigonella foenum-graecum*) is a PDS authorized by the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) to fight powdery mildew of grape vine (*Erysiphe necator*) and powdery mildew of melon (*Podosphaera fuliginea and Golovinomyces cichoracearum*) (ANSES agreement no. 2012-1685 and 2013-0227). Fenugreek, especially its seeds and its leaves, has been used for centuries in India and North Africa as food or in traditional medicine (4).

Similar to animals, the first step in the activation of the plant immune system is the perception of pathogens or microbe-associated molecular patterns (PAMPs/MAMPs) by pattern recognition receptors (5, 6). In both organisms, their perception induces complex cell signaling events, which result in cellular re-programming. For instance, PAMP/MAMPtriggered immunity in plants is associated with the production of phytoalexins, a class of antimicrobial metabolites, and with the reinforcement of plant cell walls. In mammals, the immune response induces, for example, the production of cytokines (interleukines, TNFα) and antimicrobial peptides. One of the inflammatory cytokine that plays a major role is IL-1β. Indeed, this cytokine is produced following inflammasome activation in monocytes, macrophages, and dendritic cells upon their stimulation by PAMP or damage/danger-associated molecular patterns. IL-1β is processed from pro-IL1β by caspase1 in several inflammasome complexes (e.g., NLRP3, NLRP1, and AIM2) (7, 8). This cytokine upregulates many other inflammatory factors such as IL6 (9) or TNFα (10).

Since plant and animal immune systems share some similarities (11, 12), we hypothesized that the PDS Stifenia could crossreact with the animal immune system. In fact, both beneficial and adverse effects have been described for fenugreek in mammals (4, 13–18) and toxicity against insects has also been reported (19). We thus tested the effects of Stifenia on human peripheral blood mononuclear cells (PBMCs) from different blood donors by quantifying the amount of the inflammatory cytokine IL-1β released by exposed cells. In parallel, we evaluated the metabolic activity (MA) of the stimulated cells using a XTT assay. We also checked the intensity of cell death induced by Stifenia. Finally, we studied the effects of this compound on the larvae of the model fish zebrafish by analyzing cytokine gene induction.

#### MATERIALS AND METHODS

#### Chemicals

Stifenia (FEN560, Société Occitane de Fabrications et de Technologies, France) was extemporaneously suspended in Roswell Park Memorial Institute medium (RPMI; for cell treatment) at 17 mg mL−1 or in autoclaved mineral Volvic water (for zebrafish treatment) at 0.1 mg mL−1 and gently shaken for 30 min. Since Stifenia is not fully soluble in water, insoluble matters were isolated by centrifugation (20,000 *g*, 30 min, room temperature) and supernatant was carefully collected in a new tube. All the concentrations indicated in this work refer to the initial concentration (17 mg mL−1 for human experimentation and 0.1 mg mL−1 for zebrafish experimentation).

Z-VAD-FMK stock solution [20 mM in 100% dimethylsulfoxide (DMSO)], purchased from Promega, was first diluted in RPMI at 85 µM. This solution was used for cell treatment. The final Z-VAD-FMK concentration was 5 µM in cell culture (0.025% DMSO). LPS from *Escherichia coli* 0111:B4 stock solution (1 mg mL−1 in pure water), purchased from Sigma-Aldrich, was diluted in RPMI to reach a final concentration of 10 ng mL−1 in cell culture. TNBS (2,4,6-trinitrobenzenesulfonic acid) stock solution (1 mg mL−1 in pure water), purchased from Sigma-Aldrich, was diluted in autoclaved mineral water (Volvic, France) at 75 µg mL−1 for zebrafish treatment.

#### Human PBMCs

Buffy coats from healthy donors were obtained from EFS Besançon, France (Agreement No. DECO-14-0124). PBMCs were prepared using Pancoll (density 1.077 g mL−1 , PAN-biotech Gmbh, Germany) and Blood Sep Filter tubes (Dominique Dutscher, France). Briefly, 15 mL of Pancoll were collected into the lower part of a Blood Sep Filter tube by a short centrifugation. Then, 25 mL of buffy coat and 15 mL of Dulbecco's phosphatebuffered saline (DPBS, PAN-biotech Gmbh, Germany) were added, gently mixed, and centrifuged (400 *g*, 30 min, room temperature) without brake for the deceleration phase. The PBMC ring was collected, washed three times in DPBS without Ca2+ and Mg2+, and centrifuged (300 *g*, 10 min, 4°C). Cells were suspended in 2–5 mL of DPBS depending on the size of the cell pellet and kept on ice. Viable PBMCs were counted using trypan blue (20), suspended in RPMI medium supplemented with 10% (bovine serum albumin, w/v) and 1% PSA (Penicilline 10,000 U mL−1 , Streptomycine 10 mg mL–1, Amphotericin B 25 µg mL–1 prepared in water), and then seeded in 96-well plate with 105 cells per well in 150 µL of medium.

Immediately after seeding, treatments were done by adding 10 µL per well of a 17-fold concentrated solution of Stifenia made by serial dilution from the stock solution described above. For anti-inflammatory studies, 30 min after Stifenia exposure, 10 µL of LPS solution was added to reach a final concentration of 10 ng mL–1. Z-VAD-FMK was added simultaneously to Stifenia treatment. The final volume of RPMI was adjusted to 170 µL for all conditions. Twenty hours after treatment, cells were centrifuged (600 *g*, 3 min). Supernatants were collected for IL-1β quantification, and cell pellets were used for measuring cell MA.

#### Cell MA

Cell MA, reflecting cell viability, was determined using the XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay (XTT sodium salt, Sigma-Aldrich, France), based on the reduction of a tetrazolium salt (XTT) by dehydrogenases of viable cells into a water-soluble orange formazan product. Briefly, PBMCs were centrifuged (600 *g*, 3 min) to remove culture medium. Then, 100 µL of RPMI without phenol red and 20 µL of a mixture of 0.9 mg mL−1 XTT and 0.01 mM phenazine methosulfate (Sigma-Aldrich, France) were added on pelleted cells. Cells were incubated at 37°C for 4 h and absorbance at 490 nm was measured with a background subtraction at 660 nm using a microplate reader (Infinites M200 PRO, Tecan, France). The results are expressed as percentage of MA compared to control non-exposed cells.

#### Quantification of Inflammatory Cytokines

Production of the inflammatory cytokine IL-1β was estimated in the culture medium using ELISA assay (Human IL-1β ELISA Ready-SET-Go! eBiosciences, France) according to the supplier's instructions.

### Quantification of Cell Death

Two hundred thousand cells were seeded in 24-well plates and treated with Stifenia during 20 h as described above. Cells contained in the culture medium were harvested by centrifugation (400 *g*, 5 min) and then suspended in 300 µL of PBS containing propidium iodide (PI) (10 µg mL−1 ). PI-stained cells were detected using a LSR II flow cytometer (BD Biosciences) and acquisitions were performed during 45 s using BD FACSDiva Software 6.1.2. Flow Jaw was used for figure drawing.

### p20-Caspase-1 Assay

Quantity of processed caspase-1 was evaluated by measuring the amount of the p20 fragment secreted in the culture medium using ELISA assay (Human Caspase-1/ICE Quantikine ELISA Kit, R&D Systems, France) according to the supplier's instructions.

#### Zebrafish Strains, Maintenance, and Treatment

According to the European Union Directive 2010/63/EU, no specific ethics approval was required for this project, as all zebrafish larvae used in this study were less than 120-h postfertilization (hpf) old (21, 22). Wild-type fishes (WIK strain) were obtained from the ZIRC (OR, USA) and kept at 28°C with a light:dark cycle 14:10 h. They were fed twice a day with dried flake food (Gemma Mirco, Skretting, France). The fish were mated and spawning was stimulated by the onset of light. Zebrafish eggs were collected immediately after being fertilized and distributed in 24-well plates (three eggs per well) containing 1 mL of autoclaved mineral water (Volvic, France). At 4 days postfertilization, water was replaced by fresh water containing the desired concentration of Stifenia or 75 µg mL−1 of TNBS. Twelve to fifteen zebrafish larvae per condition were incubated during 20 h.

### RNA Extraction and RT-qPCR

After 20 h of exposure (Stifenia or TNBS, see above), 12–15 zebrafish larvae (120 h postfertilization) were euthanized with tricaïne, collected, and disaggregated during 5 min at room temperature using 17-gage needles in 1 mL of Trizol reagent (Invitrogen, France) and vortexed. Total RNAs were extracted according to supplier's instructions. The RNA samples were treated with DNAse (TURBO DNA-free, Life Technologies) according to the manufacturer's instructions. Approximatively 15–20 µg of RNA diluted in water were washed with butanol and diethyl ether according to Krebs et al. (23). Diethyl ether was evaporated under a fume hood. The resultant water phase containing RNA was mixed with 175 µL of RNA Lysis Buffer and 350 µL of RNA Dilution Buffer and loaded on a SV Total RNA column (Promega, France) to perform on-column DNAse digestion according to supplier's instruction. Then, 1 µg of total RNA was reversed-transcribed to cDNA using the iScript™ reverse transcription supermix for RT-qPCR (Biorad, France). Analyses were performed on a thermocycler (Step One Plus, Applied Biosystems, France) using Power SYBR Green from the same purchaser. The parameters used for the PCR were 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. The relative expression ratio (experimental/control) was normalized with *Danio rerio* HPRT1 (24) according to 2−ΔΔCt method. Sequences of primers used in this study are listed in **Table 1**. RT-qPCR assays were performed in duplicates for each cDNA and each primer couple and the experiment repeated two times.

### Statistical Analysis

Data obtained were expressed as mean ± SEM. Statistical differences among treatments were evaluated by Kruskall–Wallis method. *Post hoc* tests were used to identify statistical groups as described in each figure legend.

## RESULTS

Manufacturer's instructions indicate that Stifenia has to be solubilized in water. However, Stifenia is neither fully soluble in water nor in other classical solvents such as 100% DMSO, acetone 60% in water (v/v), ethanol 100%, and RPMI medium (data not shown), because it is composed of crushed fenugreek seeds. To study its effect on human PBMCs or zebrafish larvae, we used an aqueous soluble extract obtained as described in Section "Materials and Methods." According to ANSES, the recommended use-concentration of Stifenia is 0.15–0.5% (m/v), which correspond to 1.5–5 mg mL−1 (ANSES 2012-1685, ANSES 2013-0227). In our study, we tested concentrations of Stifenia below these recommended use-concentrations.

Table 1 | List and sequences of primers used for RT-qPCR experiments.

*Dr, Danio rerio; For, forward; Rev, reverse.*

## Stifenia Induces a Dose-Dependent Release of IL-1**β** in the Culture Medium

Different concentrations of Stifenia (0.03–1 mg mL−1 ) were independently tested for 20 h on PBMC from nine different healthy human blood donors (**Figure 1A**; Figures S1 and S2 in Supplementary Material; **Table 2**). Cell MA was then measured using the XTT assay and IL-1β production was quantified in the culture medium using ELISA. A decrease of MA was observed from 0.1–1 mg mL−1 of Stifenia (**Figure 1A**). This decrease was observed with eight out of nine blood donors but to a different extend (Figure S1 in Supplementary Material). For these blood

Figure 1 | Effect of Stifenia on human peripheral blood mononuclear cell (PBMC) metabolic activity (MA) and IL–1**β** production. PBMCs were stimulated by different concentrations of Stifenia added in the culture medium. Cell MA (white bars) was estimated by the XTT assay and IL-1β (gray bars) was measured in the culture medium. (A) PBMCs were exposed to Stifenia for 20 h. (B) PBMCs were stimulated by addition of 10 ng mL−1 of LPS 30 min after the beginning of Stifenia exposure. Results are obtained from the blood donor CHR026\_91. Bars represent the mean of eight technical replicates. Different letters (lowercase for the XTT assay, capitals for IL-1β) indicate statistical differences between groups (*p* < 0.05). If two conditions share one or several letters, there are no statistical differences. Statistical differences were determined using a Kruskal–Wallis test followed by a comparison with the Steel–Dwass–Critchlow–Fligner method. CTR, control non-treated cells.

donors, it ranged from 9.5 to 33.2% when 0.3 mg mL−1 of Stifenia is used (**Table 2**).

In culture medium from unexposed cells, IL-1β was either not detected or very close to background level (**Figure 1A**). In this cell batch CHR026\_91, the lowest Stifenia concentration used (0.03 mg mL–1) did not induce IL-1β production. Higher concentrations progressively increased IL-1β release, which peaked at 3,240 pg mL–1 with 1 mg mL–1 of Stifenia. This quantity is equivalent to the one induced by 10 ng mL–1 of LPS. Although the pattern of IL-1β induction was always similar among the different PBMC batches tested, we noticed great variations in the quantities of IL-1β released (**Table 2**; Figure S1 in Supplementary Material). Thus, the lowest quantity found was 5 pg mL–1 after treatment with 0.3 mg mL–1 of Stifenia while the highest was 3,268 pg mL–1 (**Table 2**). The LPS-induced IL-1β production was also systematically measured and was also variable among the different blood donors. LPS (10 ng mL–1) induced the release of 18–2,984 pg mL–1 of IL-1β in the culture media, depending on the donor. No clear relationship was found between LPS- and Stifenia-induced IL-1β productions (**Table 2**; Figure S1 in Supplementary Material). In an exploratory experiment, we evaluated the production of others cytokines in the culture medium of Stifenia-exposed PBMC using a Multiplex assay (Table S1 in Supplementary Material). As observed with ELISA, Stifenia induced a dose-dependent production of IL-1β. We also detected a slight induction of TNFα and of the anti-inflammatory cytokine IL-10. No modulation of IFNγ, IL2, or IL-12p70 concentrations was observed.

### Stifenia Does Not Inhibit LPS-Induced Inflammation in PBMC

Literature frequently reported anti-inflammatory effects of fenugreek seed extracts (13, 25, 26). We thus tested the hypothesis that Stifenia could decrease the LPS-induced IL-1β production in PBMC. **Figure 1B** shows representative results of experiments conducted on PBMC isolated from six different blood donors. Cells were exposed with Stifenia as previously described and then stimulated 30 min later with 10 ng mL–1 LPS. IL-1β was measured 20 h later. Stifenia pretreatment did not reduce LPS-induced IL-1β production (**Figure 1B**; Figure S2 in Supplementary Material). For each Stifenia concentration tested, the IL-1β produced was either higher or equal to the LPS alone-induced IL-1β synthesis. Regarding MA, while LPS alone never reduced it, Stifenia preexposure (0.3–1 mg mL–1) decreased the MA in a dose-dependent manner as observed when used alone (**Figure 1B**; Figure S2 in Supplementary Material).

## Stifenia Does Not Contain Contaminating Microorganisms

In order to check if inflammatory activity of Stifenia was not due to contamination by microorganisms, we compared results obtained from a Stifenia preparation that was either directly used for cell treatment or previously filtered on 0.22 µm pore size membrane. In both cases, a dose-dependent induction of the cytokine production was found. IL-1β induction was not significantly different between filtered and non-filtered Stifenia although it seems that filtered Stifenia induce more IL-1β (**Figure 2**).

Table 2 | Comparison of Stifenia or LPS exposure on metabolic activity (MA) and induced IL–1**β** production of peripheral blood mononuclear cells from nine different healthy human blood donors.

*Cell MA was estimated by the XTT assay, and IL-1*β *was measured in the culture medium 20 h after the addition of Stifenia or LPS. Results represent the mean between eight technical replicates.*

*CTR control non-treated cells.*

Figure 2 | Comparison of filtrated and non-filtrated Stifenia preparation on IL-1**β** production. Results are obtained from the blood donor CHR026\_128. IL-1β was measured in the culture medium 20 h after the addition of filtered (white bars) and non-filtered (gray bars) Stifenia. Each bar corresponds to four technical replicates. Statistical differences were determined using a Kruskal–Wallis test followed by a comparison with the Conover–Iman method. Similar letters indicate no significant differences (*p* < 0.05). CTR, control non-treated cells. This experiment was repeated once with peripheral blood mononuclear cell from another blood donor (CHR026\_135) with similar results (data not shown).

### Inflammatory Effect of Stifenia Is Inhibited by Caspase Inhibitor

IL-1β is processed to its active released form by caspase-1 through the inflammasome multiprotein complex NLRP3 (7, 27). We used a pan-inhibitor of caspases in order to test whether IL-1β released upon Stifenia stimulation was generated *via* an inflammasome complex. Sixty per cent of the cytokine production induced by Stifenia was abolished by Z-VAD-FMK. As expected, Z-VAD-FMK also strongly reduced LPS-induced IL-1β production (**Figure 3A**; Figure S3 in Supplementary Material). We then checked the involvement of caspase-1 by ELISA on the production of IL-1β induced by Stifenia. In unexposed cells, detection of p-20 caspase-1 fragment in the culture medium was close to the detection threshold. Stifenia 0.3 mg mL −1 induced a huge release of extracellular p-20 Caspase-1 fragment that is much higher than with LPS exposure alone (**Figure 3B**).

### Stifenia Induces Cell Death of PBMC

Using flow cytometry and PI, we checked if a 20-h Stifenia exposure can induce PBMC cell death (Figure S4 in Supplementary Material). In our experiments performed on two independent blood donors, the lowest concentrations of Stifenia tested (0.03 and 0.1 mg mL−1 ) did not induced cell death. However, treatment of PBMC with 0.3 or 1 mg mL−1 induced cell death that ranged from 22.8 to 38.6% depending of the blood donor. LPS (10 ng mL−1 ) alone did not significantly induced cell death as described by others. Furthermore, LPS treatment did not modify or potentiate the toxicity of Stifenia.

### Stifenia Induces Cytokine Gene Expression in Zebrafish Larvae

To analyze the inflammatory effect of Stifenia on a whole organism, we used zebrafish larvae. Using RT-qPCR, we investigated the effect of Stifenia on IL-1β and TNFα gene expression. Twelve to fifteen zebrafish larvae were exposed to different concentrations of Stifenia for 20 h and then harvested for total RNA extraction. TNBS used as a reference inflammatory compound induced a slight IL-1β gene expression compared to unexposed fishes as previously described (24, 28). The lowest concentration of Stifenia induced a higher IL-1β gene expression than TNBS. Furthermore, we observed a dose-dependent effect of Stifenia on IL-1β gene expression. For TNFα gene expression, an increase was observed from 0.01 mg mL−1 of Stifenia exposure (**Figure 4**).

### DISCUSSION

### Inflammatory Effect of Stifenia

In this study, we demonstrated that the commercial plant protection product Stifenia decreased the MA of PBMC and affected cell viability. Stifenia also induced IL-1β production in all the PBMC batches tested, but to a different extent depending on

Figure 3 | Stifenia-induced IL-1**β** production is mediated by caspase1. (A) Peripheral blood mononuclear cells were stimulated by 0.3 mg mL−1 of Stifenia or 10 ng mL−1 of LPS. Cell metabolic activity (MA) (white bars) was estimated by the XTT assay and IL-1β (gray bars) was measured in the culture medium 20 h after treatment. Five micromolars Z-VAD-FMK (Z-VAD) were added simultaneously to Stifenia in the culture medium. LPS was added 30 min after Z-VAD-FMK treatment. The results showed are obtained from the blood donor CHR026\_93. Bars represent the mean between eight technical replicates. Lowercase and capital letters indicate statistical groups for MA and IL-1β concentration, respectively. Different letters indicate statistical differences between groups (*p* < 0.05). If two conditions share one or several letters, there are no statistical differences. CTR, control non-treated cells. (B) Quantity of p-20 Caspase-1 fragment was measured using ELISA in the culture medium 20 h after the addition of 0.3 mg mL−1 of Stifenia or 10 ng mL−1 of LPS. Each bar corresponds to a mean of eight technical replicates. Results are obtained from the blood donor CHR026\_93. This experiment was repeated once on another blood donor with similar results (data not shown). (A,B) Statistical differences were determined using a Kruskal–Wallis test followed by a comparison with the Steel–Dwass–Critchlow–Fligner method. Similar letters indicate no significant differences (*p* < 0.05). CTR, control non-treated cells.

the blood donor. IL-1β is an inflammatory cytokine circulating at very low levels (0.5–10 pg mL−1 ) in blood stream of healthy people (29, 30). In our culture conditions, unstimulated PBMCs produce similar concentration of IL-1β in the culture medium. When PBMCs were exposed to Stifenia 0.1 mg mL−1 , they released around 500 pg mL−1 of IL-1β, which corresponds to a 50- to 1,000-fold increase of IL-1β concentration compared either to control conditions or to the concentrations of IL-1β found in blood of healthy people. It could be hypothesized that such an increase of IL–1β concentration induced by Stifenia could have physiological effects.

of duplicates from one representative experiment out of two.

The inflammatory effect of Stifenia contrasts with other published data indicating that fenugreek seed extracts exhibit anti-inflammatory properties [reviewed by Goyal et al. (13)]. We did not detect any anti-inflammatory properties when Stifenia was used as pretreatment before LPS stimulation. A similar protocol was, however, efficient when used with other natural or synthetic compounds (31, 32). The discrepancy between our data and published ones could be explained. First, we used an aqueous extract of Stifenia while other studies used organic extracts. For instance, Mandegary et al. (25) performed a methanol extraction of fenugreek seeds followed by a fractionation using water and different organic solvents. They showed that the aqueous fraction mostly contains flavonoids and inhibits carrageenan-induced paw edema in mice. Furthermore, ethanol extract of fenugreek seeds reduced Freund's complete adjuvant-induced arthritis in rats by lowering cytokine induction (26). We suspect that the anti-inflammatory effect found in these extracts (25, 26) could be hidden or lowered by other components present in our aqueous Stifenia extract. Second, the biological/experimental models used to demonstrate the anti-inflammatory effects were different from the human PBMC model used in this study. However, the use of an aqueous extract in our study is of relevance because it reflects what is done by farmers when Stifenia is prepared according to the manufacturer's instructions. In other words, the aqueous extract we tested is similar to what is sprayed by farmers on crops.

We showed that caspase-1 was involved in the inflammatory response induced by Stifenia suggesting a role for the inflammasome NLRP3. IL-1β is produced by immune cells as a response to NLRP3 inflammasome activation when cells are confronted to PAMP but also to different danger signals (DAMP) of metabolic origin (27, 33). It could be suspected that some compounds contained in the Stifenia extract could stimulate the NLRP3 inflammasome and lead to caspase-1 activation that is responsible for pro-IL-1β processing.

Because Stifenia induced IL-1β production in a caspase-1-dependant pathway, one may suggest that Stifenia effects are partly due to a contamination with LPS which induce pro-IL-1β gene transcription after its perception by TLR4, a phenomenon known as priming. Thus, the presence of LPS in Stifenia aqueous extract has to be considered. To test this hypothesis, we checked for the presence of 3OH-fatty acids that compose LPS in the Stifenia aqueous extract using an HPLC/MSMS method (34). Interestingly, we detected 3OH-C10:0, 3OH-C12:0, 3OH-C14:0, and 3OH-C16:0 revealing a LPS contamination of Stifenia (data not shown). After filtration of the Stifenia aqueous extract on a poly-lysine column, LPS contamination was decreased by about 40%. However, this extract was still efficient in inducing IL-1β production on PBMC (data not shown). Furthermore, PBMC harvested from two blood donors, CHR026\_135, and CHR026\_152 (Figures S1D,E in Supplementary Material), are not or very slightly susceptible to LPS while they are highly reactive to Stifenia. We also showed that Stifenia exposure induced cell death of PBMC, whereas LPS did not. All these data suggest that Stifenia-induced IL-1β production and Stifenia-induced cell death are unlikely due to a direct effect of LPS, but we cannot rule out that LPS present in Stifenia aqueous extract could prime pro- IL-1β gene transcription.

### Are Inflammatory Effects Relevant for Human Health?

The ANSES has assessed the acceptable exposure level of Stifenia for operators (0.3–2.9 mg kg−1 of body weight/day), workers (3–9 mg kg−1 of body weight/day), and neighborhood (0.01–0.09 mg kg−1 of body weight/5 min of continuous exposition) (ANSES 2012-1685 and 2013-0227). Even if these estimated contaminating doses seems to be low, recurrent or longer exposures have not been tested so far. Our results demonstrated some inflammatory properties and toxicity of Stifenia on blood mononuclear cells. Of importance, the potential toxicity of fenugreek was recently highlighted by some studies both in human and animals. Thus, a survey in Moroccan maternity hospital has linked consumption of fenugreek by pregnant women to congenital malformations (14, 35). In mice, feeding females during the entire period of pregnancy with a lyophilized aqueous extract from fenugreek seeds affects their reproduction and shows teratogenic and foetotoxic effects (4). Khalki et al. (15) also reported growth retardation and altered neurobehavioral performance of mice prenatally exposed to fenugreek seed extracts. Antifertility effects of fenugreek seed extract has also been reported in rabbits (17), and other toxic effects have been described in mice, rats and rabbits (16, 18). However, since fenugreek seeds have been used for centuries in traditional medicine or in food, many tests were not included in the risk assessment of Stifenia (36). Our results pointed out the possible danger of an extensive use of the plant defense stimulator Stifenia at the level of human health.

### Environmental Toxicity

Our data demonstrated *in vivo* inflammatory effect of Stifenia on zebrafish larvae. We observed an induction of IL-1β and TNF-α gene expression that started at Stifenia concentration as low as 3 and 10 µg mL−1 , respectively. The predicted environmental concentration (PEC) of Stifenia in surface water was established by the ANSES at 0.160 µg mL–1 (ANSES 2012-1685). This is only 20-fold lower than the 3 µg mL−1 concentration that induced significant IL-1β gene expression in our experiments. This raises the issue of whether a longer exposure of zebrafish larvae to the PEC concentration could have similar inflammatory effects to those we observed. However, the methods used to predict PEC in surface water are controversial and could not reflect the actual concentrations of the molecule in the environment (37). Recently, it has been shown that the PEC calculated for several insecticides are underestimated by the procedure used by the European Union (38). Regarding the toxicity of fenugreek on animals, a study showed that topical application of 6 µg of fenugreek acetonic seed extract on two coleopteran species, *Acanthoscelides obtectus* and *Tribolium castaneum*, decreased their fertility and induced their mortality. These authors also showed that the presence of fenugreek seeds in the immediate environment of insects was sufficient to kill them (19). All these results suggest that the use of Stifenia could have adverse effects on non-target organisms.

## CONCLUSION

Our results demonstrated unexpected effects of a plant protection product on human and animal health. As written by Burketova et al. (2), "although bio-based products are of natural origin, direct toxicity of these products to human, animals, insect, microbe communities, or even plants must be studied carefully to avoid toxicity as observed with classical pesticides." The human cell-based approach developed in this work revealed a high sensitivity concerning inflammatory properties of a plant protection product. These tests could be routinely used to screen the potential adverse effects of this type of compounds that can potentially cross-react with human innate immunity. This should be the first step before engaging more expensive studies on animal models and according to the European legislation, and this approach fits with the goal of reducing studies on animal models (No. 1107/2009).

## ETHICS STATEMENT

For Zebrafish larvae, there is no need of ethics committee because they were euthanized at 120-h postfertilization [EU Directive 2010/63/EU, Strähle et al. (21) and Geisler et al. (22)]. For human PBMC, there is no need of ethics committee. PBMCs were obtained from Etablissement Français du Sang (EFS) according to agreement No. DECO-14-0124 and EFS do not indicate any information on PBMC donors.

## AUTHOR CONTRIBUTIONS

LT, OL, and JLC designed this work. LT, JCo, OL, and JLC performed most of the experiments. JCh supervised zebrafish experiments. LT, JCo, SD, JCh, DW, OL, and JLC contributed substantially to the completion of this work. LT, OL, and JLC wrote the manuscript. All the authors read and approved the final manuscript.

### ACKNOWLEDGMENTS

The authors thank Anaïs Gérard, Siham Hichami for technical help, Tatiana Lopez for zebrafish breeding, Dr. Jean-Paul Pais-De-Barros (Lipidomic platform, LipSTIC LABEX, Dijon, France) for LPS analysis, Arlette Hammann, Dr. Anabelle Seiqueira-Legrand (cytometry platform, University of Burgundy, France), and Dr. Gaëtan Jego (UMR INSERM u866, Dijon, France) for critical advices on cytometry, and colleagues for critical reading of the manuscript and help.

### FUNDING

This work was supported by a grant from FEDER (Phytosafe, présage 39703). LT was supported by a grant from the Conseil Régional de Bourgogne (JCE funding).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fpubh.2017.00074/ full#supplementary-material.

#### Table S1 | Cytokine concentrations in culture medium of Stifenia-

exposed peripheral blood mononuclear cells. Cytokines were measured in the culture medium 20 h after the addition of Stifenia at the indicated concentrations using a multiplex kit (ProcartaPlex human simplex, Affymetrix, France) according to the manufacturer's instructions on blood donor CHR026\_91. C, concentration; IF, induction factor; CTR, control non-treated cells.

Figure S1 | Effect of Stifenia IL-1**β** production and metabolic activity (MA). Peripheral blood mononuclear cells were exposed to different concentrations of Stifenia added in the culture medium for 20 h. Cell MA (white bars) was estimated by the XTT assay, and IL-1β (gray bars) was measured in the culture medium. Results are obtained from six blood donors (A–F). Bars represent the mean of eight technical replicates. Different letters (lowercase for the XTT assay, capitals for IL-1β) indicate statistical differences between groups (*p* < 0.05). Statistical differences

### REFERENCES

were determined using a Kruskal–Wallis test followed by a comparison with the Steel–Dwass–Critchlow–Fligner method. CTR, control non-treated cells.

#### Figure S2 | Effect of Stifenia on LPS-induced IL-1**β** production and

metabolic activity (MA). Peripheral blood mononuclear cells were stimulated by addition of 10 ng mL−1 of LPS 30 min after the beginning of Stifenia exposure. Cell MA (white bars) was estimated by the XTT assay, and IL-1β (gray bars) was measured in the culture medium. Results are obtained from five blood donors (A–E). Bars represent the mean of eight technical replicates. Different letters (lowercase for the XTT assay, capitals for IL-1β) indicate statistical differences between groups (*p* < 0.05). Statistical differences were determined using a Kruskal–Wallis test followed by a comparison with the Steel–Dwass–Critchlow– Fligner method. CTR, control non-treated cells.

#### Figure S3 | Effect of ZVAD on Stifenia- or LPS-induced IL-1**β** production

and metabolic activity (MA). Cell MA (white bars) was estimated by the XTT assay, and IL-1β (gray bars) was measured in the culture medium 20 h after treatment. Five micromolars Z-VAD-FMK (Z-VAD) were added simultaneously to Stifenia (0.3 mg mL−1 ) in the culture medium or 30 min before the addition of LPS (10 ng mL−1 ). Results are obtained from blood donors CHR026\_95 (A) and CHR026\_153 (B). Bars represent the mean between eight technical replicates. Different letters (lowercase for the XTT assay, capitals for IL-1β) indicate statistical differences between groups (*p* < 0.05). Statistical differences were determined using a Kruskal–Wallis test followed by a comparison with the Steel–Dwass– Critchlow–Fligner method. CTR, control non-treated cells.

#### Figure S4 | Peripheral blood mononuclear cell (PBMC) viability after

Stifenia treatment. Cell viability was estimated by propidium iodide (PI) staining 20 h after PBMC treatments. The results shown are obtained from blood donors CHR026\_153 (A,B) and CHR026\_152 (C,D). (A,C) Cell death was expressed as a percentage of PI-stained cells vs. total cells. Bars represent the mean between four technical replicates. Different letters indicate statistical differences between groups (*p* < 0.05). Statistical differences were determined using a Kruskal–Wallis test followed by a comparison with the Dunn's method. (B,D) Flow cytometry diagram of PI-stained cells 20 h after treatment. For each treatment, a representative diagram out of four technical replicates is shown for the blood donor CHR026\_153 (B) or the blood donor CHR026\_152 (D).

and *Acanthoscelides obtectus* (Say) (Coleoptera: Bruchidae). *J Stored Prod Res* (1997) 33:209–17. doi:10.1016/S0022-474X(97)00007-6

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Teyssier, Colussi, Delemasure, Chluba, Wendehenne, Lamotte and Connat. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Complex Outcomes from Insect and Weed Control with Transgenic Plants: Ecological Surprises?

Thomas Bøhn1 \* and Gabor L. Lövei 2, 3

1 GenØk-Centre for Biosafety, Tromsø, Norway, 2 Department of Agroecology, Flakkebjerg Research Centre, Aarhus University, Slagelse, Denmark, 3 Institute of Applied Ecology, Fujina Agricultural and Forestry University, Fuzhou, China

Agriculture is fundamental for human survival through food production and is performed in ecosystems that, while simplified, still operate along ecological principles and retain complexity. Agricultural plants are thus part of ecological systems, and interact in complex ways with the surrounding terrestrial, soil, and aquatic habitats. We discuss three case studies that demonstrate how agricultural solutions to pest and weed control, if they overlook important ecological and evolutionary factors, cause "surprises": (i) the fast emergence of resistance against the crop-inserted Bt-toxin in South Africa, (ii) the ecological changes generated by Bt-cotton landscapes in China, and (iii) the decline of the monarch butterfly, Danaus plexippus, in North America. The recognition that we work with complex systems is in itself important, as it should limit the belief in reductionist solutions. Agricultural practices lacking eco-evolutionary understanding result in "surprises" like resistance evolution both in weeds and pest insects, risking the reappearance of the "pesticide treadmill"—with increased use of toxic pesticides as the follow-up. We recommend prioritization of research that counteracts the tendencies of reductionist approaches. These may be beneficial on a short term, but with trade-off costs on a medium- to long-term. Such costs include loss of biodiversity, ecosystem services, long-term soil productivity, pollution, and reduced food quality.

#### Edited by:

Robin Mesnage, King's College London, United Kingdom

#### Reviewed by:

Nicolas Defarge, ETH Zurich, Switzerland Md. Asaduzzaman, University of Dhaka, Bangladesh

> \*Correspondence: Thomas Bøhn<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Environmental Health, a section of the journal Frontiers in Environmental Science

Received: 04 May 2017 Accepted: 13 September 2017 Published: 26 September 2017

#### Citation:

Bøhn T and Lövei GL (2017) Complex Outcomes from Insect and Weed Control with Transgenic Plants: Ecological Surprises? Front. Environ. Sci. 5:60. doi: 10.3389/fenvs.2017.00060 Keywords: Bt-toxins, ecology, herbicides/pesticides, glyphosate, GM crops, non-target organisms, resistance evolution

### THE USE OF PESTICIDES IN AGRICULTURAL PRACTICE WITH TRANSGENIC PLANTS

Genetic modifications of crop plants have great potential and promises, but current growing practices are overwhelmingly restricted to four crop species and two kinds of GM modifications.  The majority of these crops (except cotton) are grown on largescale industrial–style farms, mostly in North and South America (James, 2016). The two dominant modifications, herbicide tolerance (HT) and insect resistance (IR), can make crops tolerant to selected herbicides, or toxic to specific groups of herbivorous insects, respectively. Most of the GM soybean and oilseed rape are HT, while transgenic maize and cotton are mostly IR. An increasing number of GM cultivars are "stacked" and/or "pyramided," containing both kinds of modifications, and several constructs.

HT GM plants open the formerly narrow time window when herbicides could be sprayed on crop fields without risk to the crop itself. Farmers planting HT crops can use herbicides, even at higher concentrations than previously, without damaging their crop. As a result, the global share of the few herbicides that can be used on such GM crops has increased dramatically. The commercially most successful application is linked to herbicides with glyphosate as the active ingredient (Roundup products), whose use only in the USA increased from 3.6 million kg in 1987 to 108 million kg in 2014 (Myers et al., 2016).

To make cultivated plants toxic to herbivores, the most commonly used method is the insertion of activated toxin genes from the soil-living bacterium Bacillus thuringiensis (Barton et al., 1987). Numerous strains of this bacterium have been isolated, characterized, manipulated and inserted into a variety of crop plants (Bravo et al., 2011), but the majority of fieldgrown insect resistant GM plants are maize and cotton. In the case of maize, the aim was to defend the plant from the attack of two pests that are important in the USA: the nonnative European corn borer Ostrinia nubilalis (Lepidoptera: Pyralidae), and a native beetle, the corn rootworm (Diabrotica virgifera, Coleoptera: Chrysomelidae). In the case of cotton, the primary target pest was the cotton bollworm (Helicoverpa armigera, Lepidoptera: Noctuidae), an important pest in Eurasia, Africa, and the Americas. The presence of the bacterial toxin in the plant defends it from damage by these main pests. After the introduction of Bt-plants a reduction in the amount of insecticides sprayed was observed, especially on cotton, the most intensively insecticide-treated crop plant in the world (Deguine et al., 2008).

GM plants represent an important trajectory of modern agriculture, with a strong focus on weed and insect control. Moreover, in GM plants, whether HT or IR, permit new ways to use pesticides in agriculture. This has interesting practical as well as ecological and evolutionary consequences in comparison to other agricultural practices.

There is an ongoing and rapid change in GM crop plants. In 1996, the first year of commercial planting, GM crop plants predominantly expressed a single transgene of bacterial origins, either a cry1Ab Bt gene in IR crops, or a cp4 epsps gene (glyphosate tolerance) in HT plants. Twenty years later, an increasing number of GM crop plants can express up to 6 different Bt-toxins and up to 3 different herbicide tolerance traits (Hilbeck and Otto, 2015; Venter and Bøhn, 2016). We may even expect up to 14 different transgenes in a single GM plant by 2020 (Hakim, 2016). What is the driving force for "stacking" all these traits on top of each other?

In order to forecast the environmental consequences of field-growing GM plants, it is important to stress that genes alone do not determine the outcomes: the gene-organismenvironment ("The Triple Helix") interactions are crucial for the understanding of any biological system (Lewontin, 2000). If we ignore the dynamic responses of nature, "surprises" and failures will be the order of the day. In contrast, by using ecological and evolutionary theory, we would have been able to foresee and possibly avoid many unwanted outcomes that we are experiencing today.

Pesticides are an integral, nearly unavoidable, part of the dominant current agricultural practices. Their dominance can be traced back to the period during and after the Second World War. Problems emerged gradually, and started to be voiced in the early 1960s with the "Silent Spring" of Rachel Carson (Carson, 2002). Since then, pesticides have been under tighter and tighter regulation, and were increasingly recognized as the mixed blessing they are. Notwithstanding the technological advances with pesticides and their applications, the serious global health effects caused by hazardous pesticides has recently made the UN formulate a new set of recommendations. These includes that (i) pesticide use must be closely monitored, regulated and reduced worldwide, and that (ii) non-chemical alternatives must be considered first, e.g., use (agro)ecological methods to naturally supress pests (United Nations, 2017).

In this article, we aim to illustrate the dynamics of ecological/evolutionary responses to field growing of transgenic plants, an important component of modern agriculture. We present three case studies and use these to discuss dynamic ecological and evolutionary responses related to insect and weed control with GM crop plants. Our center of attention concerns the sustainability of this agricultural practice, and whether the ignoring of ecological complexities may lead to new, mistaken technological solutions, resistance evolution and further pesticide use.

#### CASE STUDY 1. RESISTANCE EVOLUTION IN BUSSEOLA FUSCA TO CRY1AB TOXIN, SOUTH AFRICA

Several important insect pests of maize are internal feeders, and thus not easy to control by traditional pesticides. This was a strong motive to develop transgenic GM maize lines that can express an insect toxin in planta, thus presenting the potential to control such internal feeders. In South Africa, the main target insect pest, Busseola fusca (Lepidoptera: Noctuidae), is such a pest, whose larvae are boring inside the maize plant. They were successfully controlled by Bt-maize expressing the Cry1Ab-toxin (MON810) after its introduction in 1998, for a period of about 6 years. In the 2004/5 season, the first reports on resistant insects were coming in: B. fusca larvae could be found feeding on Bt maize plants (van Rensburg, 2007; **Figure 1A**). By 2010, the area where such resistant insects were found increased to cover most of the maize growing areas in the country (Kruger et al., 2012; Van den Berg et al., 2013). Where resistant insects appeared, farmers responded by re-starting the previous practice of spraying insecticides—now on the transgenic MON810 variety (**Figure 1B**).

This resistance evolution of B. fusca in South Africa triggered the replacement of the original, single toxin-expressing MON810 with MON89034, a plant that expresses two toxins: Cry1A.105 and Cry2Ab2 (Van den Berg et al., 2013). Thus, the emerging resistance in pest insects led to the stacking of two insect toxins in the same plant. This may resemble the start of the "pesticide treadmill," where the typical response to emerging resistance to an insecticide was to start using cocktails of various ones, with

Photos taken in the Vaalharts region in 2012 by Thomas Bøhn.

the argument that these combinations will remain effective for longer (Nicholls and Altieri, 1997). For sprayed pesticides, this has not been a sustainable solution, because multiple resistances emerged, making the problem more severe.

### CASE STUDY 2. HELICOVERPA ARMIGERA REPLACED BY MIRID BUGS AS MAJOR PESTS, CHINA

China is among the biggest cotton producers of the world. On five of the six major cotton-growing regions, mostly smallholders grow cotton, using it as a cash crop. Before 1997, the use of pesticides to control insect pests, the cotton bollworm H. armigera in particular, was very high and included 14–28 treatments per season (Pemsl and Waibel, 2007). Resistance was widespread, and the increased frequency of sprayings did not provide a lasting solution, even if the serious human safety issues are disregarded. The introduction of Bt-cotton after a government decision in 1996 resulted in quick take-up of the technology, and improved the conditions for the farmers considerably. Not only was there a decline in pest attacks on cotton, especially in the early season; other important crops harmed by the polyphagous H. armigera also started to show reduced pest densities (Wu et al., 2008). The reason was that the typically small cotton fields were dispersed in the landscape and cotton, the previously preferred crop for H. armigera, was replaced by Bt-cotton and suddenly became unusable as a habitat for that insect species. The efficient killing of the target pest had turned cotton plots into a population sink at the regional/landscape level (Lu et al., 2010). This shows the potential dynamics in source and sink populations, i.e., conditional sources and sinks (Loreau et al., 2013), altered by context dependent conditions, here the introduction of Bt-toxin in cotton, and the particular distribution of cotton plots in the overall cultivated landscape.

Had the story ended here, it would have been a great success story of the Bt-cotton (Wu et al., 2008). The fact that Bt-cotton led to a regional decline in a major pest species and therefore also diminished the damage caused by H. armigera on multiple crop plants also coincided with a reduced load of toxic pesticides (Pemsl and Waibel, 2007).

However, due to ecological responses and interactions, the full story is more interesting. As Bt-cotton became less suitable to H. armigera, large resources, both in terms of pesticide-free habitat and cotton plant biomass became available to species less susceptible to Bt-toxins. Additionally, the reduced pesticide pressure also, at least initially (Carrière et al., 2016), eased the pesticide pressure on other insect groups. A formerly secondary pest group, mirid bugs (Miridae, Heteroptera) now increased in numbers, and became an important pest on cotton (Lu et al., 2010). With the high densities of mirids on Bt-cotton, these plots now became population sources of mirids that subsequently migrated to other crops. The outcome was that the higher proportion of cotton was Bt-transgenic in the landscape, the higher became the densities of mirids on other crops in the region (Lu and Wu, 2011). Thus, the management regime of Btcotton, including the change in pesticide use triggered by the use of the GM cultivars, made the same cotton patches sources of another herbivorous pest, and caused the subsequent spread of a non-target, secondary pest at the landscape level (Lu et al., 2010).

Case study 2 illustrates how the single-species focus can backfire when a "technological solution" is employed against a single species, and ecological complexity gets ignored. Crops usually have numerous target pests, or potential target pests, some of which may be minor due to various reasons; this does not mean that they do not have the potential to cause large damage their damage potential is only suppressed by the dominating pest species. Change the density of this major pest, and these other potential pests may quickly respond due to competitive release (Zeilinger et al., 2016).

### CASE STUDY 3. THE MONARCH BUTTERFLY AND LANDSCAPE LEVEL EFFECTS OF HERBICIDE USE, NORTH AMERICA

One of the most fascinating insect migrations is performed by the North American monarch butterfly, Danaus plexippus (Lepidoptera: Danaidae), that migrates from its summer distribution area, covering a large part of North America, to winter in a small part of central Mexico, in spectacular masses (Stenoien et al., 2016). For this reason, as well as for its spectacular coloration, the monarch has become a symbol of beauty and freedom—one of the conservation icons of North America. The monarch larvae feed on milkweed, Asclepias syriaca, which is a common weed, especially in maize and soybeans fields (Oberhauser et al., 2001). In 1999, an article in Nature reported that the larvae of the monarch butterfly suffered high mortality in laboratory trials where they fed on milkweed leaves dusted with a high density of transgenic Btmaize pollen (Losey et al., 1999). This study generated heated discussions, but also triggered a large research project examining the possible consequences of planting transgenic maize over vast areas in the summer distribution range of the monarch butterfly. The summary conclusion was that the risk of being exposed to significant amounts of Bt-toxin from maize plants through pollen in the field was negligible (Sears et al., 2001). However, Oberhauser and co-workers cautioned that the issue needed to be looked at from a larger perspective, including weed control and the use of pesticides by farmers (Oberhauser et al., 2001).

By the mid-2010s it became obvious that the cautions were justified: the monarch population densities were dramatically reduced with a loss of an estimated 40 million individual butterflies per year (about 9% decline per year) after 1993/1994 (Williams and Brower, 2016; Oberhauser et al., 2017). The lowest recordings of the monarch densities on the wintering ground are from 2013 to 2015 (Rendón-Salinas and Tavera-Alonso, 2015), and the probability that the fascinating migration of the eastern monarch will go extinct within 20 years is estimated to be 11– 57% (Semmens et al., 2016). Alarmed by these perspectives, initiatives across borders (US, Mexico, Canada) have been started to conserve the monarch butterfly. However, management action needs an understanding of the causes of the decline in order to improve the situation. So what are the key causal factors to explain the monarch decline?

The exponential growth of hectares with GM plants after 1996 in the US increased the acreage under Bt-transgenic plants, but even more those of glyphosate tolerant GM plants. From 2004 and until 2015, plants with "stacked" traits, i.e., both with Bttoxins and herbicide tolerance increased from <10 million ha to about 60 million ha (James, 2015). As a result, non-target organisms including monarchs would interact, not only with Bttoxins and herbicides directly, but also with the indirect effects of these factors at a landscape level.

A subtle but highly important indirect effect on the landscape level is related to herbicide tolerant GM crops and the massive use of broad-spectrum herbicides. The increased use of glyphosate products have caused a dramatic decline in the dominant host plant for the monarch. This is, seen from the monarch point of view, a serious habitat destruction of its highly specialized habitat. What is efficient weed management for the farmers can be fatal for a butterfly depending on a dominant host plant that grows between the rows of HT GM plants.

Monarch butterflies have experienced a dramatic decline in the availability of their host plant, the milkweed. In Iowa, milkweed was present in 51% of the fields in 1999, but only in 8% a decade later. In addition, even in the fields where still observed, milkweed density was reduced to about 10% of its original value (Hartzler, 2010). In sum, the decline in milkweed amounted to a near-complete elimination in the core of the breeding range of the monarchs (Pleasants, 2015). The maize and soybean fields were turned into milkweed deserts.

At the landscape level over the whole mid-western USA, the decline in milkweed has been almost 40%. However, since the monarch butterflies on average lay 3.9 times more eggs on milkweed stems in agricultural fields, where the reduction of milkweed is most severe, the capacity to support the monarch as a species is reduced by 71% (Pleasants, 2017).

The threat to the monarch triggered various conservation responses, including a restoration goal of reaching "six ha of overwintering monarchs" (i.e., six ha of trees covered by monarchs). To succeed, 1.6 billion additional milkweed plants would be needed, a number higher than the estimated current total population (1.34 billion plants) for the whole Midwest (Pleasants, 2017). Restoration of the milkweed seems to be crucial and the use of HT GM plants is identified as a key for the milkweed decline (Pleasants and Oberhauser, 2013; Zalucki et al., 2016).

## DISCUSSION

The three case studies described above exemplify that agricultural ecosystems, even if arguably simplified, retain complexity, and that solutions to agricultural problems should be scrutinized from an ecological point of view. Ignoring ecological interactions tends to undermine the overly simple solutions, here exemplified with insect and weed control by the dominating GM plant traits and associated technology. When a pest insect or weed species overcomes a suggested "solution" to hold their density low, strong selective advantages will play out. Under such conditions, natural selection may be effective in a timespan of a few years, and threaten to undermine the efficiency of our weed and insect control, and thus also the goal of improved agricultural productivity.

When we look at the dominant technologies currently accompanying GM plants, there are particular challenges related to resistance evolution, both for herbicide tolerance traits and for insect resistance.

### Herbicide Tolerant Crops and Weed Resistance Evolution

From 1995 to 2014, the global agricultural use of glyphosate rose 14.6-fold, from 51 million kg to 747 million kg and HT GM crops have been a major driver for this change. Moreover, by 2016, about 56% of the global use of glyphosate was connected to HT GM crops (Benbrook, 2016). Specific for the HT GM plants is that herbicides can be sprayed in higher doses and repeatedly during the growth season of the plants. The vast "experiment" that was initiated with HT GM crops and glyphosate as a stand-alone herbicide on millions of hectares of cropland, imparted tremendous selection pressure on the weed populations. This has been a key factor for the resistance evolution, now documented for 37 species of weeds globally. Such development may lead to the familiar "treadmill" where resistance triggers more applications/higher doses, which leads to stronger selection pressure for resistance, etc. and eventually the use of additional herbicides like atrazine and 2,4D (Binimelis et al., 2009).

Unfortunately, the glyphosate resistant crops were not integrated into a total weed management program, rather it replaced all of the other programs (Shaner et al., 2012). In hindsight, this was not a wise move and showed us that no herbicide is invulnerable to resistance. At least three different mechanisms of resistance is identified: (i) alteration of the target site; (ii) changes in sequestration and/or translocation of the herbicide, and (iii) changes in the rates of metabolism of the herbicide (reviewed in Shaner et al., 2012).

For the farmers, resistant weeds are a difficult practical obstacle to handle. Although farmers often have a longterm perspective on their farming activity, they may also be attracted to quick-fix solutions, including pesticides and growing monocultures. Unfortunately, crop and herbicide monocultures create conditions for resistance development (Beckie, 2011).

The magnitude of resistance problems should be incentive enough to further explore the plurality of methods that can be used under integrated pest management, not only to delay resistance but to promote alternative and preferably nontoxic pest control systems (United Nations, 2017). Chemical treatments, coupled with the unavoidable resistance development are major blocking factors to a sustainable agriculture.

The use of herbicides like glyphosate also has the potential to affect ecosystem, animal and human health. The massive use of glyphosate, totaling 852 million kg globally by 2014 (Benbrook, 2016), which directly or indirectly will expose non-target biodiversity in terrestrial, soil and aquatic communities (Venter and Bøhn, 2016), represent a major source of environmental pollution. In addition, glyphosate is shown to accumulate in (i) soils that have a history of glyphosate use (Duke et al., 2017), and (ii) in HT soybeans (Bøhn et al., 2014), more when the plant is sprayed later in the season (Duke et al., 2003). This will bring glyphosate residues into the global food and feed chains (Bøhn et al., 2014).

The increased awareness of glyphosate toxicity, coupled with the increased volume used, should lead to stronger restrictions, for example lower acceptance level for glyphosate residues in food and feed (Cuhra et al., 2016). In this context it is perplexing why the maximum residue level (MRL) for glyphosate was raised 200-fold from 0.1 to 20 mg/kg in Europe, and to 40 mg/kg in the US (Cuhra, 2015). This set of events has been termed "The Glyphosate Paradox" (Cuhra et al., 2016). The WHO/IARC categorization of glyphosate as probably carcinogenic to humans (Guyton et al., 2015), although disputed by EFSA (EFSA, 2015), is underlining the significance of the controversy around the glyphosate-based herbicides.

Glyphosate is now also implicated in the decline of the monarch butterfly (Stenoien et al., 2016), further illustrating the various kinds of environmental damage that reliance on a few plant protection chemicals may bring. However, the monarch may not be the only species at risk for similar reasons—a total of 39 protected European lepidopteran species have maize weeds in their host plant range (Lövei et al., 2016).

### Insect Resistant Crops and Resistance Evolution

GMO related "internal" pesticides such as Bt-toxins have a particular problem related to resistance evolution. The GM plants express Bt-toxins continuously, also when the "pests" are not a problem due to their low density. Pesticides expressed continuously, as in current insect resistant crops, simply raises the bar and offers continuous "trial and error testing" within potential pest populations, with a huge fitness reward on individuals that acquire resistance.

That evolution will eventually result in resistance developing in the target pest populations was foreseen before Bt-transgenic plants were grown commercially, and different strategies have been suggested and adopted to delay undesirable pest adaptation. For transgenic Bt- plants, the high-dose/refugia strategy is the most frequently recommended (Carrière et al., 2016). The role of the non-GM refugia is to secure the reproduction of susceptible insects and assure that the genes that make the target sensitive to the Bt-toxin do not disappear from the population. Thus, the high-dose Bt should remain effective, killing insects that have resistance alleles from one of the parents, and keeping the target population heterozygous.

The South African case with B. fusca showed that farmers initially did not follow the recommendation; only 8% of them established a refuge. By 2008, however, most or all farmers had established refugia (Kruger et al., 2009). This may have been too late, it seems likely that the initial non-compliance played a role in the fast appearance of the resistance (Van den Berg, 2016). The other key factor in promoting field-evolved resistance to Bt-toxin is that the high-dose standard is not met. The Cry1Ab maize used in South Africa in the relevant period did not fulfill this criteria for B. fusca (Van den Berg, 2016). Finally, the hypothesis of functionally recessive inheritance of resistance in the insect, meaning that when resistant and susceptible parents mate, the offspring will be susceptible, was rejected by experimental data in the South African B. fusca (Campagne et al., 2013).

The B. fusca case illustrates that the positive effect of reduced amounts of insecticides sprayed (e.g., Marvier et al., 2007; Osteen and Fernandez-Cornejo, 2013) may not last, or lead to the use of stacked events with multiple Cry toxins inserted. A recent review (Carrière et al., 2016) concluded that under the current way of growing, the pyramiding of Bt-toxins is not a stand-alone solution to the resistance development problem.

Another reason for pyramiding different Cry toxins in the same plant is to protect the plant from pest insects from different taxonomic groups, e.g., from both Lepidoptera (Cry1 and Cry2 toxins) and Coleoptera (Cry3 toxins). In maize, up to six different Cry toxins are combined in the same plant, as in hybrid MON 89034 × 1507 × MON 88017 × 59122, from Monsanto and Dow AgroSciences, which expresses cry1A.105, cry1F, cry2Ab2, cry3Bb1-, cry34Ab1, and cry35Ab1. Clearly, the added range of targeted pests is likely to produce stronger effects on non-target communities as well (Then, 2009).

When two toxins are active against the same insect species, resistance may be delayed. In North Carolina, transgenic cotton with two Cry-toxins resulted in much higher mortality (96 vs. 44%) in the pest Helicoverpa zea compared to cotton with a single Cry-toxin (Carrière et al., 2016). At the same time, using several toxins at the same time is analogous to the use of multiple antibiotics in the same treatment. The risk is that fewer agents are left unused when resistance appears. In a long-term perspective, resistance will inevitably develop, even if a range of pest management practices will delay the process. Reliance on pesticides, does not represent a sustainable agricultural practice. Therefore, the UN is recommending (i) proactive measures to reduce or eliminate harmful pesticides, and (ii) to consider nonchemical alternatives first (United Nations, 2017).

The risk that control measures against target pest insects are lost due to resistance evolution can be tracked back more than 100 years, and is particularly relevant if there is a continuous selection pressure for resistance (Andow, 2008). Nevertheless, most pest populations have remained susceptible to Bt toxins, but 5 out of 13 major pest species have already acquired field-based resistance (reviewed in Tabashnik et al., 2013).

A pesticide could best solve a pest problem if it was perfectly specific for the target species, and killed only that harmful organism. In that case, effects on other species would be limited to the altered ecological interactions in the ecosystem, most plausibly on species directly linked to the target species in the food web. However, known chemical and biological pesticides are either very broad, harming all insects with an exoskeleton (as for DDT), or are more specific because their action requires certain conditions (like high pH, cleavage by enzymes, etc. as with Bt-toxins). This may limit the range of species/taxa harmed, although there still may be several species in the harmed group. For example, the order Lepidoptera, the main target group of Cry1 and Cry2 toxins, contains 126 families and some 180,000 known species (Capinera, 2008). Moreover, a single plant species may be a host to numerous species. Maize and cotton are registered as hosts for 776 and 872 lepidopteran species, respectively (Robinson et al., 2010).

When the criteria of specificity to the pest species is not fulfilled, harmful effects on non-target species (biodiversity) can be expected. A range of factors contributes to potential negative effects on non-target species.

Firstly, the toxicity of the pesticides will be crucial, which is typically taxa/species/age- and context- dependent.

Secondly, the break-down rate will modify and reduce the toxicity over time. Pesticides that are decaying slowly may accumulate in the food web and have serious long-term effects, such as the PCBs (Gobas et al., 2016). Pesticides that do not break down may be a part of the food or feed produced and have further effects on humans or animals along the food/feed chain. The break-down rate of chemicals depends on environmental factors like pH, soil type, binding to other particles etc., which adds to the complexity.

Thirdly, the timing and dosage of applications are important for potential unwanted effects. Treatments with toxic pesticides are ideally precisely timed to hit when the problem is severe. The option to time the application of a pesticide can therefore be a good thing. With pesticides that are expressed continuously from the plant genome, as in Bt-plants, such flexibility is lost.

In the context of negative effects on biodiversity, the sensitivity of non-target organisms, most of the species in soil and aquatic communities have never been tested for their vulnerability to Bttoxins. Several aspects of the fate of Bt-toxins are not well-known, including amounts, break-down rates and effects. Further, studies of tri-trophic relationships and food web interactions may provide insight to community responses (Yu et al., 2014).

## Can Stacked Traits Act as Multiple Stressors?

The understanding of resistance evolution and stacking/pyramiding of traits must be linked to potential combinatorial effects on non-target organisms. The use of stacked events represents: (i) increased doses/more applications of herbicides per season, and (ii) a broader range of Cry toxins in insect resistant GM plants. Both these effects trigger positive feedback loops with stronger selection pressures and further resistance evolution. Since these toxins/chemicals/traits will meet and interact, also with other stressors in the environment, the co-exposure and potential combinatorial effects need to be studied (Nørgaard and Cedergreen, 2010; Bjergager et al., 2011). Combinatorial effects between Bt-toxins and herbicides may enhance toxicity (Then and Bauer-Panskus, 2017). For example, Bøhn and co-workers showed that Cry1Ab and Cry2Aa toxins act in combination (additively), indicating that "stacked events" may increase negative effects on non-target organisms (Bøhn et al., 2016). However, combinatorial effects represents a major knowledge gap in the scientific literature (Venter and Bøhn, 2016).

### Sustainability of Agriculture

Agriculture has been fundamental for the rise of human civilization (Diamond, 1999) and continues to be vital for human survival through food production. However, many modern agricultural innovations relied on non-renewable resources that are not sustainable (Gliessman, 2015). We need a strong prioritization of resources for research to build knowledge to ensure that future food production is sustainable. In particular, there is a need to counteract agricultural practices that are beneficial on a short term, but with trade-off costs on a medium to long term scale. Such costs includes loss of biodiversity, ecosystem services, soil productivity, pollution and reduced food quality.

## CONCLUSIONS

The currently dominant agricultural practice has changed the natural spatial distributions of plant species that provide food, fiber, and other important resources for us (classified as provisioning ecosystem services), and resulted in habitats that are less diverse than the original habitats that were converted to croplands. Nonetheless, these are biological entities, supporting and interacting with various plant, animal, fungal, and microbial communities in complex ways. The recognition that when trying to manage agricultural fields and landscapes, we work with a complex biological system is in itself important, as adopting this view should limit the belief in reductionist solutions.

Moreover, from an evolutionary perspective on sustainable food production, chemical pesticides, both insecticides such as Bt-toxins and sprayed herbicides, carry problems that are hard to solve. Dominant technologies in transgenic plants rely on new ways of using pesticides. These practices does not utilize and often contradicts ecological understanding and are therefore likely to exacerbate current problems. It is not likely that pesticides can be eliminated from our dominant agricultural practices in the near future. Key factors to uphold or improve their efficiency would be to increase the precision (specificity, timing) while minimizing the amount used. This would reduce pollution, lower their accumulation in the environment as well as in food, both of which are positive outcomes for ecosystem and human health. We also have to place the analysis of possible environmental consequences into an agroecological context, because that approach inherently considers the possibility of multiple stressor interaction, sublethal effects, non-linear, and synergistic outcomes that are so characteristic of biological systems. The

#### REFERENCES

Carson, R. (2002). Silent Spring. Boston, MA: Houghton Mifflin.

examples discussed in this article also underline the importance to incorporate landscape-level ecological knowledge into the evaluation practice, because spatially explicit analysis of potential impacts are important tools in making agricultural practice more sustainable.

In the three case studies discussed, the GM plants associated with simple pesticide-solutions were unable to solve complex agricultural problems. We argue that the resulting resistance development and increased use of herbicides arose because basic ecological and evolutionary theory was overlooked. Had such knowledge been included, we would have foreseen and possibly been able to avoid some of the unwanted outcomes we are experiencing today.

#### AUTHOR CONTRIBUTIONS

TB and GL conceived the study and wrote the paper.

#### FUNDING

The study was funded by GenØk—Centre for Biosafety, Fujian Agriculture and Forestry University, and Aarhus University.

United Nations (2017). Report of the Special Rapporteur on the Right to Food.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Bøhn and Lövei. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Specificity and Combinatorial Effects of Bacillus Thuringiensis Cry Toxins in the Context of GMO Environmental Risk Assessment

Angelika Hilbeck 1 \* and Mathias Otto2

*1 Department of Environmental Systems Science, Swiss Federal Institute of Technology, Institute for Integrative Biology, Zurich, Switzerland, 2 Federal Agency for Nature Conservation, Bonn, Germany*

Stacked GM crops expressing up to six Cry toxins from *Bacillus thuringiensis* (Bt) are today replacing the formerly grown single-transgene GM crop varieties. Stacking of multiple Cry toxins not only increase the environmental load of toxins but also raise the question on how possible interactions of the toxins can be assessed for risk assessment, which is mandatory for GM crops. However, no operational guidelines for a testing strategy or testing procedures exist. From the developers point of view, little data testing for combinatorial effects of Cry toxins is necessary as the range of possibly affected organisms focuses on pest species and no evidence is claimed to exist pointing to combinatorial effects on non-target organisms. We have examined this rationale critically using information reported in the scientific literature. To do so, we address the hypothesis of narrow specificity of Cry toxins subdivided into three underlying different conceptual conditions (i) "efficacy" in target pests as indicator for "narrow specificity," (ii) lack of reported adverse effects of Cry toxins on non-target organisms, and (iii) proposed modes of action of Cry toxins (or the lack thereof) as mechanisms underlying the reported activity/efficacy/specificity of Cry toxins. Complementary to this information, we evaluate reports about outcomes of combinatorial effect testing of Cry toxins in the scientific literature and relate those findings to the practice of environmental risk assessment of Bt-crops in general and of stacked Bt-events in particular.

#### Edited by:

*Robin Mesnage, King's College London, UK*

#### Reviewed by:

*M. Jahangir Alam, University of Houston College of Pharmacy, USA Yue-Wern Huang, Missouri University of Science and Technology, USA*

> \*Correspondence: *Angelika Hilbeck<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Environmental Health, a section of the journal Frontiers in Environmental Science*

Received: *26 August 2015* Accepted: *15 October 2015* Published: *09 November 2015*

#### Citation:

*Hilbeck A and Otto M (2015) Specificity and Combinatorial Effects of Bacillus Thuringiensis Cry Toxins in the Context of GMO Environmental Risk Assessment. Front. Environ. Sci. 3:71. doi: 10.3389/fenvs.2015.00071* Keywords: Bt toxins, non-target organisms, target organisms, synergistic effects, mode of action, adverse effects

## BACKGROUND

Today, many crop plants have been genetically modified (GM) to contain transgenic DNA sequences from the bacterium Bacillus thuringiensis (Bt) coding for the expression of socalled Cry toxins from 3 to 4 different classes (Cry1, Cry2, Cry3, and to a very limited extent Cry9 class) (Schnepf et al., 1998; Sanchis, 2011; Sansinenea, 2012; van Frankenhuyzen, 2013). These microbial toxins engineered into GM crops plants aim to control certain target pest species which differ depending on the regions where the crops are grown. In Bt plants, the Cry toxins are present persistently and usually in all plants parts from germination to harvest of the crops. Specifically, it is through genetic engineering that also the pollen of GM plants can express these bacterial toxins. Bt-toxins in pollen are not degraded by UV light and remain bioactive when, for example, deposited on host plants (Ohlfrest et al., 2002). Toxic pollen is rare in nature as, typically, there is little selective advantage for such a trait to evolve.

When used as sprayable, externally applied B. thuringiensis based insecticides, the risk for non-target organisms to ingest the Cry toxins is low due to the limited persistence of these sprays in space and time as the Bt toxin is quickly degraded by UV light and removed from plants by rain (Behle et al., 1997). Risks to non-target insects are further reduced because sprayable B. thuringiensis products usually consist of Cry toxins in their inactive crystalline form (hence their abbreviation "Cry") that need to undergo a complex activation process before becoming active (see below for details). With the introduction of Cry toxin producing GM crop plants into industrial agricultural systems, a whole new dimension of spatio-temporal exposure to Cry toxins opened up involving a far broader range of non-target organisms below- and above ground (Hilbeck, 2001, 2002). Thus, the potential for chronic longterm effects became more likely to occur than with short-lived, inactive sprayable B. thuringiensis insecticides. Consequently, it was with the introduction of Cry toxin producing GM crops that the likelihood of potential adverse effects (i.e., risks) on non-target organisms, in particular beneficial insects like natural enemies of pests, pollinators, and species of conservation concern, came on the research agenda (Hilbeck, 2001, 2002).

Since about one decade, there is a marked increase in the commercial approval and adoption of GM plants carrying multiple transgenes coding for the simultaneous expression of several insecticidal Cry toxins (and also transgenes for herbicide resistance), so called "pyramided" or "stacked" events (Fernandez-Cornejo et al., 2014; United States Department of Agriculture and Economic Research Service (ERS), 2014). In 2014, stacked GM cotton reached almost 80% of cotton plantings up from around 25% 10 years earlier. Stacked GM maize made up 76% of the planted maize area in 2014 up from less than 10% a decade ago (Fernandez-Cornejo et al., 2014; United States Department of Agriculture and Economic Research Service (ERS), 2014). Stacking Bt transgenes was triggered by spreading resistance among target pest populations against GM crops expressing only single Cry toxins (Tabashnik et al., 2013). The underlying assumption is that pest species are less likely to develop resistance simultaneously against multiple Cry toxins because of their somewhat different modes of action. Whether or not this assumption will hold true for the currently employed Cry toxins that still share significant similarities in their modes of action (Hernández-Rodríguez et al., 2013) is not subject of our evaluation in this paper but deserves a separate analysis.

In this review, we are concerned with the drastically increased Cry toxin load in stacked or pyramided Bt crop varieties resulting in persistent exposure of a wide range of non-target organisms in terrestrial and aquatic ecosystems. In SmartStax R maize, for example, combining six Cry toxins, maximum amounts of >250µg/g fresh weight in leaves, > 90µg/g fresh weight in roots, and > 150µg/g fresh weight in pollen was reported (**Table 1**; Stillwell and Silvanovich, 2007; Phillips, 2008). Potential combinatorial effects of these multiple Cry toxins have been recognized by the European regulatory authorities only few years ago (EFSA, 2010a), and are still intensely discussed by regulators and in the scientific community alike (de Schrijver et al., 2014). Yet, most regulators, including EFSA, still operate under the current controversial paradigm that limits the environmental risk assessment to focusing on the added novel substance only which is tested as single purified protein, produced by microbes, in isolation of the GM plants, following testing schemes developed for the regulatory approval of synthetic insecticides (for more details see Hilbeck et al., 2011). The results of these tests are then used in all risk assessments for Bt crops that express these Cry toxins but irrespective of the GM event (e.g., Garcia-Alonso et al., 2006; Romeis et al., 2008; Dolezel et al., 2011; Hilbeck et al., 2011). Consequently, for stacked GM crop plants combining multiple Cry toxins, developers of GM plants are arguing for minimal regulatory oversight of stacked events, if any at all, on the basis that "previously approved GM events that have been combined by conventional plant breeding and contain GM traits that are not likely to interact in a manner affecting safety should be considered

\**Source: compilation based on Phillips (2008) and Stillwell and Silvanovich (2007).*

to be as safe as their conventional counterparts" [Pilacinski et al., 2011, similar arguments by Raybould et al. (2012) and CropLife International (2015)]. EFSA (2010b) largely follows this view and has approved stacked events based on the above arguments exemplary statements from an opinion for approval of a stacked event expressing three Cry toxins are as follows:

"The safety of Cry1A.105 and Cry2Ab2 proteins expressed in maize MON 89034, the Cry1F and PAT proteins expressed in maize 1507, and the CP4 EPSPS and CP4 EPSPS L214P proteins expressed in maize NK603 have been assessed for their safety previously and no safety concerns were identified for humans and animals.... the EFSA GMO Panel considers it unlikely that interactions between the single maize events will occur that may impact on the food and feed safety and nutritional pro perties." [excerpts from EFSA (2010b)—similar justifications and wording has been used in other approvals of stacked events].

In this paper, we examine the two claims that serve as justification to minizime or omit the testing of combinatorial effects of multiple Cry toxins expressed in stacked GM Bt crop plants: (1) Due to narrow specificity of Cry toxins, no species outside of the primary class of target pest organisms are affected and (2) as long as single Cry toxins do not elicit adverse effects, they will not do so in combination with other Cry toxins or other naturally occurring compounds.

Lastly, we will reflect on the regulatory practice of risk assessment of stacked Bt-crops in the EU and will provide recommendations for improvements of the current testing practice.

### DEFINITIONS AND DATA USED IN THIS REVIEW

We carried out a data base search of the Web of Science for peer reviewed international publications in English language for the following key word categories: Category I:"Bacillus thuringiensis" or "Bt" or "toxin" or "protein" combined with Category II: "synergistic" or "interaction" or "combined/combinatorial" "effects." We then selected those studies that reported about in vivo tests only, e.g., bioassays with target and non-target organisms. On occasion, these reports contained also data on in-vitro tests with cell lines that we disregarded for comparability reasons. We restricted our evaluation to studies that reported on combinatorial effects between Cry proteins and some naturally occuring plant-, bacteria-, or insect-compounds as they can be encountered by non-target organisms feeding on stacked GM Bt plants in their natural environment. Studies on combinatorial or complementary effects with herbicides and other pesticides, such as neonicotinoid residues (Douglas and Tooker, 2015), have been omitted and left for future evaluations. In **Table 2**, we compiled the selected key reports to allow for a quick overview of the involved compounds, test organisms, and proposed mechanisms.

We report our findings according to the claims outlined above. The definitions for various types of combinatorial effects followed those by Tabashnik (1992):

Synergistic effects in the context of this paper entails effects e.g., mortality rates—of combined toxins exceeding those found for the individual toxin with the highest activity. If synergistic effects occur, this means that the toxicity of a mixture cannot be predicted from the individual ingredients. Antagonistic effects in the context of this paper are effects contrasting synergistic effects: when a mixture of toxins leads to less toxicity than found for individual toxins with the lowest activity. If potency is less than expected.

Additive effects in the context of this paper entail effects e.g., mortality rates—of combined toxins not exceeding those found for the individual toxin with the highest activity. If additive effects occur, this means that the toxicity of a mixture could be predicted from the individual ingredients.

### RESULTS

The first claim of narrow specificity rests on two premises: (i) "efficacy" determined in economically important target pests is a reliable indicator for "narrow specificity" of Cry toxins, and (ii) the mode of action of Cry toxins supports the claim of narrow specificity. In the following, we will evaluate the data base for these two premises.

Furthermore, we will summarize the scientific literature documenting effects of single and multiple combined Cry toxins and their postulated mechanisms to examine whether the reported data and methodologies confirm the conclusion of predictability (likelihood) of combinatorial effects.

### NARROW SPECIFICITY OF Bt PROTEINS

Since the claimed narrow specificity of Bt toxins serves as the first line of justification to forego the testing of non-target organisms, the premises this claim rests upon should be well-supported with scientific evidence. Below, we, firstly, scrutinize the current definition of specificity and its application within risk assessment of Bt-crops and non-target organisms. Secondly, we summarize the current state of knowledge regarding the proposed mode of action of Cry toxins.

#### Assumptions vs. Evidence

B. thuringiensis bacteria express and deposit a multitude of proteineous, insecticidal toxins in various crystalline forms in the bacterial mother cell (called Cry proteins). When the insecticidal properties of these deposited Cry proteins were discovered a century ago, very quickly their potential utility for pest control was recognized and research and development of B. thuringiensis based sprayable insecticides began. Today, several commercial B. thuringiensis based insecticide formulations are broadly available commercially and used in organic crop production as one of the few permitted sprayable insecticides. Hence, ever since their discovery, research and development efforts have focused almost exclusively on studying their efficacy on economically important, herbivorous pest species (e.g., Hoefte and Whiteley, 1989; Schnepf et al., 1998; Sanchis, 2011; van Frankenhuyzen, 2013). Naturally, for practical pest control purposes, the most efficacious B. thuringiensis-produced Cry proteins are considered to be those that induce maximum—if possible 100%—mortality in a given target pest population with the least amount of toxin

#### TABLE 2 | Some key publications highlighting different types of combinatorial effects on insects reported in the scientific literature.

#### TABLE 2 | Continued

#### TABLE 2 | Continued

*LE, Level of effect; "–", antagonistic; "0", neutral; "*+*", additive (definitions see text).*

ingested in the shortest period of time. We refer to this as the concept of "quick kill." Based on this economically motivated concept of "efficacy," most Cry toxins affect most efficaciously only a relatively narrow range of so called "target" pest species. However, this pest control-focused concept underlies the generalization of narrow specificity of all Cry toxins applied to all non-target species and the assumption that Cry toxins are unlikely to affect other species outside of their range of primary (target) organisms (e.g., Soberón et al., 2009; Sanchis, 2011; Pardo-López et al., 2013).

It was with the introduction of GM crops expressing activated Cry toxins constitutively, meaning in all tissues of the GM plants throughout their entire lifespan, that ecologists urged to revisit the validity of the economic definition of "specificity," or "quick kill," and began testing various Cry toxins on non-pest nontarget organisms (Hilbeck, 2001, 2002). Today, with the vastly expanded spatio-temporal exposure of Cry toxins from GM crop plants, easily also reaching beyond the crop field (Hofmann et al., 2014), the need arose to investigate ecologically relevant adverse effects beyond the narrow scope of a small group of economically relevant herbivores considered pests in crop fields and also beyond the single economic parameter of "quick kill." Ecological parameters including cumulative lethal effects, i.e., "slow kill," and sublethal impacts (e.g., developmental time, weight gain, behavioral changes) have now gained importance. Such effects could cause as severe or even more severe ecological consequences for a terrestrial or aquatic ecosystem as a "quick kill" could.

The most comprehensive data source concerning the specificity of Bt proteins is the Bt Toxin Specificity Database (http://www.glfc.cfs.nrcan.gc.ca/bacillus/; van Frankenhuyzen and Nystrom, 2002). Analyses from this database have been published by van Frankenhuyzen in 2009 and 2013 with a recent update in de Schrijver et al. in 2014. While, originally, Cry toxin activity was assumed to be restricted to the insect order of Lepidoptera, this has successively been expanded to include today up to six arthropod orders for which so-called cross-active Bt toxins have been reported (van Frankenhuyzen, 2013). Notably, today, the most widely employed and studied Cry toxins, such as Cry1Ab or Cry1Ac, have been reported to affect species from different insect orders or even phyla (Cry1Ab: Coleoptera, Lepidoptera, Diptera, Hemiptera; Neuroptera, Trichoptera and Nematoda; Cry1Ac: Lepidoptera, Diptera, Hemiptera). But van Frankenhuyzen (2013) found that only a small fraction (17%) of Cry toxins have ever been tested with species from more than 1 or 2 insect orders. Despite this restriction to certain tested pest species, approximately 40% of all Bt toxins tested across two or more orders did show cross-activity (de Schrijver et al., 2014). Yet, even for the most tested lepidopteran-active Cry1 toxins, only a little more than one third has ever been experimentally tested outside of that order. In total, van Frankenhuyzen (2013) compiled evidence for cross-activity of 27 Bt toxins and 69 insect taxa. We expect that the number of reported cross-activities will likely rise as more experiments with non-target organisms emerge and the old definition of order-specifity of Bt-toxins (van Frankenhuyzen, 2013; de Schrijver et al., 2014) may no longer be regarded as a functional concept—cross-activity may actually become rather a common phenomenon than an execption, certainly under an ecological definition of "efficacy" or "specificity." Apart from few exceptions, the majority of available data from the van Frankenhuyzen-database rely on mortality as the measured endpoint. However, sub-lethal effects such as growth inhibition, changes in developmental time or other parameters which may affect fitness can be expected to occur at far lower effect-doses than those inducing a "quick kill."

#### Proposed Mode(s) of Action (Mechanism)

Much of the claimed specificity of Bt-toxins rests on what is known about the mode of action of Cry toxins from research with this narrow set of herbivorous target pest insects and from studying predominantly one Cry toxin class only, Cry 1. By comparison, modes of action of Cry 3 and Cry 2 toxins have received far less attention (Schnepf et al., 1998; Whalon and Wingerd, 2003; Vachon et al., 2012). Some authors think that specificity rests equally on the solubilization-activation process as on the receptor-binding and pore-formation process (Smouse and Nishiura, 1997), while others postulate specificity to rest mainly on (affinity to) specific receptors (Schnepf et al., 1998). Today, quite a controversy exists over the mode of action of Cry toxins which is owed to the fact that there is less scientific certainty about it today than there was when cry transgenes from B. thuringiensis were first engineered into GM plants roughly 30 years ago. Until about a decade ago, the predominant and most agreed model for the mode of action of Cry proteins (the "classical model") as produced by B. thuringiensis went as follows: ingested, inactive crystalline (Cry) proteins must be solubilized in an insect gut environment with a high pH (>10). The solubilization of crystalline proteins yields a still inactive socalled full-length protoxin (ca. 130 kDa Cry1 class and ca 73 kDa Cry3 class) that requires further biochemical cleavage to produce a small toxic fragment. This toxic fragment (ca. 65 kDa Cry1 class and 55 kDa Cry3 class) then must bind to certain receptors located in the midgut ephithelium and, thereby, induce pore formation (also called ion channel forming) and lysis of the gut resulting in septicemia subsequently killing the insect (Schnepf et al., 1998; Whalon and Wingerd, 2003; Vachon et al., 2012). Different sized fragments of the same Cry class have been shown to exhibit different activities in different ranges of affected insects (Haider and Ellar, 1987). A whole range of membrane binding proteins have been suggested as receptors, including numerous cadherins, aminopeptidases, and alkaline phosphatases as well as glycolipids (Pigott and Ellar, 2007; Sanchis, 2011; Vachon et al., 2012).

Today, additional models for Cry toxin mode of action have been suggested with supporting new data. In a recent review, Vachon et al. (2012) describe and critique three models: the "classical" model, the "sequential binding" model, and the "signaling pathway" model. The first two models have in common that the mode of action of activated Cry toxins hinges on the binding of activated (i.e., cleaved) toxic fragments of the original Cry protein to receptors in the midgut epithelium in insect larvae. The sequential binding model proposes a more complex sequence of events with more binding steps involving more receptors and the removal of an alpha helix from the Cry toxin leading to a required oligomerization step of the Cry toxins before inserting into the gut membrane and inducing pore formation in the gut of the insect (Soberon et al., 2012). Consequently, in addition to the cadherin receptors of the "classical" model, which are now called to be "primary" receptors, so called "secondary" receptors, GPI (glycosylphosphatidyl-inositol)-anchored receptors, are suggested to have a significant role in pore formation (Soberón et al., 2009; Soberon et al., 2012; Pardo-López et al., 2013). While in the classical model it was believed that monomeric Cry1 toxins can bind to cadherin receptors and induce pore formation, in the sequential model it is proposed that cadherin-bound monomeric Cry1 toxins cause conformational changes favoring proteolytic cleavage that allows the rest of the toxin to oligomerize (Jiménez-Juárez et al., 2007; Soberon et al., 2012; Vachon et al., 2012). The Cry1 toxin oligomers subsequently bind to the GPI-anchored receptors and only then pore formation is induced. Mechanisms of resistance in target pests were found to be often associated with mutations affecting the binding to these cadherin receptors. Hence, a proposed solution to overcome such cadherin-based resistance was "the rational design of improved toxins" (Sóberon et al., 2007, 2009). This led to the development of modified Cry1 toxins, so called CryMod toxins, lacking an alpha-helix that circumvented the cadherin-binding step by oligomerizing simply in the presence of trypsin and, subsequently, continue to induce pore formation requiring only binding to "secondary" receptors (Sóberon et al., 2007). Vachon et al. (2012) challenge the validity of this model and also Pigott and Ellar (2007) pointed out that "other explanations of the data are possible." The credibility of the sequential binding model was further eroded because of later, admitted manipulations of images of the gels, including the removal of stains from the blot and shifting positions of the bands of the blot (e.g., Jimenez-Juarez et al., 2013), which were offered as evidence for the sequential binding model in a total of 11 publications. We conclude that the sequential binding model is, therefore, in need of independent validation and experimental reconciliation with the critique by Vachon et al. (2012) and Pigott and Ellar (2007).

Yet another model has been suggested by Zhang et al. (2005, 2006a) that differs from the two above in that pore formation is not an essential feature anymore in the cause of death of the insect. Instead the signal transduction model proposes that binding of Cry toxin monomers to cadherin activates an intracellular cell death mechanism (Smouse and Nishiura, 1997; Zhang et al., 2005, 2006a). This model also has been questioned by Vachon et al. (2012) and also by Soberón et al. (2009) who developed the competing "sequential binding model." In contrast, Jurat-Fuentes et al. (Jurat-Fuentes and Adang, 2006), suggested a combination of the Zhang et al. (2005, 2006a) and the "sequential binding" model to be at work, and Kumar and Kumari (2015) consider both modes of action to act in a complementary fashion. Most authors, however, seem to agree that much still needs to be learned about the modes of action of Cry proteins. Pigott and Ellar (2007) expect that "as more toxin receptors are discovered and as our understanding of toxin-receptor interactions increases, it will be interesting to see the extent to which Cry toxins utilize a common mode of action."

Further complexity has been added to the presented new proposals of modes of action of Cry toxins by research suggesting that Cry toxins require the interaction with gut microbes in order to exert their lethal effects in target pest organisms (Broderick et al., 2006, 2009). When highly susceptible lepidopteran larvae were fed with antibiotics prior to being offered Cry toxin-spiked diet, the Cry toxins lost entirely their activity and no adverse effects on survival could be observed. The hypothesis proposed is that the Cry toxin induced pore formation of the midgut of susceptible larvae allows pathogenic gut bacteria to enter the hemocoel, allowing the bacteria to multiply and kill the host larvae via septicemia. Without the presence of such bacteria Cry toxins alone do not kill the larva (Broderick et al., 2006, 2009). When Enterococcus faecalis bacteria were added again to the diet, susceptibility to the Cry toxins was restored and high mortality observed. Hence, E. faecalis, which is a commensal bacteria in an intact gut, can become a pathogen when invading the hemocoel. A process the authors referred to as the "commensalto-pathogen" switch (Mason et al., 2011). Similarly, Jung and Kim (2006) reported that while B. thuringiensis subsp. aizawai (Bta) did efficiently kill third instar Spodoptera exigua larvae, it did not cause high mortality of fifth instar larvae. But when adding nematodes to Bt fed fifth instar larvae, it resulted in significant synergistic effects. They also suggested that this was due to Bta damaging at least somewhat the midgut cells of the fifth instars allowing the nematodes to enter the hemocoel. While the controversy remains regarding whether or not midgut microbiota (bacteria or nematodes) is essential for Cry toxins to kill susceptible insect larvae (Johnston and Crickmore, 2009; Raymond et al., 2009), it does highlight that the modes of action of Cry toxins are far from conclusive to date (Graf, 2011), and that co-factors which naturally occur in the environment impact the efficacy and specificity of Cry toxins which may help explaining some of the effects of Cry toxins on non-target organismes reported in the literature.

### SIGNIFICANT EFFECTS OF SINGLE CRY TOXINS ON NON-TARGET ORGANISMS

Interestingly, there are indeed many documented cases of crossorder activities of Cry toxins on non-target organisms including reports about a range of lethal and non-lethal developmental (e.g., Lövei and Arpaia, 2005; Hilbeck and Schmidt, 2006; Marvier et al., 2007; Lövei et al., 2009; van Frankenhuyzen, 2009, 2013) or behavioral effects (e.g., Meier and Hilbeck, 2001; Zemkova Rovenska et al., 2005) challenging the narrow specificity narrative of Cry toxins, in particular when expressed as activated Cry toxins in the GM plants (**Table 2**). These reports include significant adverse effects of Cry toxins on nontarget coccinellid species, Harmonia axyridis and Henosepilachna vigintioctomaculata (Stephens et al., 2012; Song et al., 2012) with research groups reporting significant cross-order effects of lepidopteran active Cry1 proteins on coleopteran coccinellid predators both when administered directly (Dhillon and Sharma, 2009; Schmidt et al., 2009; Hilbeck et al., 2012a) and via unaffected and affected prey (Zhang et al., 2006a,b,c; **Table 2**). In some cases reports of adverse effects even on non-arthropod species have been published. When studying Cry toxins expressed in GM maize material with the snail species Cantareus aspersus, the researchers found 25% lower growth rate than in the control treatment (Kramarz et al., 2009). Just recently Shu et al. (2015) reported about significant effects of Cry1Ab from Mon810 maize on compost worm Eisenia fetida. Furthermore, several researchers reported about adverse effects of Cry toxins from GM host plant material on several aquatic organisms. In laboratory feeding trials, Rosi-Marshall et al. (2007) and Chambers et al. (2010) showed that consumption of single Cry toxin maize plant material reduced growth and increased mortality of the nontarget stream insects Lepidostoma liba and Helicopsyche borealis, respectively. Also Bøhn et al. (2008; Bøhn et al., 2010) reported that mortality was higher, a lower proportion of females reached sexual maturation, and the overall egg production was lower in Daphnia magna that were fed Cry1Ab toxin producing GM maize compared to D. magna fed control maize. The authors argued specifically that the combination of reduced fitness with earlier onset of reproduction of D. magna fed Cry toxin maize indicated a direct toxic effect. Other aquatic taxa for which negative effects on single species have been observed include crane flies (Isopods; 19% growth reduction; Jensen et al., 2010), Chironomids (Prihoda and Coats, 2008), and even crayfish (Linn and Moore, 2014).

The reported diversity of lethal and sublethal, chronic effects may sum up and lead to shifts in species composition at the community level. For example, Campos and Hernandez (2015) reported significant differences in dung beetle species composition—an important functional group—possibly leading to impaired ecosystems services such as feces removal, seed dispersal, edaphic aeration, and incorporation of organic matter. For aquatic habitats, Axelsson et al. (2011) reported that the composition of aquatic insect communities colonizing the litter from Cry3Aa expressing GM trees was significantly affected in unanticipated ways. Similarly, Rosi-Marshall et al. (2007) raised concerns that effects of Bt pollen and debris may negatively affect caddisflies and the food-web. Although this seems not to be the case for highly degraded industrial agricultural habitats (Chambers et al., 2010), the risk could not be clarified for more natural terrestrial and aquatic habitats which play an important role in ecosystem functioning.

In light of the above reports, it is clear that the claim of no reported adverse effects of single Cry toxins on cross-order nontarget organisms is not supported by the scientific evidence in the scientific literature. In fact, there is an increasing body of evidence suggesting significant effects of Cry toxins far beyond the originally postulated primary taxa of herbivorous target pest organism are possible.

### COMBINATORIAL EFFECTS OF CRY PROTEINS

Combinatorial (including synergistic) effects of Bt toxins were reported already decades ago (e.g., Wu et al., 1985). Best known are the synergistic effects of spores of B. thuringiensis subsp. kurstaki to increase toxicity of Cry toxins in susceptible and resistant larvae of the diamondblack moth, Plutella xylostella (Dubois and Dean, 1995; Tang et al., 1996; Liu et al., 1998). Cry toxins combined with spores "can be toxic even though the toxins and spores have little or no independent toxicity" (Liu et al., 1998). Tang et al. (1996) observed synergistic effects among spores and the three Cry toxins Cry1Aa, Cry1Ab, and Cry1Ac. They also reported about synergistic effects between spores and Cry1C toxins on P. xylostella but, interestingly, not between spores and Cry2A toxin. However, since spores play no role in stacked or pyramided Bt crops—although they may still be around naturally—combinatorial effects of Cry toxins with other compounds encountered in nature are at the center of this review. These include combinatorial effects with (a) other Cry toxins, (b) bacteria-derived compounds, (c) plant-derived compounds and (d) insect-derived compounds. In the following, we address the reported effects (phenomena) and the suggested modes of actions (mechanisms) separately. In **Table 2**, we compiled the data from some widely cited key reports to allow for a quick overview of reported combinatorial effects, the involved compounds, test organisms, and proposed mechanisms.

### Combinatorial Effects

#### Combinatorial Effects of Different Cry Toxins

Of the eight studies listed in **Table 2** that tested various Cry toxin combinations, seven reported significant combinatorial effects involving lepidopteran and dipteran species. Lee et al. (1996) reported a synergistic effect for a combination of Cry1Aa with Cry1Ac but an antagonistic effect for Cry1Aa and Cry1Ab and no combinatorial effect for Cry1Ab and Cry1Ac in Lymantria dispar. Interestingly, when keeping Cry1Aa stable (at 1) but increasing Cry1Ac two-fold (1:2), susceptibility increased from 49.9 to 34.9 ng ID (growth inhibition dose). But when increasing Cry1Ac more (1:4, 1:6 up to 1:12), susceptibility dropped substantially. Yet, none of these combinatorial effects was observed in Bombyx mori, the other test organism (Lee et al., 1996). More recently, Sharma et al. (2010) reported synergistic effects of various Cry1A toxins in Chilo partellus larvae. Poncet et al. (1995) found synergistic and additive effects of combined Cry toxins in three different mosquito species. Both Ibargutxi et al. (2008) and Yunus et al. (2011) reported synergistic effects of Cry1Ac and Cry2A toxins on Earias insulana, the spotted bollworm. In contrast, in two studies involving Helicoverpa armigera larvae and testing a similar combination of Cry1Ac and Cry1F, Ibargutxi et al. (2008) found no synergistic interaction while Charkrabarti et al. (1998) did (**Table 2**).

#### Combinatorial Effects of Cry Toxins with Bacteria-derived Compounds [Cytolytic (Cyt) Toxins]

Rincon-Castro et al. (1999) tested Cry1Ac toxins and Cyt1A1 toxins from engineered bacteria on Trichoplusia ni cell lines and larvae and found antagonistic effects. In contrast, Cyt1A proteins were found to synergize toxicity of Cry4A and Cry4B toxins, Cry10Aa and Cry11Aa in mosquito larvae (Chang et al., 1993; Wu et al., 1994; Wirth et al., 2004; Fernández-Luna et al., 2010). In fact, Wirth et al. (2004) reported that when Cry toxins from Bti were combined with B. sphaericus, in the presence or absence of Cyt1Aa, synergistically increased toxicity and an expanded host range were observed. Also Promdonkoy et al. (2005) reported that Cry4Ba toxins were toxic to A. aegypti larvae but virtually inactive to C. quinquefasciatus larvae. Cyt2Aa2 exhibited moderate activity against A. aegypti and C. quinquefasciatus larvae. But the combination of both toxins dramatically increased toxicity to both A. aegypti and C. quinquefasciatus larvae. Chitinases produced by bacterial B. thuringiensis increased activity of the produced Cry toxins more than two-fold in Spodoptera exigua larvae (Liu et al., 2002, **Table 2**). Chitinases are widely produced in many bacterial B. thuringiensis strains and in some cases enhanced the toxicity of the produced Cry toxins (Ramírez-Suero et al., 2011; Hu et al., 2013). It was proposed that they could be used to enhance efficacy of Bt toxins for pest control (Liu et al., 2002).

#### Combinatorial Effects of Cry Toxins with Insect-derived Compounds

Chen et al. (2007) reported that a peptide fragment of a toxin-binding cadherin isolated from Manduca sexta guts and expressed in E. coli synergistically enhanced toxicity of Cry1 toxins in other lepidoptera species (**Table 2**; Chen et al., 2007). Similarly, a fragment of a cadherin from A. gambiae was found to enhance the toxicity of Cry4Ba mosquitocidal toxins. For both types of co-factors, effects of individual proteins were often lower and non-lethal while in combination observed effects were stronger and lethal.

#### Combinatorial Effects of Cry Toxins with Plant-derived Compounds

Mohan et al. (2008) report synergistic effects of Cry2A toxin with plant defense compounds like Mir1-cysteine protease (**Table 2**) in maize varieties from Antiqua (Carribean). The combinatorial effects observed were lethal and, as in the example above, much stronger than the sub-lethal effects caused by Mir-CP alone. They were discovered in exotic maize varieties from Antiqua (Carribean) and bred conventionally into local varieties. Also here, effects of individual proteins were at best sublethal but when administered in combination effects were more dramatic and lethal (**Table 2**).

### Proposed Mechanisms for Combinatorial Effects

Below, we summarize the diversity of possible mechanisms proposed for the various observed combinatorial effects as this information will contribute to understanding whether combinatorial effects can be predicted.

#### Combinatorial Effects of Different Cry Toxins

Three different hypotheses are proposed. One hypothesis suggests that individual pores are formed by each Cry1A toxin individually and may act cooperatively, together inducing higher toxicity. A second theory proposes the formation of additional hetero-oligomers which may have better insertion ability than a homo-oligomer complex (Charkrabarti et al., 1998). A third theory suggests that the toxin mix might enhance toxicity by preventing non-productive binding (Schnepf et al., 1998). However, all of these hypotheses presume that all toxin molecules interact similarly with the BBMVs following more or less the "classical model." Sharma et al. (2010) found that all three Cry toxin combinations showed increase in binding and direct positive correlation between increased binding and mortality. Many reports involve a great deal of speculation.

#### Combinatorial Effects of Cry Toxins with Cytolytic (Cyt) Toxins

As possible mechanism of the observed antagonistic effect between a Cry1 toxin and the cytolitic Cyt1A1, the forming of a complex blocking one or more binding sites or the competition for space instead of receptors was offered as explanation. However, none of this has been confirmed yet. For the observed synergistic effects between Cry11Aa and Cyt toxin, Pérez et al. (2005) suggest that the Cyt1Aa toxin acts as receptor for Cry11Aa. Wirth et al. (2004) propose an interplay between different affinities of the varying toxins for receptor binding sites as mechanism—either masking or enhancing toxicity through competition, blocking or preferential binding dynamics working in conjunction.

#### Combinatorial Effects of Cry Toxins with Insect-derived Compounds

For combinatorial effects with insect-derived compounds like various cadherin fragments or chitinolytic proteases different mechanisms have been suggested supported by data to some degree. Some researchers suggested that the presence of cadherin binding sites, i.e., fragments of cadherin receptors isolated from different target pest organisms allowed for increased oligomerization of activated, monomeric Cry toxins which in turn increased the ability of a Cry toxin-CR complex to insert into the midgut membrane and induce pore formation (Chen et al., 2007; Park et al., 2009). Chitinolytic proteases are known to affect the peritrophic matrix (PM) and, thus, like cysteine proteases, allow greater access for Cry toxins to epithelial cells where pore formation takes place. The PM is an extracellular matrix of chitin, glycoproteins and proteoglycans that lines and protects the midgut epithelium from damage and assists in nutrient uptake. Through greater (affinity) or faster access, their efficacy is likely enhanced and toxicity increased, meaning a smaller dose of Cry toxins can induce the formation of more pores quickly.

#### Combinatorial Effects of Cry Toxins with Plant-derived Compounds

For combinatorial effects with plant-derived compounds like the Mir-cysteine proteases (Mir-CP), it was suggested that they increase the permeability of the PM which in turn facilitates the movement of Cry toxins through the PM to allow greater access to epithelial cells where pore formation takes place.

### DISCUSSION

The objective of this review was to evaluate the scientific basis of the claims serving as the rationale for minimizing or omitting the testing of combinatorial effects of multiple Cry toxins expressed in stacked GM crop plants. To do so we compiled and evaluated published experimental evidence.

### Narrow Specificity Narrative Depends on Definition of Efficacy and Reference Systems