Researchers who work on GMO crops are developing special “artificial diet systems”. The stated purpose of these new diets is to standardise the testing of the Cry toxins, often used in GMO crops, for their effects on non-target species. But a paper published last month in the journal Toxins implies a very different interpretation of their purpose. The new diets contain hidden ingredients that can mask Cry toxicity and allow them to pass undetected through toxicity tests on beneficial species like lacewings (Hilbeck et al., 2018). Thus the new diets will benefit GMO crop developers by letting new ones come to market quicker and more reliably. Tests conducted with the new diets are even being used to cast doubt on previous findings of ecotoxicological harm.
GMO Cry toxins
Cry toxins are a family of highly active protein toxins originally isolated from the gut pathogenic bacterium Bacillus thuringiensis (Latham et al., 2017). They confer insect-resistance and up to six distinct ones are added to GMO corn, cotton, and other crops (Hilbeck and Otto, 2015).
The resulting crops are usually called Bt crops. Cry toxins kill insects that eat the GMO crop because the toxin punches a hole in the membranes of the insect gut when it is ingested, causing the insect to immediately stop feeding and eventually die of septicaemia.
Cry toxins are controversial. Although the biotech industry claims they have narrow specificity, and are therefore safe for all organisms except so-called ‘target’ organisms, plenty of researchers disagree. They suspect that Cry toxins may affect many non-target species, even including mammals and humans (e.g. Dolezel et al., 2011; Latham et al., 2017; Zdziarski, et al., 2018).
The Cry toxin mode of action, we and others have noted, does not necessarily discriminate between species. Any organism with a membrane-lined gut is, in principle, vulnerable if it consumes the GMO Bt crop. In these Bt crops the leaves, straw, roots, nectar, and pollen, all typically contain Cry toxins. Therefore, most organisms in agricultural landscapes will at some point in their life-cycle be exposed to GMO plant material. As pollinator declines and a more generalised insect apocalypse have revealed, the question of the effects of such crops on biodiversity is far from trivial.
The biotech industry is also very much aware of the steady stream of research, from evidence of allergenicity, to toxicity, of their Cry proteins towards so-called ‘non-target’ organisms. Organisms affected by Cry toxins include monarch butterflies, swallowtail butterflies, lacewings, caddisflies, bees, water fleas, and mammals (Losey et al., 1999; Bøhn et al., 2008; Ramirez-Romero et al., 2008; Schmidt et al., 2008; Sabugosa-Madeira et al., 2008; Mezzomo et al., 2015; Zdziarski, et al., 2018). Much of this research does not get the attention it deserves (e.g. COGEM 2014), but if swallowtail butterflies can succumb to just 14 pollen grains of Syngenta’s BT-176 corn (Lang and Vojtech, 2006) the industry is aware it can hardly truthfully market GMOs as environmentally beneficial.
As we have reported, one response of the biotech industry has been to try to bake approval into regulatory decisions. That is, make regulatory processes operate such that no possible future findings of unexpected harm by Cry toxins towards non-target organisms, no matter where in the risk assessment process they are observed, can derail approval. Thus, in “The Biotech Industry Is Taking Over the Regulation of GMOs from the Inside” we showed how “tiered risk assessment”, a regulatory procedure being promoted by the crop biotech industry, functions, in practice, as an “approval” system (Romeis et al. 2008). That is, regulatory denial of an application, under tiered risk assessment is nigh impossible.
How to mask Cry toxicity
The latest development, which adds further to the improbability that GMO Cry toxins will fail risk assessment, comes to light thanks to Hilbeck and colleagues (Hilbeck et al., 2018).
They report that these new “artificial diet systems” for raising non-target organisms contain surprisingly large amounts of antibiotics (Li et al., 2014; Ali et al., 2016a; and Ali et al. 2016b). The significance of this is that antibiotics are known to act as antidotes to Cry toxins (Broderick et al., 2006, Mason et al., 2011). By masking the harm caused by the toxin, antibiotics can give the unsuspecting reader a false impression of Cry harmlessness.
This effect of antibiotics was first shown in 2006 by researchers at the University of Wisconsin. They and others showed that gut bacteria are required for Cry toxins to achieve their full effect (Broderick, et al., 2006; Mason et al., 2011). Broderick et al wrote:
“Here, we report that B. thuringiensis does not kill larvae of the gypsy moth in the absence of indigenous midgut bacteria. Elimination of the gut microbial community by oral administration of antibiotics abolished B. thuringiensis insecticidal activity” (Broderick et al., 2006)
What Hilbeck et al., note about the diets, which their inventors claim are “needed” for reproducible testing of Cry toxins on carnivorous insects such as green lacewings and ladybirds, are the very large quantities of antibiotic compared to the amounts necessary to prevent spoilage (Li et al., 2014; Ali et al. 2016a; and Ali et al. 2016b). The diets developed by these authors contain the antibiotics Streptomycin (130mg) and Cephalosporin (50mg) per 100g (Li et al.; 2014); or Streptomycin (400mg) and Penicillin (400mg) per 100g (Ali 2016a); and Streptomycin (400mg) andPenicillin (400mg) per 100g (Ali 2016b).
Such large antibiotic quantities are questionable on several grounds. Previous authors have specifically noted the need to minimise antibiotic use in test diets intended to measure the toxicity of Cry proteins. As Porcar et al. (2010) wrote:
“Antibiotics were deliberately excluded from the diet composition since bacteria occurring in the insect midgut naturally might be critical for sensitivity (Broderick et al., 2006).” (Porcar et al. 2010)
Furthermore, a very similar diet developed by other authors, also in China, for green lacewings (reared for different reasons), used no antibiotics (Cheng et al., 2017). Third, Li et al. (2014) claimed their diet is a development of one from Cohen and Smith (1998). Yet the diet developed by Li et al. contains almost six times as much streptomycin and two and a half times the level of a second antibiotic, cephalosporin (though Cohen and Smith used tetracycline). No mention or explanation of the raised antibiotic levels was made.
In other words, the problem of antibiotics acting as antidotes to Cry proteins is widely known to Cry toxin researchers but is ignored by the authors of these three papers. Less surprisingly, using their antibiotic-laden diet Li et al. in 2014 found “no detrimental impact of these Cry proteins on any of the C. sinica(Green lacewing) life-table parameters measured”, as also did Ali et al. (Ali et al., 2016a)
Such papers as these have multiple harmful effects. Their results contradict (in strong probability erroneously) previous findings that GMO Cry toxins harm non-target insects like ladybeetles and lacewings; thus placing earlier findings in doubt. Second, future experimenters who adopt these diets will also likely, wittingly or unwittingly, obtain falsely negative results. Third, if the claimed “need” for them is any guide, these artificial diet systems will be promoted (with industry help) to be adopted as gold-standards for future research, just like tiered risk assessment.
Unsurprisingly perhaps, researching the background of these authors one finds that Joerg Romeis is one of them (Li et al., 2014). Romeis is a Swiss academic who has for many years been closely associated with the biotech industry. Romeis has called studies that find harmful effects of Cry toxins “bad science”; he has published many papers with industry authors that he believes refute evidence of effects on non-target organisms, and he led their tiered testing project (Romeis et al., 2008; Romeis et al., 2013).
Who is really doing bad science?
Ali, I.; Zhang, S.; Cui, J.J. (2016a) Bio-safety evaluation of Cry1Ac, Cry2Ab, Cry1Ca, Cry1F and Vip3Aa on Harmonia axyridis larvae. J. Appl. Entomol. 141, 53–60.
Ali, I.; Zhang, S.; Luo, J.-Y.; Wang, C.-Y.; Lv, L.-M.; Cui, J.-J. (2016) Artificial diet development and its effect on the reproductive performances of Propylea japonica and Harmonia axyridis. J. Asia-Pac. Entomol. 19, 289–293.
Bøhn T., Primicerio R., Hessen D.O., Traavik T. (2008) Reduced Fitness of Daphnia magna Fed a Bt-Transgenic Maize Variety. Arch. Environ. Contam. Toxicol. 55: 584-92.
Broderick, N.A.; Raffa, K.F.; Handelsman, J. Midgut bacteria required for Bacillus thuringiensis insecticidal activity. Proc. Natl. Acad. Sci. USA 2006, 103, 15196–15199.
Cheng, Y., Zhi, J., Li, F., Jin, J., & Zhou, Y. (2018). An artificial diet for continuous maintenance of Coccinella septempunctata adults (Coleoptera: Coccinellidae). Biocontrol Science and Technology, 28(3), 242–252.
Cohen A C, L K Smith (1998) A New Concept in Artificial Diets for Chrysoperla rufilabris: The Efficacy of Solid Diets.Biological Control Volume 13, Issue 1, September 1998, Pages 49-54.
Dolezel, M., Miklau, M., Hilbeck, A., Otto, M., Eckerstorfer, M., Heissenberger, A., … Gaugitsch, H. (2011). Scrutinizing the current practice of the environmental risk assessment of GM maize applications for cultivation in the EU. Environmental Sciences Europe, 23, 33. doi:10.1186/2190-4715-23-33.
Hilbeck, A., & Otto, M. (2015). Specificity and combinatorial effects of Bacillus thuringiensis Cry toxins in the context of GMO environmental risk assessment. Frontiers in Environment Science. doi:10.3389/fenvs.2015.00071
Hilbeck A, N Defarge, T Bøhn, M Krautter, C Conradin et al. (2018) Impact of Antibiotics on Efficacy of Cry Toxins Produced in Two Different Genetically Modified Bt Maize Varieties in Two Lepidopteran Herbivore Species, Ostrinia nubilalis and Spodoptera littoralis. Toxins , 10: 489;
Latham, J.R.; Love, M.; Hilbeck, A. The distinct properties of natural and GM Cry insecticidal proteins. Biotechnol. Genet. Eng. Rev. 2017, 33, 62–96.
Losey J. E. , L. S. Rayor & M. E. Carter (1999) Transgenic pollen harms monarch larvae. Nature 399: 214.
Li, Y.; Hu, L.; Romeis, J.; Wang, Y.; Han, L.; Chen, X.; Peng, Y. Use of an artificial diet system to study the toxicity of gut-active insecticidal compounds on larvae of the green lacewing Chrysoperla sinica. Biol. Control 2014, 69, 45–51.
Mason, K.L.; Stepien, T.A.; Blum, J.E.; Holt, J.F.; Labbe, N.H.; Rush, J.S.; Raffa, K.F.; Handelsman, J. From commensal to pathogen: Translocation of Enterococcus faecalis from the midgut to the hemocoel of Manduca sexta. mBio 2011, 2, e00065-00011.
Mezzomo, BP, Miranda-Vilela,AL and Grisolia CK (2015) Toxicological Evaluation of a Potential Immunosensitizer for Use as a Mucosal Adjuvant—Bacillus thuringiensis Cry1Ac Spore-Crystals: A Possible Inverse Agonist that Deserves Further Investigation. Toxins (Basel). 2015 Dec; 7(12): 5348–5358.
Porcar, M.; Garcia-Robles, I.; Dominguez-Escriba, L.; Latorre, A. (2010) Effects of Bacillus thuringiensis Cry1Ab and Cry3Aa endotoxins on predatory coleoptera tested through artificial diet-incorporation bioassays. Bull. Entomol. Res. , 100, 297–302.()
Ramirez-Romero R.; Desneux N.; Decourtye A.; Chaffiol A.; Pham-Delègue M.H. (2008) Does Cry1Ab protein affect learning performances of the honey bee Apis mellifera L. (Hymenoptera, Apidae)? Ecotoxicol Environ Saf. 70: 327-33.
Romeis, Jörg; Bartsch, Detlef; Bigler, Franz; Candolfi, Marco P; Gielkens, Marco M C; et al. (2008) Assessment of risk of insect-resistant transgenic crops to nontarget arthropods. Nature Biotechnology; 26: 203-8.
J Romeis, MA McLean, AM Shelton (2013) When bad science makes good headlines: Bt maize and regulatory bans. Nature Biotechnology 31: 386–387.
Rosi-Marshall, E.J.; J. L. Tank; T. V. Royer; M. R. Whiles; M. Evans-White; C. Chambers; N. A. Griffiths; J. Pokelsek and M. L. Stephen (2007) Toxins in transgenic crop byproducts may affect headwater stream ecosystems. Proc Natl Acad Sci USA 104: 16204–16208.
Sabugosa-Madeira B.; Abreu I.; Ribeiro H. and Cunha M. (2007) Bt transgenic maize pollen and the silent poisoning of the hive. Journal of Apicultural Research 46: 57-58.
Schmidt J.E.; Braun C.U.; Whitehouse L.P.; Hilbeck A. (2008) Effects of Activated Bt Transgene Products (Cry1Ab, Cry3Bb) on Immature Stages of the Ladybird Adalia bipunctata in Laboratory Ecotoxicity Testing. Arch Environ Contam. Toxicol. 56: 221-228
Zdziarski, I M., J.A. Carman, J.W. Edwards (2018) Histopathological Investigation of the Stomach of Rats Fed a 60% Genetically Modified Corn Diet. Food and Nutrition Sciences, 9, 763-796.