Bt maize is a genetically modified crop that produces insecticidal proteins derived from the bacterium Bacillus thuringiensis . The proteins act against susceptible herbivorous pests, including lepidopteran insects. Wind-dispersed maize pollen can settle on larval host plants, where caterpillars may ingest Bt proteins and experience additional mortality or sublethal adverse effects.

European Union rules require environmental risk assessment of genetically modified organisms before market placement, and controlled laboratory tests form a central part of assessing potential hazards to non-target organisms. Because thousands of European butterfly and moth species exist, however, it is not feasible to test every species that could be exposed. The researchers began with the LEPIDAT database of the German Federal Agency for Nature Conservation, which contained 8,670 entries for lepidopteran taxonomic units, including genera, species and subspecies.

A preliminary screen retained 1,478 entries for which information was available on altitude, habitat, larval feeding period and feeding type. Most “Micro-Lepidoptera” were excluded before the formal selection because their biological information was insufficient, although the diamondback moth, Plutella xylostella , was retained as an exception. The researchers regarded it as an abundant farmland moth, sensitive to Bt, and easy to breed in the laboratory.

The first formal step excluded 350 listed species in the family Noctuidae, some of which are now classified in Nolidae, because members of this group had been reported to be comparatively insensitive to Bt toxins. The next steps removed species that did not occur in Germany and those unlikely to be exposed to Bt maize pollen. The latter exclusions covered species restricted to elevations above 700 metres, where maize is not cultivated in Germany; species whose larval periods did not overlap with the approximate German maize pollen-shedding period from late June to late August; and species whose larvae feed within host plants or below ground on roots, where deposited pollen would not normally reach them.

These filters reduced the candidate pool to 652 potentially exposed species. The remaining species were classified into broad habitat types: farmland, dryland, wetland, woodland and settlements. A species could qualify for more than one habitat category.

The researchers selected candidates found concurrently in at least four habitat types, including farmland, to favor widespread species capable of representing a range of landscapes surrounding maize cultivation. This stage retained 54 species. Laboratory practicality then provided the next filter.

Candidate species needed evidence that females could lay eggs under laboratory conditions and that their larvae could be reared indoors on host-plant leaves or an artificial diet. Twenty-two species without such evidence were removed, leaving 32 species considered suitable for laboratory experiments. Conservation status and expert judgment shaped the final choice.

Protected species were ranked highly because risk assessment must consider potential harm to populations that are conservation targets under European legislation. The researchers then assessed taxonomic variety, the geographic and ecological range represented by the candidates, and differences in larval body size. These criteria produced a tentative panel of 15 focal species from four lepidopteran superfamilies.

Breeding feasibility was supported by a separate literature review covering nine superfamilies and 30 families of European Lepidoptera. The search identified 548 publications, including handbooks, reviews, traditional breeding guides, modern rearing studies and artificial-diet protocols. Some records dated from more than a century ago, while others described controlled methods and detailed environmental requirements.

Although the review found evidence for laboratory culture across a broad taxonomic range, it also showed that an individual species may require time and adaptation before a reliable method can produce enough healthy larvae for testing. Natural host plants most closely reproduce the circumstances in which caterpillars would encounter deposited maize pollen, and the source study notes that they are the recommended approach for Bt maize effect tests. Artificial diets can nevertheless aid standardization, reproducibility or rapid screening.

Common formulations contain flour from legumes or cereals, protein, yeast, cholesterol, vitamins, minerals and agar as a binder; some also include dried plant material or sucrose to stimulate feeding. Such diets are not universally accepted, especially by newly hatched larvae, and larvae that have already fed on natural food may not transfer successfully. Artificial food must be replaced before it dries or develops mould, and the cited guidance advises against allowing larvae to pupate within it.

Laboratory culture also requires control of several biological and physical factors, including host-plant identity and availability, temperature, humidity, photoperiod, diapause, mating conditions, oviposition substrates and population density. Regular replacement of food, adequate ventilation and routine disinfection of containers and handling equipment help limit condensation, mould and disease. Colonies maintained over several generations may require measures to avoid inbreeding and, in some cases, supplementation with field-collected individuals.

Eggs or pupae may be disinfected before mass rearing, while field-collected food plants can introduce pathogens or parasitoid eggs. These practical requirements are why the researchers treated successful breeding as a central selection criterion rather than a detail to be addressed only after choosing test organisms. The procedure is presented as a selection protocol, not as evidence that particular butterflies or moths are hazardous or protected from Bt exposure.

Susceptibility to Bt maize pollen remains poorly known for most non-target lepidopterans, which is why it was not used as the main screening factor. The final two stages also relied on expert assessment, although that judgment was applied within the documented sequence. The German list cannot be transferred unchanged to other regions: researchers would need to adapt altitude limits, pollen-shedding periods, habitat information and the species present in the relevant receiving environment.

Some excluded species may also deserve testing because information was missing rather than because they were unsuitable. Even with those limitations, the framework offers a systematic way to turn a large regional fauna into a diverse, ecologically informed and practically testable set of candidate non-target insects.

Subject of Research: Selection of non-target butterflies and moths for laboratory assessment of Bt maize pollen effects.

Article Title: Selection of non-target Lepidoptera species to test Bt maize effects in the laboratory: which species and how to breed them?

Article References: Lang, A., Dolek, M., Lee, M. S., Freese-Hager, A., & Otto, M. (2020). Selection of non-target Lepidoptera species to test Bt maize effects in the laboratory: which species and how to breed them?. BioRisk, 15, 45-65. https://doi.org/10.3897/biorisk.15.59823

Image Credits: AI Generated

DOI: 10.3897/biorisk.15.59823

Keywords: Bt maize, non-target Lepidoptera, environmental risk assessment, laboratory breeding, maize pollen, butterflies, moths, species selection, insect conservation, artificial diets, Selection, non-target