Research Article |
Corresponding author: Andreas Lang ( andreas.lang@unibas.ch ) Academic editor: Josef Settele
© 2020 Andreas Lang, Matthias Dolek, Marina S. Lee, Anja Freese-Hager, Mathias Otto.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Lang A, Dolek M, Lee MS, 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
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Bt maize targeting Lepidopteran pests poses potential risks for non-target (NT) butterflies and moths which are addressed in the environmental risk assessment of genetically modified crop plants. For this purpose, eco-toxicological tests are often conducted with specific NT species in the laboratory in order to assess possible adverse effects. As only a limited number of surrogate species can be addressed, the choice of focal species to be tested is an important decision. However, practical and standardised selection procedures have hardly been developed and applied for NT Lepidoptera, so far. Here, we present a transparent and systematic selection process of suitable test species for Germany, involving selection criteria such as exposure to Bt maize, habitat range and laboratory maintenance of the species. As a result, we compiled a list of 15 lepidopteran species particularly appropriate for testing the adverse effects of Bt maize in the laboratory. In addition, we collected and reviewed published reports for breeding methods of Lepidoptera, which provides essential information on maintaining lab stocks of NT Lepidoptera. The presented selection procedure allows focusing on the relevant test species in a transparent and reproducible way, and supplies the breeding knowledge required to breed and maintain them, which will be of great utility for the future assessment on possible risks of Bt maize cultivation to non-target Lepidoptera.
breeding, Bt maize, ecotoxicity, GMO, Lepidoptera, risk assessment, species selection, test species
Transgenic maize is one of the major genetically modified (GM) crops cultivated today (
In the EU, the assessment of any GMO is carried out on a case-by-case basis including, amongst other aspects, all receiving environments because a high number of NT species are potentially exposed to GM crops in the field. So far, only a limited, non-representative number of NT Lepidoptera has been studied with regard to the potential hazard of Bt maize (
Several papers have been published dealing with NT Lepidoptera species to be considered for the assessment of possible harmful effects of Bt maize. Most of these studies recorded and compiled lists of lepidopteran species that occur near maize fields or in arable land during maize anthesis, and are thus potentially exposed spatially and/or temporally to maize pollen dispersal (
To our knowledge, only two studies conducted a comprehensive, systematic and standardised attempt to select and prioritise NT Lepidoptera species for their likelihood to be affected by Bt maize, applying selection criteria and resulting in a list of a limited number of focal species to concentrate on in ERA of Bt maize (
Therefore, the objectives of this study were
The aim was to compile a list of potential non-target Lepidoptera species, whose larvae appear generally appropriate as test organisms for studying the effects of Bt maize on Lepidoptera in the laboratory. The selection process was carried out by developing and applying a systematic, consistent and transparent selection sequence on the NT Lepidoptera species present in Germany. For this purpose, the national LEPIDAT database of the German Federal Agency for Nature Conservation (BfN) was used to identify suitable species (see also
An initial, pre-selection screening was applied focussing on “Macro-Lepidoptera” for which sufficient biological information was available regarding altitude and habitat type of occurrence as well as for larval feeding periods and feeding type. In this pre-selection step, all “Micro-Lepidoptera” and other species with too little biological information were excluded prior to the actual selection procedure, with one exception. We kept the micro-moth Plutella xylostella (Plutellidae), because P. xylostella is a well-known and abundant moth in farmland, is very sensitive to Bt, and can be bred in the lab easily.
Then, the selection sequence was run with the remaining 1,478 entries for “Macro-Lepidoptera” in order to identify potential test species by applying a step by step process, including expert knowledge in a final step (see below). To begin with, several criteria were checked assessing the exposure of Lepidoptera larvae to Bt maize pollen dispersal, i.e. all species that are not exposed were eventually ruled out. Subsequently, the remaining species were prioritised according to the number and type of habitats that they could concurrently occupy. Species were then prioritised according to breeding feasibility in the laboratory by excluding species that cannot be reared well in captivity. The resulting species were then prioritised by conservation status and then, finally, expert knowledge was used to select a representative diversity of species in terms of different habitat types, taxonomic variability, body size distribution and species of protection values.
After the above described pre-selection leaving 1,478 data entries, the subsequent selection process was carried out through the following steps:
Principally, the species-specific susceptibility of lepidopteran larvae to Bt maize pollen is a relevant parameter. But as this is still unknown for the majority of the species (cf.
When selecting NT species for ERA of GM crops, the local receiving environment should be considered; for this reason this study was carried out considering the German maize cultivation areas. Therefore, only species actually occurring in Germany were taken into account (according to LEPIDAT).
Species where the larvae are not likely to be exposed to Bt maize pollen shedding were excluded (according to the information given in LEPIDAT), i.e.,
We aimed at selecting widespread species which occur in various different habitats so that the species selected are representative of the range of possible Bt maize cultivation environments. So, the specific habitat types as given by LEPIDAT were assigned to each species. Habitat requirements of the species are described in fine detail in the LEPIDAT database, however, for the current approach the species were assigned to the gross habitat classifications: farmland, dryland, wetland, woodland, settlements (if deemed necessary, the specific habitat types can still be ascribed ex post). Double entries were possible, e.g., species occurring in two habitat types could be noted for both habitats. Then, we selected all species which can be found in at least four different habitat types concurrently, including farmland.
Species were selected that could be bred and kept in the laboratory easily according to available knowledge. A literature search was conducted in order to compile information on breeding Lepidoptera families and species, and thus assess the feasibility of breeding each species. Species were defined in three breeding categories: P1 = can be bred in the lab from egg to adult as a stable colony over several generations; P2 = egg laying of adult females is possible in the lab, larvae subsequently can be reared in the lab; P3 = eggs or larvae must be collected in the field but can be kept in the lab thereafter; P4 = keeping and rearing is difficult (no oviposition and problematic keeping in the lab); P5 = unknown. Species that had no record of successful oviposition and rearing in the laboratory were excluded (P3, P4, P5).
For the risk assessment, protected species are of special concern as they represent a protection goal by EU legislation (
In the following steps, the species selection was further fine-tuned with regard to a representative distribution of:
A screening for existing breeding methods of Lepidoptera was carried out through a literature search, supplemented with expert interviews. A general internet search using google and google scholar did not produce many valuable results, although some non-academic information exists on different internet sources. Relevant literature was mostly retrieved from general biological databases such as BIOSIS (http://isiknowledge.com/biosis), but also from two specific databases (www.entomologische-literatur.de; www.zobodat.at). “Entomologische Literatur” is a private database on publications on Lepidoptera from Germany, whereas “Zobodat” is the publication database of the Biologiezentrum Linz, Austria. These two more specific databases offer access to publications from the beginning and the middle of the 20th century, mostly in the German language. Many of the publications cover “traditional breeding” defined as breeding without controlled (microclimatic) conditions. The most valuable search strings for the German/Austrian databases were <Zucht> (German for breeding/rearing) and family names. Searching BIOSIS was rendered most efficient by using the Lepidoptera family name together with <laboratory rearing> or <artificial diet>.
The search on breeding focused on the superfamilies and families presented in Table
All publications selected using the above procedure were listed in a table (see Suppl. material
Superfamilies and families of Lepidoptera screened for breeding information (taxonomy according to www.fauna-eu.org). Number of European species according to
Superfamily | Family (species number in Europe) |
---|---|
Bombycoidea | Brahmaeidae (7), Endromidae (2), Saturniidae (11), Sphingidae (42) |
Cossoidea | Brachodidae (15), Castniidae (1), Cossidae (37), Sesiidae (117) |
Drepanoidea | Cimeliidae (3), Drepanidae (22) |
Geometroidea | Geometridae (1,092), Uraniidae (1) |
Hepialoidea | Hepialidae (18) |
Lasiocampoidea | Lasiocampidae (48) |
Noctuoidea | Erebidae (372), Euteliidae (2), Noctuidae (1,306), Nolidae (48), Notodontidae (59) |
Papilionoidea | Hesperiidae (48), Lycaenidae (151), Nymphalidae (261), Papilionidae (16), Pieridae (61), Riodinidae (1) |
Zygaenoidea | Epipyropidae (2), Heterogynidae (13), Limacodidae (5), Somabrachyidae (1), Zygaenidae (68) |
Pre-selection: overall, the LEPIDAT database contained 8,670 entries for Lepidoptera taxonomic units (genera, species, sub-species). Excluding all taxa for which the database did not contain the information required by the following selection steps resulted in 1,478 taxa for selection step 1 (Fig.
Selection step 1 (excluding unsusceptible species): species known to be insensitive to Bt were excluded, in this case all 350 listed species belonging to the family Noctuidae (some now in the family Nolidae), leaving 1,128 taxa (Fig.
Selection step 2 (excluding non-resident species) and step 3 (excluding unexposed species): of the remainder, 476 taxa were eliminated when applying the exclusion criteria of steps 2 and 3, i.e. species unlikely to be exposed to Bt maize in Germany were excluded; either because the species are not native (= step 2), or because they are not exposed due to their altitudinal distribution, phenology or feeding habits (= steps 3a–3c). In consequence, a total of 652 Lepidoptera species remained as potentially adequate test species (Fig.
Selection step 4 (excluding species with restricted distribution): assigning habitat types to the remaining 652 species produced a list of 479 “woodland species”, 304 “farmland species”, 271 “dryland species”, 207 “wetland species”, and 161 “settlement species” (multiple nominations of species to habitat categories were possible). From this pool, we selected all species which can be found in four different habitat types concurrently including farmland, as this covers widespread species representative for several different habitat types. This resulted in a list of 54 species (Fig.
Schematic presentation of the selection process to choose focal Lepidoptera species for the testing of Bt maize effects in the laboratory.
Selection step 5 (excluding species that are difficult to breed in the lab): here, we assessed the species’ suitability for lab culturing, i.e. whether the species are relatively easy to breed. All species were selected that oviposit in the lab and/or can be reared as larvae indoors (P1 and P2) according to the defined breeding categories. This resulted in a list of 32 species (Table
Selection steps 6 (prioritising conservation status) – 7 (ensuring a representative variety of species): All 32 species (Table
List of 32 Lepidoptera species suitable for laboratory experiments to test the adverse effects of Bt maize. A tentative list of 15 focal species is marked in bold. Breeding categories: “P1” = can be bred in the lab from egg to adult as a stable colony over several generations; “P2” = egg laying of adult females possible in lab, larvae can be reared on host plant leaves or artificial diet; see methods for details. Red list classifications: “EN” = endangered, “VU” = vulnerable”, “NT” = near threatened, “LC” = least concern (
Species | Family | Red List Germany | Breeding category |
---|---|---|---|
Arctia caja | Arctiidae | NT | P1 |
Diacrisia sannio | Arctiidae | LC | P1 to P2 |
Diaphora mendica | Arctiidae | LC | P1 to P2 |
Euplagia quadripunctaria | Arctiidae | LC | P1 to P2 |
Parasemia plantaginis | Arctiidae | NT | P1 |
Ematurga atomaria | Geometridae | LC | P1 to P2 |
Peribatodes rhomboidaria | Geometridae | LC | P1 to P2 |
Scopula immutata | Geometridae | LC | potentially P1/P2 |
Pyrgus malvae | Hesperiidae | NT | P1 |
Lycaena tityrus | Lycaenidae | LC | P1 |
Lycaena virgaureae | Lycaenidae | NT | P1 to P2 |
Aglais io | Nymphalidae | LC | P1 |
Aglais urticae | Nymphalidae | LC | P1 |
Aphantopus hyperantus | Nymphalidae | LC | P1 |
Argynnis adippe | Nymphalidae | VU | P1 |
Argynnis aglaja | Nymphalidae | NT | P1 |
Boloria selene | Nymphalidae | NT | P1 |
Coenonympha pamphilus | Nymphalidae | LC | P1 |
Erebia medusa | Nymphalidae | NT | P1 to P2 |
Euphydryas aurinia | Nymphalidae | EN | P1 |
Hipparchia semele | Nymphalidae | VU | P1 |
Maniola jurtina | Nymphalidae | LC | P1 |
Melitaea athalia | Nymphalidae | VU | P1 to P2 |
Minois dryas | Nymphalidae | EN | P1 to P2 |
Anthocharis cardamines | Pieridae | LC | P1 |
Pieris brassicae | Pieridae | LC | P1 |
Pieris napi | Pieridae | LC | P1 |
Pieris rapae | Pieridae | LC | P1 |
Hamearis lucina | Riodinidae | VU | P1 to P2 |
Deilephila elpenor | Sphingidae | LC | P1 |
Hyles galii | Sphingidae | LC | P1 to P2 |
Plutella xylostella | Plutellidae | LC | P1 |
The literature search for breeding and rearing European Lepidoptera resulted in a list of 548 publications including a number of handbooks and reviews, which are all compiled and listed in Suppl. material
In the laboratory, Lepidoptera are often reared in the traditional way, i.e. on natural host plants under room conditions without control of the microclimate. Keeping larvae on their natural host plants is also the recommended approach for testing Bt maize effects (
Factor | Larvae | Adults |
---|---|---|
Population origin | Is it possible to obtain caterpillars or other immatures from a laboratory population? | How can the adults be obtained (light trapping, netting…)? |
How can immatures be obtained from the field? | ||
Food | Which plants and plant parts serve as food? | Do adults require feeding in order to mate/oviposit? |
Is the food permanently available? | Is forced feeding possible or necessary? | |
What particular requirements do the food plants need to meet (water, nutrition)? | What type of food do adults require? | |
Is food permanently available (i.e. flowers)? | ||
Can caterpillars be reared on artificial diets (see below)? | Which plants or substrates are needed for oviposition? | |
Climatic conditions | What is the temperature range for an optimal development? | What are conditions for adults to mate and oviposit (photoperiod, sunlight, temperature)? |
What is the relative humidity needed for development and to minimise disease incidence? | ||
Which day/night cycle is needed? | ||
Which conditions should be avoided to prevent dormancy or diapause? | ||
Rearing containers | What are the required dimensions or other characteristics of the rearing containers? | What requirements do adults need for eclosion? |
What dimensions are necessary in order to achieve mating? | ||
Population density | How many caterpillars can be reared together (competition, infections)? | How many adults (female:male ratios) should be placed together to achieve mating? |
Life cycle | What are the development times for the different life stages and at what temperatures? | Do adults show dormancy or diapause and what are the triggers? |
Handling | Is there any special consideration regarding handling (i.e. larval stress, damaging moulting stages)? | How often will it be possible to obtain large numbers of eggs? |
How many larvae can be reared at a time by the workforce (number of working hours available)? | ||
Disease/predator control | What are the most frequent diseases and how can they be prevented or reduced, can eggs or pupae be disinfected and how? Is it necessary to wash/disinfect the host plant? | Is it necessary to take any particular measures for adult feeding or egg laying, e.g. washing or disinfecting wildflowers or oviposition substrate? |
Others | Are there special requirements for the larvae to pupate (e.g. substrate)? | Is hand-pairing necessary and feasible? |
Are there any particular other aspects of the species that need to be considered (e.g. myrmecophily)? | Is it possible to preserve adults, pupae or eggs in the refrigerator, for how long? |
For testing Bt maize pollen effects,
Several publications exist that give an overview of different artificial diets and/or list successfully reared species, e.g.
Depending on the natural feeding habits, artificial diets must be presented in different ways to mimic natural conditions (
Here we present a list of potential, non-target Lepidoptera species for assessing the effects of Bt maize pollen on lepidopteran larvae in the laboratory, and develop a systematic selection process allowing to identify the suitable test species in a transparent manner. A number of criteria have been proposed for selecting adequate non-target species of various taxonomic groups to assess the potential environmental risks of cultivating GM plants (e.g.,
Importantly, a crucial selection criterion was step 5, the possibility to breed the species in the laboratory, which is of relevance when keeping specimens for testing purposes and establishing lab cultures of test organisms (
In contrast to the initial systematic selection criteria (steps 1–5), the last selection steps 6 and 7 were done according to expert assessment, as was proposed by other published selection protocols (
In our study we used LEPIDAT, a database compiled by the BfN (
It has to be noted that our selection procedure resulted in the general identification of representative species suitable for laboratory experiments and does not aim to recognise a specific hazard to certain species in advance. In fact, the potential hazard has to be studied in the subsequent lab trials, which is also why the still mostly unknown susceptibility of single lepidopteran species to Bt maize pollen was not a selection criterion for choosing the test species in our study. Any selection procedure for test species must identify and take into account the species of the local, corresponding environment, i.e. the species that would be exposed in the area where the Bt maize is to be grown. If applying our selection process to regions in Europe other than Germany, the conditions determining the magnitude of the exposure of certain lepidopteran species to Bt maize will differ, and the selection process should be adapted accordingly, e.g. with respect to the altitude ranges of maize cultivation or times of maize pollen shedding.
In view of the thousands of lepidopteran species in Europe it is indispensable to focus on only a limited range of species for the assessment of adverse effects of Bt maize on lepidopteran larvae. On the other hand, it is also paramount to consider a sufficient number of species to assure a reasonable representativeness of the tested species group. Our stepwise selection procedure provides a systematic, transparent and generic approach to create a representative list of NT Lepidoptera for Bt maize testing. This is a major achievement as a standardised protocol on how to select the relevant indicator species did not exist up to now. The selection process is generic in the sense that it can be simply adapted to other locations as well as to specific requirements and objectives. In our case, the resulting species list is highly representative and exceeds the so far limited range of studied test species for the risk assessment of Bt maize in Central Europe. Breeding feasibility is of crucial importance for maintaining laboratory cultures of test species, on which there is a large body of literature. For all species-rich Lepidoptera families many reports on breeding for several species exist; this often includes rearing on an artificial diet. In other words, laboratory testing of NT Lepidoptera species appears feasible for a very broad taxonomic range, which is summarised here for the first time. Thus, any risk assessment involving the breeding of butterfly and moth larvae will benefit from the information presented. In particular, NT testing of Lepidoptera in Europe for the assessment of Bt maize will greatly profit from following the reported approach, information and results.
We are grateful to Ingo Heide for his great support with the LEPIDAT database. The study was supported by the German Federal Agency for Nature Conservation, BfN, Bonn (FKZ 3515890100) with funds from the Federal Ministry for the Environment, Nature Conservation and Nuclear Safety (BMU).
Reference list: breeding and rearing Lepidoptera
Data type: Literature list (Excel table)
Explanation note: List of publications reporting on breeding Lepidoptera with short description of study contents.
Review breeding information
Data type: Breeding info (pdf)
Explanation note: Short review of how to breed Lepidoptera of various families in the laboratory.