another view on bt proteins – how specific are they and...

* Corresponding author: E-mail: [email protected]

Another View on Bt Proteins – How Specific are They and WhatElse Might They Do?

A. HILBECK* AND J.E.U. SCHMIDTInstitute of Integrative Biology, ETH Zurich, Switzerland

Biopestic. Int. 2 (1): 1-50 (2006)

ABSTRACT The entomopathogenic bacterium Bacillus thuringiensis (Bt) and its toxins areextensively used for pest control purposes in agriculture, forestry and public health programmessince the 1930. In addition to spray formulations, transgenic plants containing Bt genes forthe expression of the toxins (Bt plants) are commercially available since the mid 1990s and aregrown on an increasing percentage of the global agricultural area. A main reason for the importanceof Bt as a pesticide is the assumed environmental safety concluded from the high specificityof its endotoxins (Cry proteins) towards a limited number of target organisms, mostly distinctgroups of pest insects. While the mode of action of the Cry toxins in these susceptible targetinsects is well studied, Bt experts claim that several details are still not understood well enough.Although there is considerable experience with the application and the environmental safetyof Bt sprays, a number of research papers were published in the past that did report adverseeffects on non-target organisms. These and the widespread use of transgenic Bt plants stimulatedus to review the published laboratory feeding studies on effects of Bt toxins and transgenicBt plants on non-target invertebrates. We describe those reports that documented adverseeffects in non-target organisms in more detail and focus on one prominent example, the greenlacewing, Chrysoperla carnea. Discussing our findings in the context of current molecularstudies, we argue firstly that the evidence for adverse effects in non-target organisms is compellingenough that it would merit more research. We further conclude from our in-depth analysis thatthe published reports studying the effects of Bt toxins from Bt pesticides and transgenic Btplants on green lacewing larvae provide complementary and not contradictory data. And, finally,we find that the key experiments explaining the mode of action not only in this particular affectednon-target species but also in most other affected non-target species are still missing. Consideringthe steadily increasing global production area of Bt crops, it seems prudent to thoroughlyunderstand how Bt toxins might affect non-target organisms.

KEY WORDS : Bacillus thuringiensis, Cry proteins, mode of action, specificity, transgenic Btplants, unexpected effects

INTRODUCTION

Bacillus thuringiensis – A Microbial InsecticideBacillus thuringiensis Berliner, commonly

abbreviated as Bt, is a gram-positive, facultative

aerobic, rod-like and motile bacterium, which hasgained outstanding significance as a microbialpesticide throughout the 20th century (Entwistle etal., 1993; de Maagd et al., 2003). While about 100bacteria were identified as exo- and endopathogens

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2 Biopesticides International Vol. 2, no. 1

of arthropods (Thacker, 2002), only a few are used inpest management (e.g. Bacillus popilliae, Bacillussphaericus, Serratia entomophila) and only one,namely Bt, has achieved significant attention andcommercial success (Rodgers, 1993; Brar et al., 2006).Bt has been reported to occur in samples fromvarious and mostly insect-rich environments, suchas grain stores and stored products, differentcomposts and soils, the phylloplane of differentplants, insect cadavers, and faeces of herbivorousvertebrates, but also from aquatic environments(Bernhard et al., 1997; Martínez and Caballero, 2002).Because of its apparent opportunity to colonise suchdiverse habitats, Bt is often referred to as ubiquitousand globally distributed (de Maagd et al., 2001).Within the Bacillaceae family, close genetic relationsbetween Bt and the mammal pathogen Bacillusanthracis, and especially to Bacillus cereus, abacterium that causes food poisoning, are considered(Priest, 2000; de Maagd et al., 2003).

The growth cycle of Bt consists of a vegetativeand a stationary phase (Lambert and Peferoen,1992). Cells can grow in a vegetative mode as longas nutrients are sufficiently available, but formendospores within sporangia under unfavourableenvironmental conditions. The spores are able tosurvive until the conditions have improved andvegetative reproduction becomes feasible again.Coinciding with the sporulation process, Bt producescrystalline parasporal inclusion bodies that consistof a large amount of one or more proteins of thecrystal (Cry) or the cytotoxic (Cty) type (Crickmoreet al., 1998; de Maagd et al., 2003). Apart fromthese proteins, which have attracted most attentionfor their insect toxicity, the chemical arsenal of Btstrains is much broader and includes diversesubstances with different characteristics, specificitiesand modes of action (Lereclus et al., 1993; Schnepfet al., 1998; de Maagd et al., 2003). To date, 335different Cry d-endotoxins have been described(Crickmore et al., 2005), most of which share astructure consisting of three globular domains andhave a size of either ca. 130 kDa or 70 kDa (Deanet al., 1996; Schnepf et al., 1998; de Maagd et al.,2003). The three domains have distinct roles aspart of the commonly accepted mode of action intarget pest species (see below), but details of theirfunctions are still being investigated.

Despite considerable research and literatureabout Bt, the evolutionary role of the toxins stillremains subject of discussion (de Maagd et al.,2001). While dead insects, which were killed by thetoxins, provide a suitable nutrient source for thespores to germinate and continue vegetative growth,a possible role of the toxins in interactions betweenmicroorganisms in soil or in dead organic matter wasalso proposed (Addison, 1993).

Brief History of the Use of Bt Spray For-mulations

Bt was first isolateded in 1901 by bacteriologistS. Ishiwata as “Bacillus sotto” after being recognizedas the cause of the sudden-collapse disease (“sotto”)in larvae of the silkworm moth Bombyx mori (L.)(Lepidoptera: Bombycidae) (Beegle and Yamamoto,1992). At that time, it was considered a threat toJapan’s silk industry and its potential as a microbialpest control agent was not yet realized (Glare andO’Callaghan, 2000). Only in 1915, Bt was scientificallydescribed and given its valid name by Germanscientist Ernst Berliner, who had discovered it in 1911in dead flour moth caterpillars killed by the“Schlaffsucht” disease. As it had now been foundin a pest insect, its insect pathogenic characteristicsmade the bacterium attractive for use as a pesticide.Efforts to develop methods for its culture andapplication against flour moths started soon afterBerliner’s publication. Later, trials were conductedto explore the suitability of Bt as a microbialinsecticide against the European corn borer, Ostrinianubilalis (Glare and O’Callaghan, 2000). In 1938, thefirst commercial formulations of Bt consisting mainlyof sporulated cells, were available under the productname “Sporeine” in France (Lambert and Peferoen,1992). However, the mode of action in target species(see below) was not described before 1956 (Crookand Jarrett, 1991), when Bt attracted more and moreinterest because of the increasing environmentalproblems with synthetic insecticides. For many years,Bt, namely the potent subspecies B. thuringiensiskurstaki (Btk), was only used to control Lepidoptera.Strains of Btk still form the basis for many sprayformulations and had an important role in thecreation of transgenic Bt plants. Screening programs,however, have identified many other subspecies and

2006 Hilbeck and Schmidt : Another View on Bt Proteins 3

strains of Bt, with aizawai, israelensis, tenebrionisbeing the most important ones (Crickmore et al.,1998; de Maagd et al., 2003), and additional possi-bilities for its use were discussed (Feitelson et al.,1992). In particular, the isolation of coleopteran anddipteran-active strains was important in thesubsequent development of control strategies againstbeetle pest species in agriculture and against dipterandisease vectors in public health programmes (Kellerand Langenbruch, 1993; Becker and Margalit, 1993).

In all Bt strains, the genes that produce theproteinaceous crystal are located on plasmids. Btproteins were originally classified into one of fourclasses known as CryI, CryII, CryIII and CryIVaccording to their insecticidal activities (Höfte andWhiteley 1989): CryI and CryII proteins are activeagainst lepidopteran and/or dipteran species, CryIIIare active against Coleoptera and CryIV are activeagainst Diptera. Crickmore et al. (1998) introduced anew nomenclature based on relationships in theamino acid sequences of the toxins. However, thespectrum of Cry proteins and their assumed specifictoxicity is much more diverse, including otherarthropods, nematodes, flatworms and protozoa(Feitelson et al., 1992). Most Bt strains are able toproduce several different crystal proteins and thesame protein can be found in different strains orsubspecies (Koziel et al., 1993; Schnepf et al., 1998).The diversity of Bt toxins is used for controllingimportant pests in agriculture and forestry (mainlyherbivorous larvae of moth and beetle species) anddisease vectors (mosquitoes). However, short-comings of the use of Bt are low persistence of thetoxins under UV light and difficulties to controlcertain important pests like stem borers such as theEuropean corn borer, Ostrinia nubilalis (Hübner)(Lepidoptera: Pyralidae), in maize in Europe and NorthAmerica, but also other species in tropic regions(Rice and Pilcher, 1998; Hilbeck, 2002).

TRANSGENIC Bt PLANTSThe interactions between insects and plants

have long been recognized as complex and veryimportant for ecosystems (Schoonhoven et al., 1998).To protect themselves against the damage ofherbivorous arthropods, plants have developed aplethora of defence strategies ranging from

mechanical to chemical. Various substances that areproduced by secondary metabolic pathways are toxicto insects, but their biosynthesis is often poorlyunderstood or too complex to be used for geneticengineering of insect-resistant plants. Severalstrategies have been followed to generate resistancein crop plants involving conventional plant breeding,and, more recently, in vitro techniques (e.g.electrofusion of protoplasts) and genetic engineeringto create plants that express insecticidal orentomopathogenic proteins (Jouanin et al., 1998). Ingenetic engineering genes from other species areisolated and transferred into the plant genome. Thisinvolves the use of the gall-forming bacteriumAgrobacterium tumefaciens as vector for geneticinformation or the application of ballistic methodslike so-called “gene-guns”.

Two main approaches to create plantsexpressing insecticidal proteins were followed(Jouanin et al., 1998), both targeting the digestivesystem of insect pests (Schuler et al., 1998). Oneapproach is based on plant-derived genes, e.g. thegenetic information that code for enzymes likeproteinase inhibitors, amylase inhibitors, cholesteroloxidase or lectines (e.g. the snowdrop lectin (GNA)of Galanthus nivalis L.; Amaryllidaceae). The otherapproach uses genes of B. thuringiensis that codefor their insect-toxic proteins – mostly the cry-genes(Schuler et al., 1998). Only this approach has led tothe commercially available transgenic insecticidalcrop plants that are currently cultivated. The mostcommon Bt toxins expressed by these plants includethe lepidopteran-active Cry1Ab in maize (Zea maysL., Poaceae), the lepidopteran-active Cry1Ac in maizeand cotton (Gossypium spp., Malvaceae) and thecoleopteran-active Cry3Bb in maize (Andow andHilbeck, 2004). More and more transgenic crop plantscarry now two or more different Bt transgenescombined in their genomes. In addition to thecommercially available Bt crop plants, various Bttoxins are expressed in transgenic varieties of an arrayof other crop plant species from several families thatare not yet approved (Hilbeck, 2001; e.g. Cry3Bb ineggplants, Solanum melongena L., Solanaceae,Arpaia et al., 1997). Since the first commercialreleases 10 years ago, the area of agricultural landplanted with transgenic insect resistant plants for


pest control has increased considerably (James,2005).

One reason for the interest in transgenic Btplants is that they are often assumed to be harmlessto beneficial insects, including predators andparasitoids, and to other non-target species basedon the commonly accepted mode of action knownfrom bacterial Bt toxins (Shelton et al., 2002). Otherbenefits claimed for transgenic plants are: less effortrequired for monitoring of target pests (Obrycki etal., 2001), reduced applications of broad-spectruminsecticides and increased or more secure yields dueto season-long control of important pest species.Transgenic Bt plants are different from conventionalBt spray formulations. First, while Bt toxins can onlypersist for a short time on the surface of plants afterspray applications, transgenic Bt plants express theBt toxins throughout their entire lifetime. Bt sprayformulations contain bacterial cells, spores andinactive protoxins which must be activated in acomplex biochemical process as described below,during which the molecular weight of the proteins isreduced, e.g. from 130-140 kDa to 60-65 kDa for theCry1 toxins. The structure of some activated proteinshave been described (de Maagd et al., 2003). Incontrast, transgenic plants express Bt toxins in a moreactivated form of differing molecular weights (69 kDain the toxin Cry1Ab) (Hilbeck, 2001).

Effects of Bt plants on non-target speciesbecame a major concern after some publicationsreporting adverse effects on organisms outside ofthe known range of target insects (Table 3). Severalreviews were published recently where the authorsevaluated often the same studies but arrived atdifferent conclusions (Lövei and Arpaia, 2005;O’Callaghan et al., 2005, Romeis et al., 2006).

In this paper, we also review recent peer-reviewed literature, in which effects of Bt toxinsand transgenic Bt plants on non-target organismswere investigated. However, we focus on thosestudies, that reported statistically significantdifferences between a Bt treatment and thecorresponding control. While we do acknowledgethat there are several studies, concerned withpossible implications of Bt toxins on vertebrates, inthis review paper, we address invertebrates only asthey fall in our area expertise.

MODE OF ACTION OF Bt CRY TOXINSIN INVERTEBRATESCurrent Understanding in Target Pest Insects

According to the commonly acceptedunderstanding of its mode of action, the insecticidalactivity of Bt is triggered when spores and toxincrystals are ingested (Höfte and Whitley, 1989; Gillet al., 1992; Knowles and Dow, 1993; Knowles, 1994;Schnepf et al., 1998; Whalon and Wingerd, 2003).To solubilise the toxin crystal, pH conditions in themidgut must be suitable, whereas differences insolubility are known for the different toxin families.Cry1 and Cry2 proteins need a higher pH, which isrealised in the more alkaline gut milieus of their targetinsect groups, Lepidoptera and Diptera, whilecoleopteran active Cry3 proteins are solubilized at apH closer to neutrality, reflecting the typical gutconditions in their target species. Protease enzymesof the insect gut digest a portion of the solubilizedprotoxin by removing amino acid sequences from itsC- and N-terminal ends and release a proteaseresistant polypeptide, the so called d-endotoxin,which represents the biologically active fragment.Through this process, the mass of the originalprotoxin (ca. 130-140 kDa) is reduced to ca. 60-65kDa in the active toxin. The C-terminal domain of thebiologically active toxin binds to specific receptorson the membranes of brush border epithelium cellsof the target insect’s midgut followed by the insertionof the hydrophobic region of the toxin molecule intothe cell membrane. This induces a change in themembrane permeability and the osmotic balance, theformation of transmembrane pores and, subsequently,cell lysis in the gut wall, which allows gut contentsto leak into the haemocoel (Dean et al., 1996).

The infested target insect then dies fromstarvation and lethal septicaemia and, if bacterialspores are present, the abundance of nutrientsstimulates germination and the beginning ofvegetative bacterial growth. The most frequentlyexpressed advantage of using Bt for pest control isits presumed specificity to certain insect species, theso-called target species, while all other organisms(referred to as “non-target species”) are not affected.This specificity in activity is assumed to be causedby the existence of the toxin specific receptors inthe brush border epithelium and the pH content in


the gut (Höfte and Whiteley, 1989). Different targetspecies exhibit different numbers of binding sitesand the toxin affinity does not appear to be constantfor all insects (van Rie et al., 1989)

‘Specificity’ and ‘Susceptibility’The known mode of action of Bt toxins stems

largely from investigations with economicallyimportant target pest species known to besusceptible at least to a significant degree in thecontext of crop production or forestry, such aslepidopteran and coleopteran pest species (e.g.Heliothis virescens, Ostrinia nubilalis, Lymantriadispar, Leptinotarsa decemlineata). Out of thistradition, terms like ‘susceptibility’ and ‘selectivity’were defined for an economic, not an ecologicalcontext. The ‘economic’ definition only characterizes‘susceptible’ species that can be killed quickly withfew or one application measure only or – with regardto transgenic Bt plants – when taking a few bites ofthe Bt tissue. This requires an acute, lethal effect ofa sufficiently high dose of Bt toxin. An ‘ecological’definition of ‚susceptibility’ or ‚selectivity’ would alsoinclude species that exhibit long-term, sublethal andlethal effects. With the persistent and constitutiveexpression of activated Bt-toxins in transgenic Btplants, ecological long-term effects became muchmore relevant for environmental risk assessment thanwith short-lived, inactive Bt-protoxins in microbialsprays. Chronic, sublethal effects can also causesevere adverse effects, even in a pest managementcontext of crops. For instance, if the sublethal effectof prolonged development in a parasitoid or predatorspecies causes temporal disruption in an importantnatural enemy – prey/host relationship, this can leadto serious consequences, possibly even more seriousthan a certain low-level lethal short-term effect.Research on modes of action in non-target insects,however, is an important gap of knowledge whenconsidering the large areas planted to transgenic Btcrops and their persistent presence in the above-and below-ground agroecosystems.

Indications of Additional or Alternative In-ter-actions

While the mode of action of natural and

transgene produced Bt toxins is well documentedin target pest species, authors of recent studies havepointed out that many of its details are still notthoroughly understood and that the interactionsbetween Bt toxins and invertebrates may be morecomplex than thought before (see below).Summarizing recent molecular studies on Bt toxinsand invertebrates conducted with nematode-activeBt toxins and the nematode Caenorhabditis elegansand with model Lepidoptera species, Crickmore (2005)indicates that modern approaches have revealednovel receptors and possible signal transductionpathways induced within the host followingintoxication. Most notably, the author emphasisesthat (i) Bt toxin activity can be modulated by alteredactivation, referring to studies with midgut proteases;(ii) Bt toxin specificity is determined not only by theability to bind to appropriate receptor molecules, butalso by the ability to subsequently oligomerize andinsert into the membrane; (iii) subtle differences intoxin structure could affect binding and that thesedifferences could account for host specificity and(iv) sequestration of the peritrophic membrane couldalso apply for Bt toxins.

Very few studies have addressed the fate ofingested Bt toxin in non-target invertebrates. Brandtet al. (2004) conducted biochemical and immuno-cytochemical experiments with the bug Lygushesperus, which is not susceptible to the Bt toxinsCry1Ac and Cry2Ab. When both toxins were fed tospecimens in their activated form, proteolyticprocessing of the toxin within the digestive systemof L. hesperus was observed, but excreted toxinsretained their lepidopteran-active characteristics. Onthe other hand, Cry1Ac did not associate with L.hesperus tissues, while Cry2Ab did. The authorsconclude, that binding alone is not sufficient fortoxicity. Similar studies with invertebrate species,which exhibited non-target effects, would be ofespecially high value for understanding theseobserved effects. One crucial aspect in this contextare possible structural changes of the ingested toxinsin non-target species, which might, even if they areminor, change their binding affinity to membranemolecules (Crickmore, 2005) or other characteristics.

On the other hand, studies on possibleadditional or alternative effects of Bt toxins on target


species are also rare. Cerstiaens et al. (2001) injecteddifferent activated Bt toxins into the haemocoel ofLymantria dispar and Neobelleria bullata and foundlethal or sublethal effects depending on the toxinapplied. The authors indicate that the mode of actionwhich is responsible for these results, must bedifferent from the one occurring after ingestion. In-vitro toxicity of the Bt toxin Cry1C for L. disparneuronal cells was demonstrated in this study and aconnection with the observed effects is discussed.While such additional effects of Bt toxins might bemasked by the immediate death of target species,they are important for a thorough understanding ofthe interactions between Bt toxins and invertebrates.

ADVERSE EFFECTS OF Bt TOXINS ANDTRANSGENIC BT PLANTS ON NON-TARGETINVERTEBRATES

Published Studies on Non-target Effects ofBt Toxins in Biopesticides

Early feeding studies of invertebrates with Bttoxin conducted throughout the 1950 were mainlyconcerned with finding new susceptible pest speciesfor a limited number of Bt strains (mostlylepidopteran-active). The first laboratory trials thataimed to assess the effects of non-targetinvertebrates started in the 1960s and involvedbeneficial species of economic significance like honeybees and earthworms (e.g. Smirnoff and Heimpel,1961; Wilson, 1962). A tritrophic feeding experimentwith Bt toxins under laboratory conditions was firstperformed by Yousten (1973) using the preyingmantids Tenodera sinensis fed with B. thuringiensissubsp. kurstaki (Btk) fed cabbage looper,Trichoplusia ni, larvae. Moreover, first field studieswere conducted to assess impacts on non-targetarthropod (mainly lepidopteran) communities of areastreated with Bt sprays (Jaques, 1965).

Considerable laboratory testing of non-targeteffects was conducted with commercial sprayformulations based on Btk which produces toxinsof the Cry1 family (Krieg and Langenbruch, 1981;MacIntosh et al., 1990), including commercialproducts and solutions that contained varyingcombinations of spores, crystals and toxins of

different Bt subspecies. The resulting reports andpublications were subsequently reviewed by severalauthors (e.g. Flexner et al., 1986; Melin and Cozzi,1990; Navon, 1993; Glare and O’Callaghan, 2000).Glare and O’Callaghan (2000) list more than 300species of Lepidoptera and other invertebrates towhich Btk was toxic. In general, the reported effectsare contradictory: some studies documented a lackof effects while others found lethal or sublethaleffects on various non-target invertebrates, includingpredators and, most notably, parasitoids. Sublethaleffects were observed in context with different fitnessparameters, e.g. fecundity, parasitation rate, hostconsumption, adult longevity, development time andsex ratios (Flexner et al., 1986). While many studieswere published in the form of reports or in journalsthat are difficult to access (see Flexner et al., 1986and Glare and O’Callaghan, 2000 for a review), welist some of the species, for which adverse effects ofmicrobial Bt proteins were documented (Table 1).In some of these studies, an observed toxicity wasattributed to the presence of β-exotoxins, but onlyKrieg and co-workers conducted more systematicstudies in this context for adult honey bees (Kriegand Herfs, 1963; Krieg and Kulikov 1965; Krieg,1963). Most of the older studies investigatingimpacts of Bt endotoxins on non-target speciesconcentrated on lethal effects, which were reportedonly in a few cases. Also sublethal effects werereported but, to a much lesser extent (Glare andO’Callaghan, 2000).

Published Studies on Non-target Effects ofBt Toxins Expressed in Transgenic Plants

It was not until the mid and late 90s that effectsof Bt toxins from transgenic plants on non-targetorganisms outside of the taxonomic order of thetarget pests were investigated in a meaningful way.At that time, based on the experience with microbialBt sprays, the common assumption was that the Bttoxins expressed by transgenic Bt plants would notaffect any organisms outside of the order of the targetpest species, i.e., lepidoptera and herbivorouscoleopteran (Sims, 1995). The publication of adverseeffects of Bt toxins in predaceous green lacewinglarvae (Chrysoperla carnea; Neuroptera: Chryso-pidae) (Hilbeck et al., 1998a,b and 1999) and larvae


Table 1. Summary of feeding studies with non-target invertebrate species that reported significantdifferences between a microbial Bt treatment and the corresponding control.

Herbivores:

Hymenoptera

Honey bee, Apismellifera L.(Apidae)

Diptera

Fruit fly,Drosophilamelanogaster(Meigen)(Drosophilidae)

Chironomussrassicaudatus,Chironomusdecorus, Glypto-tendipes paripes,Tanytaurus sp.(Chironomidae)

Predators:

Coleoptera

11-spot ladybird,CoccinellaundecimpunctataL. (Coccinellidae)

Bt subspecieskurstaki

Bt subspeciesthuringiensis





Bt subspeciesisraelensis

Bt subspeciesentomocidus

Increasedmortality

Increasedmortality

Total mortality

Total (supernatant)or increased(spores) mortality

Increasedmortality

Increasedmortality

Increasedmortality

Prolonged larvaldevelopment,reduced preyconsumption

Non-sporulatedcultures fed toadults

Toxin in sugarsolutions fed toadults

Diets containingthe spore-crystal-exotoxin complexor the spore-crystalcomplex fed toadult workers

Sugar solutionscontaining spores orculture supernatantwith β-exotoxins fedto adults

Non-sporulatedbroth cultures fedto adults

Toxin in substratefed to neonatelarvae

Toxin sprayedaphids fed tonewly hatchedlarvae

Presence ofβ-toxins1

Presence ofβ-toxins

Presence ofexotoxins

Presence ofexotoxins3

Presence ofα-toxins4

Krieg (1973)

Krieg and Herfs(1963)2

Martouret andEuverte (1964)

Cantwell et al.(1966)

Krieg (1973)

Krieg (1965)5

Ali (1981), Ali etal. (1981)

Salama et al.(1982)

Species Bt subspecies/product

Observed effect Experimental setup

Explanation forreported effects(if provided bythe authors)

Reference


Neuroptera

Green lacewing,Chrysoperla(Chrysopa)carnea Stephens(Chrysopidae)

Parasitoids:

Hymenoptera

Cardiochilesnigriceps Viereck(Braconidae)

Cotesia(Apanteles)glomarata L.(Braconidae)

Cotesiamelanoscelus(Ratzeburg)(Braconidae)

Cotesia rubecula(Marshall)(Braconidae)

Hyposoterexiguae (Viereck)(Ichneumonidae)

MicroplitisdemolitorWilkinison(Braconidae)

Pimpla turionella L.(Ichneumonidae)

Rogas lymantriaeWatanabe(Braconidae)


Bt subspecieskurstaki(commercial)

Bt subspecieskurstaki (Dipel)

Bt subspecieskurstaki(commercial)

Bt subspecieskurstaki (strainHD-1)




Bt subspecieskurstaki

Prolonged larvaldevelopment,reduced preyconsumption.

Shorter life spans

Increasedmortality aftertwo weeks

Increasedparasitisation(synergism);prolongeddevelopment.

Increasedmortality

Increasedmortality

Reducedreproductionpotential

Midgut epitheliumdamage (but noobserved effect onthe adults)

Sex ratio skewedtowards females

Toxin sprayedaphids or toxintreated caterpillars(Spodopteralittoralis) fed tonewly hatchedlarvae

Toxin suspensionfed to fieldcollected adults

Toxin fed toadults

Susceptible (targetspecies) host(Lymantria dispar)fed with toxincontaining diet

Susceptible (targetspecies) host(Pieris rapae) fedwith toxincontaining diet

Toxin suspensionfed to adults

Host (Spodopteralittoralis) fedwith toxincontaining diet

Toxin fed toadults

Susceptible (targetspecies) host(Lymantria dispar)fed with toxincontaining diet

Salama et al.(1982)

Dunbar andJohnson (1975)

Mück et al. (1981)

Weseloh andAndreadis (1982)

McDonald et al.(1990)

Thomas andWatson (1986)

Salama et al.(1982)

Mück et al.(1981)

Wallner et al.(1983)

Starvation?6

Slower growth andlonger persistenceat sizes suitable forparasitisation

Host sufferingfrom acuteintoxication

Spore-crystalcomplexresponsible

Result of the ICP

Females lay morefertilized eggs inlarger, untreatedlarvae.




Reference


of the Monarch butterfly (Losey et al., 1999)surprised many scientists for different reasons andgave this field more momentum. Non-target effectsof transgenic Bt plants finally made it on the agendaof mainstream research. Since then, research on sucheffects increased significantly until today. In thischapter, we will provide an overview of these studiesconducted with Bt toxins and transgenic Bt plantssince 1995 and discuss those reporting adverseeffects on non-target organisms in more detail.

Table 2 lists a total of 60 non-target invertebratespecies and two invertebrate groups of highertaxonomic order that were tested with regard totransgenic Bt plants and microbially produced Bttoxins in international peer-reviewed scientificjournals. Twenty-four herbivore species from sixinsect orders and two herbivorous mites as well as25 natural enemy species from four insect orders,three predatory mites and one spider were tested.Of these 25 natural enemy species, nine werehymenopteran primary parasitoids, one was a

hymenopteran hyperparasitoid and the remainingwere predators. Additionally, four detrivorous soilorganisms (Lumbricus terrestris, Folsomia candida,Porcellio scaber and Oppia nitens), unspecifiednematodes and protozoa, the detritivorous cockroachBlattella germanica and, as a single aquaticorganism, plankton-feeding larvae of Aedes aegyptiwere investigated. For the vast majority of theseorganisms only one publication was found, manyonly involving one experiment. Only five herbivorespecies, Spodoptera littoralis (5), Apis mellifera (4),monarch butterfly (4), spider mites (3),Rhopalosiphum padi (3) and 2 predators,Chrysoperla carnea (9) and Coleomegilla maculata(4), were subjected to experimentation in more thanone publication found (Table 2). The reasons for thisare arbitrary and based on professional preferencerather than ecological necessity and justification.Further, only very few of the studied species arerelevant for subtropical or tropical agroecosystems(e.g. Cyrtorhinus lividipennis and Parallorhogas

TrichogrammacacoeciaeMarchal (Tricho-grammatidae)

Zelechlorophthalma(Nees)(Braconidae)




Reducedparasitisationcapacity

Reducedparasitisationrate, reducedemergence, reducedreproductivepotential,retardation indevelopment.

Reducedreproductionpotential

Toxin suspensionfed to adults



Hassan & Krieg(1975)

Salama and Zaki(1983)

Salama and Zaki(1983)

Presenceof β-exotoxins

1 Sporulated cultures did not show a similar effect (Krieg and Herfs, 1963).2 High doses of the spore-endotoxin complex are toxic.3 Crystals were not harmful.4 Sporulated cultures did not show a similar effect (Krieg et al., 1980).5 No toxicity of the spore-endotoxin complex also at high doses.6 Not sure if ingestion took place.




Reference


Table 2. List of invertebrates from different trophic levels tested for non-target effects of transgenic Btplants (ticks represents the individual number of studies with the respective species published in international,peer-reviewed journals).

Trophic level (feeding type)Taxon/Species Herbivores Predators, Other feeding type

parasitoids

PROTOZOA: √ (unclear)NEMATODA: √ (unclear)ANNELIDAE:Oligochaeta Lumbricus terrestris √,√ (detritivore)ARTHROPODA – CRUSTACEA:Isopoda Porcellio scaber √,√ (detritivore)ARTHROPODA – CHELICERATA:Araneae Araneus diadematus √Acari Neoseiulus cucumeris √ Oppia nitens √ (detritivore) Phytoseiulus persimilis √ Rhizoglyphus robini √ Tetranychus urticae √,√,√ARTHROPODA – INSECTA :Collembola Folsomia candida √ (detritivore)Blattodea Blattella germanica √ (detritivore)Thysanoptera Frankliniella tenuicornis √Heteroptera Cyrtorhinus lividipennis √ Geocoris pallens √ Geocoris punctipes √,√ Lygus hesperus √ Nabis sp. √,√ Orius insidiosus √,√ Orius majusculus √ Orius tristicolor √,√ Zelus renardii √Homoptera Aphis fabae √ Macrosiphum avenae √ Macrosiphum euphorbiae √ Myzus persicae √ Nilaparvata lugens √ Rhopalosiphum padi √,√,√Neuroptera Chrysoperla carnea √,√,√,√,√,√,√,√,√ Micromus tasmaniae √


Coleoptera Adalia bipunctata √ Anthonomus grandis √ Coleomegilla maculata √,√,√,√ Diabrotica undecimpunctata √ Hippodamia convergens √,√ Leptinotarsa decemlineata √,√ Poecilus cupreus √ Propylea japonica √Hymenoptera Apanteles subandinus √ Aphidius nigripes √ Apis mellifera √,√,√,√ Athalia rosae √ Copidosoma floridanum √ Cotesia flavipes √ Cotesia marginiventris √,√ Cotesia plutellae √ Microplitis mediator √ Nasonia vitripennis √ Parallorhogas pyralophagus √ Tetrastichus howardi √Lepidoptera Acherontia atropos √ Autographa gamma √ Danaus plexippus √,√,√,√ Galleria mellonella √ Manduca sexta √ Papilio polyxenes √ Pieris brassicae √ Pieris rapae √ Plutella xylostella √ Pseudoplusia includens √ Spodoptera littoralis √,√,√,√,√Diptera Aedes aegypti √ (plankton-feeder)TOTAL:

Trophic level (feeding type)Taxon/Species Herbivores Predators, Other feeding type

parasitoids

52 insect species from 10 or-ders;

8 species from other inverte-brate taxa;

2 invertebrate groups of highertaxonomic level.

24 insect spe-cies from 6 or-ders;

2 species fromother inverte-brate orders.

25 insect speciesfrom 4 orders;

3 species fromother inverte-brate orders.

3 insect species from 3orders;

3 species from otherinvertebrate orders;

2 invertebrate groups ofhigher taxonomic level.


pyralophagus), while the vast majority occurs innorthern, temperate agro-ecosystems. In mostpublications, transgenic plant parts were used fortesting, many used pollen from transgenic Bt maizeand a few used microbially produced activated Btproteins. Most of the studies focussed on lethaleffects (parameters measured: mortality or survival),while some also reported parameters likedevelopment time, weight gain and fertility.

In 27 (50%) of the reviewed 54 studies, theauthors reported negative effects on one or more ofthe tested parameters (Table 3); this also includesstudies that reported no effects on other parameters.Positive effects were rare (Escher et al., 2000; Demlet al., 1999). Zemková Rovenská et al. (2005)reported a preference of spider mites, Tetranychusurticae, for transgenic Bt eggplants. The observedeffects were often unpredictable in terms of degreeand type of impact. Only the fact that Bt maizepollen containing the lepidopteran-specific Cry1Abtoxin adversely affected the caterpillars of theMonarch butterfly and other Lepidoptera specieswas hardly surprising. However, the actual surprisewas the exposure route that had been overlookeduntil the publication by Losey et al. (1999). Sincethen, tests for Bt effects on Monarch butterflycaterpillars became a standard for regulatoryapproval in the USA (Oberhauser and Rivers, 2003).

Although there are many studies that reportedno effects of Bt toxins and transgenic Bt plants onnon-target arthropods in the current peer-reviewedliterature, several significant examples exist whereadverse effects on very different arthropod taxa weredocumented (Table 4a,b). Most studies wereconducted with non-target herbivores, mostprominently non-target Lepidoptera and transgenicBt plants expressing the lepidopteran toxin Cry1Ab(Table 4a). This is the most proximate approach,because of the close taxonomic relation betweenthe target and the non-target lepidopteran species.The most prominent study was that of Losey et al.(1999) reporting an increased mortality of Monarchbutterfly (Danaus plexippus) larvae after feedingon their host plant, the common milkweedAscelapias syriaca, dusted with pollen from Cry1Abexpressing maize plants. Toxicity of Bt pollen toMonarch caterpillars was also reported by Jesse and

Obrycki (2000) in an independent study. As theseresults, in particular, became subject to debatebetween proponents and opponents of the technologyand because of the cultural importance of theMonarch butterfly in North America, this systemwas extensively studied from different perspectives(Oberhauser and Rivers, 2003). Some of thesubsequent experiments confirmed the toxicity ofdifferent tissues from Bt transgenic maize flowers(anthers, pollen) for caterpillars (Hellmich et al.,2001; Anderson et al., 2004), but also highlighteda lower risk for caterpillars when exposed to pollenfrom Bt maize varieties with low expression of theBt toxin in pollen. However, effects of different Bttoxins were also found in other non-targetLepidoptera (Deml et al., 1999; Felke et al., 2002;Baur and Boethel, 2003). The mode of action ofCry1 toxins in non-target Lepidotera is presumedto be similar to that in target Lepidoptera. However,additional studies seem to be necessary to confirmthis, in particular for non-target Lepidoptera thatexhibited only sublethal effects. Most notably, Demlet al. (1999), who conducted an extensive studywith native Bt toxins, found that also thecoleopteran-active Cry3A toxins can have adverseeffects on non-target Lepidoptera. Similarly, Husseinet al. (2005) and Hussein et al. (2006) reporteddeleterious effects on the polyphagous mothSpodoptera littoralis when caterpillars were fedCry3A-expressing potato foliage. In studies with S.littoralis larvae feeding on lepidopteran-activeCry1Ab expressing maize, increased mortality,prolonged development time and reduced weightwere reported in several studies (Dutton et al., 2002;Dutton et al., 2005; Vojtech et al., 2005). In otherstudies, no or little effects of Cry1Ab toxin on thisspecies were found (Höfte and Whiteley, 1989;Müller-Cohn et al., 1996). The youngest larvalstages were reported to be most sensitive (Hilbecket al., 1999; Dutton et al., 2005). Further, the effectsof transgenic Cry1Ab maize on S. littoralis mortalityand development time were more pronounced intransgenic plants compared to a commercial sprayformulation (Dutton et al., 2005).

Some non-target effects found in non-lepidopteran herbivorous species (Table 4b), suckinginsects in particular, were mostly attributed to


Tabl

e 3.

Lis

t of

inve

rteb

rate

spe

cies

fro

m d

iffer

ent

trop

hic

grou

ps t

este

d in

labo

rato

ry f

eedi

ng s

tudi

es (

rank

ed in

chr

onol

ogic

al o

rder

) w

ith r

egar

d to

diffe

renc

es b

etw

een

a no

n-B

t co

ntro

l and

mic

robi

al B

t-pro

tein

s (m

) an

d/or

tra

nsge

nic

Bt-p

lant

mat

eria

l (tg

).

1995

Anth

onom

usgr

andi

s(S

ims,

1995

)

Dia

brot

ica

unde

cim

punc

tata

(Sim

s, 19

95)

Lept

inot

arsa

dece

mlin

eata

(Sim

s, 19

95)

Myz

us p

ersi

cae

(Sim

s, 19

95)

Apis

mel

lifer

a(S

ims,

1995

)

Blat

tella

germ

anic

a(S

ims,

1995

)

Cry

1Acm

Cry

1Acm

Cry

1Acm

Cry

1Acm

Cry

1Acm

Cry

1Acm

0 0 0 0 0 0

Hip

poda

mia

conv

erge

ns(S

ims,

1995

)

Chr

ysop

erla

carn

ea(S

ims,

1995

)

Cry

1Acm

(bi-t

roph

.)

Cry

1Acm

(bi-t

roph

.)

0 0

Nas

onia

vitri

penn

is(S

ims,

1995

)

Cry

1Acm

(bi-t

roph

.)0

Aede

s aeg

ypti

(Sim

s, 19

95)

Cry

1Acm

(bi-t

roph

.)0

1996

Apis

mel

lifer

a(A

rpai

a, 1

996)

Cry

3Bm

0H

ippo

dam

iaco

nver

gens

(Dog

an et

al.,

1996

)

Cry

3tg

(tri-t

roph

)0

Year

Non

-targ

ethe

rbiv

ores

Bt

test

mat

eria

lO

bs.

Diff

.Pr

edat

ors

Bt

test

mat

eria

lO

bs.

Diff

.Pa

rasi

toid

s-H

yper

pars

itoid

sB

t te

stm

ater

ial

Obs

.D

iff.

Oth

erfe

edin

g ty

peB

t te

stm

ater

ial

Obs

.D

iff.


1997

Cole

omeg

illa

mac

ulat

a(P

ilche

r et a

l.,19

97)

Chr

ysop

erla

carn

ea (P

ilche

r et

al.,

1997

)

Ori

us in

sidi

osus

(Pilc

her e

t al.,

1997

)

0 0 0

Folso

mia

cand

ida

(Yu

et a

l., 1

997)

Opp

ia n

itens

(Yu

et a

l., 1

997)

0 0

Cry

1A

btg

(Pol

len;

bi-tr

oph.

)

Cry

1A

btg

(Pol

len;

bi-tr

oph.

)

Cry

1A

btg

(Pol

len;

bi-tr

oph.

)

Cry

1Actg

Cry

1Abtg

Cry

1Actg

Cry

1Abtg

Rhop

alos

i-ph

um p

adi

(Loz

zia

et a

l.,19

98)

1998

Cry

1Abtg

Chr

ysop

erla

carn

ea(H

ilbec

k et

al.,

1998

b)

Chr

ysop

erla

carn

ea(H

ilbec

k et

al.,

1998

a)

Chr

ysop

erla

carn

ea(L

ozzi

a et

al.,

1998

)

Cole

omeg

illa

mac

ulat

a(R

iddi

ck &

Bar

bosa

, 199

8)

- - 0 0

0C

ry1A

btg

(tri-

troph

.)

Cry

1Abm

(bi-t

roph

.)

Cry

1Abtg

(tri-

troph

.)

Cry

1Abtg

(tri-

troph

.)

Year

Non

-targ

ethe

rbiv

ores

Bt

test

mat

eria

lO

bs.

Diff

.Pr

edat

ors

Bt

test

mat

eria

lO

bs.

Diff

.Pa

rasi

toid

s-H

yper

pars

itoid

sB

t te

stm

ater

ial

Obs

.D

iff.

Oth

erfe

edin

g ty

peB

t te

stm

ater

ial

Obs

.D

iff.


Dan

aus p

lexi

ppus

(Los

ey e

t al.,

1999

)

Ostr

inia

nub

ilalis

(Dem

l et a

l.,19

99)

Ache

ront

iaat

ropo

s(D

eml e

t al.,

1999

)

Man

duca

sext

a(D

eml e

t al.,

1999

)

Auto

grap

haga

mm

a(D

eml e

t al.,

1999

)

Lept

inot

arsa

dece

mlin

eata

(Dem

l et a

l.,19

99)

Aphi

s fab

ae(D

eml e

t al.,

1999

)M

acro

siph

umav

enae

(Dem

l et a

l.,19

99)

1999

Cry

1Abtg

(pol

len)

Cry

1Acm

Cry

3Am

Cry

1Acm

Cry

3Am

Cry

1Acm

Cry

3Am

Cry

1Acm

Cry

3Am

Cry

1Acm

Cry

3Am

Cry

1Acm

Cry

3Am

Cry

1Acm

Cry

3Am

Chr

ysop

erla

carn

ea(H

ilbec

k et

al.,

1999

)

Cry

1Abm

,C

ry2A

m

(tri-

troph

.)

-Co

tesia

plu

tella

e(S

chul

er et

al.,

1999

)

0— — 0,

0

–,–

–,–

0,0

–,0

0,0

Cry

1Actg

(tri-t

roph

.)

Year

Non

-targ

ethe

rbiv

ores

Bt

test

mat

eria

lO

bs.

Diff

.Pr

edat

ors

Bt

test

mat

eria

lO

bs.

Diff

.Pa

rasi

toid

s-H

yper

pars

itoid

sB

t te

stm

ater

ial

Obs

.D

iff.

Oth

erfe

edin

g ty

peB

t te

stm

ater

ial

Obs

.D

iff.


Dan

aus p

lexi

ppus

(Jes

se &

Obr

ycki

,20

00)

Papi

lio p

olyx

enes

(Wra

ight

et a

l.,20

00)

Tetra

nych

usur

tical

e(L

ozzi

a et

al.,

2000

)

2000

Ori

us tr

istic

olor

(Arm

er et

al.,

2000

)

Geo

cori

spu

nctip

es(A

rmer

et a

l.,20

00)

Geo

cori

s pal

lens

(Arm

er et

al.,

2000

)

Lygu

s he

sper

us(A

rmer

et a

l.,20

00)

Nab

is s

p.(A

rmer

et a

l.,20

00)

Ori

us m

ajus

culu

s(Z

wah

len

et a

l.,20

00)

0 0 0 0 0 0

Porc

ellio

scab

er(E

sche

r et a

l.,20

00)

0,+

— 0, — 0

Cry

3tg

(bi-t

roph

)

Cry

3tg

(bi-t

roph

)

Cry

3tg

(bi-t

roph

)

Cry

3tg

(bi-t

roph

)

Cry

3tg

(bi-t

roph

)

Cry

1Abtg

(tri-t

roph

)

Cry

1Abtg

Cry

1Abtg

(pol

len)

Cry

1Abtg

(pol

len)

Cry

1Abtg

Dan

aus p

lexi

ppus

(Hel

lmic

h et

al.,

2001

)

2001

Ori

us i

nsid

iosu

s(A

l-Dee

b et

al.,

2001

)

0Ap

hidi

us n

igrip

es(A

shou

ri et

al.,

2001

a)

-Lu

mbr

icus

terr

estr

is(S

axen

a &St

otzk

y, 2

001)

Cry

1Abtg

(roo

tex

udat

es,

plan

tre

sidu

es)

00,

–C

ry1A

btg

(tri-t

roph

)M

ixed

Cry

toxi

nsm

(Dip

el)

(tri-t

roph

)

Cry

3Atg

(tri-t

roph

)Cr

y1A

bm,tg

Cry1

Acm

,tg

Cry

9Cm

,tg

Cry

1Fm

,tg

(pol

len)

Year

Non

-targ

ethe

rbiv

ores

Bt

test

mat

eria

lO

bs.

Diff

.Pr

edat

ors

Bt

test

mat

eria

lO

bs.

Diff

.Pa

rasi

toid

s-H

yper

pars

itoid

sB

t te

stm

ater

ial

Obs

.D

iff.

Oth

erfe

edin

g ty

peB

t te

stm

ater

ial

Obs

.D

iff.


Rhop

alos

iphu

mpa

di (M

eier

&H

ilbec

k, 2

001)

Apis

mel

lifer

a(M

alon

e et

al.,

2001

)

Mac

rosi

phum

euph

orbi

ae(A

shou

ri et

al.,

2001

b)

Chr

ysop

erla

car

nea

(Mei

er &

Hilb

eck,

2001

)

0,–

Nem

atod

es(S

axen

a &St

otzk

y, 2

001)

Prot

ozoa

(Sax

ena &

Stot

zky,

200

1)

Cry

1Abtg

(roo

tex

udat

es,

plan

tre

sidu

es)

Cry

1Abtg

(roo

tex

udat

es,

plan

tre

sidu

es)

0 0

0 0 –

Cry

1Abtg

(tri-

troph

.)

Cry

1Abtg

Cry

1Bam

Cry

3Atg

Pier

is b

rass

icae

(Fel

ke et

al.,

2002

)

Pier

is ra

pae

(Fel

ke et

al.,

2002

)

Plut

ella

xylo

stella

(Fel

ke et

al.,

2002

)

2002

Cole

omeg

illa

mac

ulat

a(L

undg

ren

and

Wie

dem

ann,

2002

)

Cole

omeg

illa

mac

ulat

a(D

uan

et a

l.,20

02)

Ori

us tr

istic

olor

(Pon

sard

et a

l.,20

02)

0 0 —

Para

llorh

ogas

pyra

loph

agus

(Ber

nal e

t al.,

2002

b)

-,0Po

rcel

liosc

aber

(Wan

dele

r et

al.,

2002

)

0; —

— — —

Cry

3Bbtg

(pol

len;

bi-tr

oph.

)

Cry

3Bb1

tg

(pol

len;

bi-tr

oph.

)

Cry

1Actg

(tri-

troph

.)

Cry

1Abtg

(tri-t

roph

.)C

ry1A

btgC

ry1A

btg

(pol

len)

Cry

1Abtg

(pol

len)

Cry

1Abtg

(pol

len)

Year

Non

-targ

ethe

rbiv

ores

Bt

test

mat

eria

lO

bs.

Diff

.Pr

edat

ors

Bt

test

mat

eria

lO

bs.

Diff

.Pa

rasi

toid

s-H

yper

pars

itoid

sB

t te

stm

ater

ial

Obs

.D

iff.

Oth

erfe

edin

g ty

peB

t te

stm

ater

ial

Obs

.D

iff.


-0 0 — -; 0

Cry

1Abtg

Cry

1Actg

Cry1

Ftg

Cry

1Actg

Apis

mel

lifer

a(H

anle

y et

al.,

2003

)

Atha

liaro

sae

(How

ald,

200

3)

Gal

leria

mel

lone

lla(H

anle

y et

al

.,20

03)

Pseu

dopl

usia

incl

uden

s (B

aur &

Boe

thel

, 200

3)

2003

Cote

siam

argi

nive

ntri

(Bau

r &B

oeth

el, 2

003)

Cop

idos

oma

florid

anum

(Bau

r & B

oeth

el,

2003

)

– –; 0

Lum

bric

uste

rres

tris

(Zw

ahle

n et

al.,

2003

Cry

1Actg

(tri-t

roph

)

Cry

1Actg

(tri-t

roph

)

Cry

1Abtg

Nila

parv

ata

luge

ns(B

erna

l et a

l.,20

02a)

Rhop

alos

iphu

mpa

di(D

utto

n et

al.,

2002

)

Tetra

nych

usur

tical

e(D

utto

n et

al.,

2002

)

Spod

opte

ralit

tora

lis(D

utto

n et

al.,

2002

)

Zelu

s ren

ardi

i(P

onsa

rd e

t al.,

2002

)

Nab

is s

p.(P

onsa

rd e

t al.,

2002

)

Cyr

torh

inus

livid

ipen

nis

(Ber

nal e

t al.,

2002

a)

Chr

ysop

erla

carn

ea (D

utto

n et

al.,

2002

)

0 0 0 0; –

0 0 0 -

Cry

1Actg

(tri-t

roph

.)

Cry

1Actg

(tri-t

roph

.)

Cry

1Abtg

Cry

1Actg

(tri-t

roph

.)

Cry

1Abtg

(tri-t

roph

.)

Cry

1Abtg

Cry

1Actg

Cry

1Abtg

Geo

cori

spu

nctip

es(P

onsa

rd e

t al.,

2002

)

Cry

1Actg

(tri-t

roph

.)—

Year

Non

-targ

ethe

rbiv

ores

Bt

test

mat

eria

lO

bs.

Diff

.Pr

edat

ors

Bt

test

mat

eria

lO

bs.

Diff

.Pa

rasi

toid

s-H

yper

pars

itoid

sB

t te

stm

ater

ial

Obs

.D

iff.

Oth

erfe

edin

g ty

peB

t te

stm

ater

ial

Obs

.D

iff.


Dan

aus p

lexi

ppus

(And

erso

n et

al.,

2004

)

Rhizo

glyp

husr

obin

i(C

arte

r et a

l.,20

04)

2004

Cot

esia

flav

ipes

(Prü

tz &

Det

tner

, 200

4)

Tetra

stich

usho

war

di(P

rütz

et a

l.,20

04)

–; 0

Cry

1Abtg

(tri-t

roph

.)C

ry1

Ab

tg

(anth

ers)

Chr

ysop

erla

carn

ea(R

omei

s et a

l.,20

04)

Cry

1Abm

(bi-t

roph

.)0

Tetra

nych

usur

ticae

(Zem

ková

Rov

ensk

á et a

l.,20

05)

Spod

opte

ralit

tora

lis(H

usse

in e

t al.,

2005

)

Spod

opte

ralit

tora

lis(D

utto

n et

al.,

2005

)

Spod

opte

ralit

tora

lis(V

ojte

ch et

al.,

2005

)

Fran

klin

iella

tenu

icor

nis

(Obr

ist e

t al.,

2005

)

2005

Mic

ropl

itis

med

iato

r(L

iu et

al.,

200

5a)

Cot

esia

mar

gini

-ve

ntri

s(V

ojte

ch et

al.,

2005

)

+1 0;—

—;—

–;0

0

Cry

3Bbtg

Cry3

Aam

,tg

Cry

1Abtg

Cry

1Abm

(Dip

el)

Cry

1Abtg

Cry

1Abtg

Phyt

osei

ulus

pers

imili

s(Z

emko

vaR

oven

ska e

t al.,

2005

)

Prop

ylea

japo

nica

(Bai

et a

l., 2

005)

Poec

ilus c

upre

us(M

eiss

le e

t al.,

2005

)

– 0; – –; 0

Cry

3Bbtg

(tri-t

roph

.)

Cry

1Abtg

(pol

len

bi-

troph

.)

Cry

1Abtg

(tri-t

roph

.)

Year

Non

-targ

ethe

rbiv

ores

Bt

test

mat

eria

lO

bs.

Diff

.Pr

edat

ors

Bt

test

mat

eria

lO

bs.

Diff

.Pa

rasi

toid

s-H

yper

pars

itoid

sB

t te

stm

ater

ial

Obs

.D

iff.

Oth

erfe

edin

g ty

peB

t te

stm

ater

ial

Obs

.D

iff.


Spod

opte

ralit

tora

lis(H

usse

in e

t al.,

2006

)

2006

Apan

tele

ssu

band

inus

(Dav

idso

n et

al.,

2006

)

0–

Cry

1Ac9

tg,

Cry

9Aa2

tg

(tri-t

roph

.)

Cry

3Aatg

Neos

eiul

uscu

cum

eris

(Obr

ist

et a

l., 2

006b

)

Neos

eiul

uscu

cum

eris

(Obr

ist

et a

l., 2

006b

)

Aran

eus

diad

emat

us(L

udy

and

Lang

,20

06)

Adal

ia b

ipun

ctat

a(S

chm

idt,

2006

)

Mic

rom

usta

sman

iae

(Dav

idso

n et

al.,

2006

)

Chr

ysop

erla

carn

ea(R

odrig

o-Si

mon

et a

l., 2

006)

0 — 0 –; 0

0 0

Cry

1Abtg

(tri-t

roph

.)

Cry

1Abtg

(pol

len;

bi-tr

oph.

)

Cry

1Abtg

(pol

len;

bi-

troph

.)

Cry

1Abm

,C

ry3B

bm

(bi-t

roph

.)

Cry

1Ac9

tg,

Cry

9Aa2

tg

(tri-t

roph

.)

Cry

1Acm

,C

ry1A

bm

Cry

2Abm

(bi-

and

tri-

troph

ic.)

Obs

. diff

: obs

erve

d di

ffer

ence

: + =

obs

erve

d di

ffer

ence

, pos

itive

for t

he te

st s

peci

es; 0

= n

o di

ffer

ence

obs

erve

d; —

= o

bser

ved

diff

eren

ce, n

egat

ive

for t

he te

st s

peci

es.

1 Pre

fere

nce

of te

sted

tran

sgen

ic B

t cul

tivar

.

Year

Non

-targ

ethe

rbiv

ores

Bt

test

mat

eria

lO

bs.

Diff

.Pr

edat

ors

Bt

test

mat

eria

lO

bs.

Diff

.Pa

rasi

toid

s-H

yper

pars

itoid

sB

t te

stm

ater

ial

Obs

.D

iff.

Oth

erfe

edin

g ty

peB

t te

stm

ater

ial

Obs

.D

iff.


pleiotropic effects of transgenic Bt plants. It wasargued that the transgenic Bt cultivars used in theexperiments provided the tested species withdifferent conditions in terms of primary andsecondary compounds than the non-transformedplants used as controls (Ashouri et al., 2001b).Pleiotropic effects are also discussed with regard todifferences in host plant preference in herbivorousmites (Zemková Rovenská et al., 2005). However,further experimental investigations to establish thecauses of the documented effects are still to beconducted. Similarly, reported effects of Cry1Ab onthe detritivorous woodlouse, Porcellio scaber weremostly discussed in the context of nutritional qualityof transgenic Bt plants (Escher et al., 2000; Wandeleret al., 2002), although Wandeler et al. (2002) alsoconsidered the possibility of Bt toxicity as anexplanation for the reduced consumption ratesobserved.

Most studies with predators tested effects ofCry1Ab toxins and Cry1Ab-fed prey on larvae ofthe green lacewing Chrysoperla carnea (Hilbeck etal., 1998a, b; Hilbeck et al., 1999; Dutton et al., 2002;Romeis et al., 2004). Their results will be reviewed inmore detail below, as this is the most prominentexample of studies revealing different outcomesdepending on different experimental methodologiesand approaches. Moreover, Ponsard et al. (2002)found a reduction in longevity in two species ofpredatory bugs, Orius tristicolor and Geocoristristicolor, when fed with caterpillars reared onCry1Ac-expressing cotton plants (Table 4b). Highermortality rates compared to the control were observedin larvae of the two-spot ladybird, Adaliabipunctata, when their food, flour moth eggs, hadbeen treated with microbially produced trypsin-activated toxins (Schmidt, 2006; Schmidt et al.,submitted). In these experiments, mortality increasedstronger in treatments with the lepidopteran-activeCry1Ab toxin than with the coleopteran activeCry3Bb. The reasons for this are unclear. However,earlier studies with earthworms reported reducedmortality at high toxins concentrations (Smirnoff andHeimpel, 1961). In another ladybird, Propylaeajaponica, Bai et al. (2005) reported an increasedlongevity of females when fed with aphids and ricepollen of one Cry1Ab expressing variety (KMD-1)

compared to females fed with aphids and rice pollenof non-Bt rice. A second Cry1Ab expressing ricevariety (KMD-2), however, did not produce similarresults. The authors could not offer an explanationfor this result.

Effects of transgenic Bt plants on parasitoidsare assumed to be more likely because of the closertrophic relationship between parasitoid larvae andtheir hosts compared to pedators and their prey(Bernal et al., 2002b). Several studies with parasitoidsreported lethal and sublethal effects of variousparameters, when parasitoids developed withinlepidopteran caterpillar hosts reared on Bt toxincontaining diets (Bernal et al., 2002b; Baur andBoethel, 2003; Prütz and Dettner, 2004; Liu et al.,2005a). Sublethal effects included prolongeddevelopment time, reduced cocoon weights, reducedlongevity, reduced fecundity and a shift in sex ratios.Liu et al. (2005b) studied the effect of a transgeniccotton variety (SGK321) expressing both a Cry1Atoxin and the insect-active protease inhibitor CpTIon the parasitoid Campoketis chlorideae Uchida(Hymenoptera: Ichneumonidae). They reportedreduced body weights of parasitoids, when their hostcaterpillars of the target species H. armigera fed onleaves of the transgenic cultivar 10-48 h afterparasitisation. If hosts fed on the transgenic cultivarfor more than 48 h, prolonged egg and larvaldevelopment and decreased pupal and adult weightwere observed. However, the reported effects cannotbe linked to one of the transgene-expressedsubstances.

Only one publication was concerned with effectsof transgenic Bt plants on a hyperparasitoid(secondary parasitoid). Prütz et al. (2004) reportedreduced parasitisation, emergence and female weightin Tetrastichus howardi when developing incocoons of the primary parasitoid Cotesia flavipes,which parasitized Bt maize fed Chilo partellus larvae.

The results of studies with predators andparasitoids are mostly discussed in context ofreduced nutritional quality of toxin-affected prey andthe known mode of action (e.g. Dutton et al., 2003;Meissle et al., 2005; Vojtech et al., 2005). It is oftenstated, that direct toxin effects are “unlikely”, becauseBt toxin specific receptors are only found in targetorganisms. However, some authors acknowledge that


Tabl

e 4a

. Sum

mar

y of

fee

ding

stu

dies

con

duct

ed w

ith n

on-ta

rget

Lep

idop

tera

spe

cies

tha

t re

port

ed d

iffer

ence

s be

twee

n a

non-

Bt

cont

rol a

nd m

icro

bial

Bt

prot

eins

and

/or

tran

sgen

ic B

t pl

ant

mat

eria

l.

Dea

thhe

adha

wkm

oth,

Ache

ront

ia a

tropo

s(L

.) (S

phin

gida

e)

Silv

er-Y

mot

h,Au

togr

apha

gam

ma,

(L.)

(Noc

tuid

ae)

Mon

arch

but

terf

ly,

Dan

aus p

lexi

ppus

(L.)

(Nym

phal

idae

)

Cry1

Ac

toxi

npr

oduc

ed fr

om B

tkstr

ain

HD

-73;

purif

ied

Cry

3A to

xin

base

d on

com

mer

cialf

orm

ulati

on(N

ovod

or).

Cry

1Ac

toxi

n pr

od-

uced

fro

m B

tk s

train

HD

-73;

pur

ified

Cry

3A t

oxin

bas

ed o

nc

om

me

rc

ia

lf

or

mu

la

ti

on

(Nov

odor

).

Cor

n (e

vent

Bt1

1)ex

pres

sing

Cry

1Ab

toxi

n.

Cor

n (e

vent

s B

t176

and

Bt1

1) ex

pres

sing

Cry

1Ab

toxi

n.

Puri

fied

(t

ryps

in-

resi

stan

t co

re)

toxi

ns(C

ry1A

b, C

ry1A

c).

Red

uced

foo

d co

n-su

mpt

ion,

re

duce

dgr

owth

4 .

Incr

ease

d m

orta

lity,

incr

ease

d fo

od co

nsu-

mpt

ion

(Cry

3A),

redu

ced

grow

th(e

xcep

t Cry

1Ab

onda

ndel

ion)

4.

Incr

ease

d m

orta

lity,

redu

ced

cons

umpt

ion.

Hig

her m

orta

lity

rate

sin

cate

rpill

ars (

afte

r 48

hour

s,

afte

r 12

0ho

urs)

.

Incr

ease

dm

orta

lity

,re

duce

dgr

owth

of

1st

inst

ar la

rvae

(or l

arva

ele

ss af

fect

ed).

10 d

ay o

ld l

arva

e fe

dw

ith to

xin

inco

rpor

ated

in P

etri

dish

es a

rtific

ial

diet

.

6, 8

, 10

day

old

larv

aefe

d w

ith to

xin

inco

rpo-

rate

d in

artif

icia

l die

t or

appl

ied

to d

ande

lion

(Tar

axac

um o

ffici

nale

)le

af di

scs i

n Pet

ri di

shes

.

Com

mon

m

ilkw

eed

(Asc

elap

ias s

yria

ca)

leaf

disc

s arti

ficia

llydu

sted

with

pol

len

fed

to c

ater

pilla

rs (

3 da

ysol

d) in

Pet

ri di

shes

.

Com

mon

mil

kwee

d(A

scel

apia

ssy

riac

a)le

afdi

scsn

atur

ally

and

artif

icia

lly d

uste

dw

ith p

olle

n fe

d to

cat

-er

pilla

rs (

1st i

nsta

r) i

nPe

tri d

ishe

s.

Toxi

ns i

ncor

pora

ted

into

artif

icia

l die

t fed

toca

terp

illar

s.

10 d

ays

durin

g 2nd

and

3rd in

star

.

6 da

ys d

urin

g 2nd

and

3rd in

star

.

4 da

ys

48 h

ours

Dur

ing

entir

e lar

val

deve

lopm

ent.

Spec

ies-

spec

ific

susc

e-pt

ibili

ty o

f th

e in

sect

sto

the t

oxin

s Red

uced

feed

ing a

nd de

crea

sed

util

izat

ion

of f

ood

cont

a-in

ing

an e

ndo-

toxi

n.

Spec

ies-

spec

ific

susc

e-pt

ibili

ty o

f the

inse

cts to

the

toxi

ns

Red

uced

feed

ing

and

decr

ease

dut

iliza

tion

of

food

cont

ainin

g an e

ndo-

toxi

n.

Effe

ct o

f B

t to

xin

inpo

llen.

Toxi

city

of B

t pol

len.

Toxi

n ef

fect

.

Dem

l et a

l. (1

999)

Dem

l et a

l. (1

999)

Lose

y et

al.

(199

9)

Jess

e an

d O

bryc

ki(2

000)

Hel

lmic

h et

al.

(200

1)

Lepi

dopt

era

spec

ies

Sour

ce o

fB

t exp

osur

eSt

atist

ical

lysig

nific

ant

diff

eren

ces c

ompa

red

to B

t-fre

e co

ntro

l1

durin

g/af

tere

xpos

ure

Expe

rim

enta

l se

t up

wit

h re

gard

to

repo

rted

effe

cts

Dur

atio

n of

Bt e

xpos

ure

Cau

se o

f rep

orte

d di

f-fe

renc

es su

gges

ted/

dis-

cuss

ed b

y th

e au

thor

s

Refe

renc

e


Puri

fied

cor

n po

llen

colle

cted

from

Bt c

orn

(eve

nt 1

76) c

onta

inin

gC

ry1A

b to

xin.

Puri

fied

cor

n po

llen

cont

amin

ated

with

cor

nta

ssel

mat

eria

l bo

thco

llec

ted

from

B

tco

rn(e

vent

s 17

6 an

dB

t11)

co

ntai

ning

Cry1

Ab

toxi

n a

nd B

tco

rn (

even

t C

bh35

1)co

ntai

ning

Cry

9C to

xin.

Cor

n (e

vent

Bt1

1)an

ther

s co

ntai

ning

Cry

1Ab

toxi

n.

Cry

1Ac

toxi

n pr

o-du

ced

from

Btk

stra

inH

D-7

3.

Cry

1Ac

toxi

n pr

o-du

ced

from

Btk

stra

inH

D-7

3;

pu

rifi

edC

ry3A

toxi

n ba

sed

onco

mm

ercia

l for

mul

ation

(Nov

odor

).

Red

uced

gro

wth

.

Incr

ease

d m

orta

lity.

Red

uced

feed

ing,

pro

-lo

nged

dev

elop

men

ttim

e, re

duce

d wei

ght i

nca

terp

illar

s Deg

ree d

e-pe

nds

on n

umbe

r of

anth

ers

prov

ided

.

Red

uced

pupa

tion

rate

, pro

long

ed d

evel

-op

men

t tim

e, r

educ

edla

rval

and p

upal

wei

ght.

Incr

ease

d m

orta

lity,

redu

ced

food

con

-su

mpt

ion,

re

duce

dgr

owth

4 .

Polle

n ap

plie

d to

com

mon

milk

wee

d (As

c-ela

pias

syria

ca) le

af di

scs

fed

to ca

terpi

llars.

Polle

n an

d ta

ssel

ma-

teria

l app

lied

to c

om-

mon

milk

wee

d (A

sce-

lapi

as s

yria

ca)

leaf

disc

s fe

d to

cat

erpi

l-la

rs.

Ant

hers

pla

ced

on le

afdi

scs

of

com

mon

milk

wee

d (A

scel

apia

ssy

riac

a) f

ed t

o ca

ter-

pilla

rs in

Pet

ri di

shes

.

Cat

erpi

llars

fed

with

toxi

n in

corp

orat

ed in

toar

tific

ial d

iet.

10 d

ay o

ld la

rvae

fed

with

toxi

n inc

orpo

rate

din

artif

icia

l die

t in

Petri

dish

es.

Dur

ing

entir

e lar

val d

e-ve

lopm

ent.

Dur

ing

entir

e lar

val d

e-ve

lopm

ent.

Entir

e la

rval

dev

elop

-m

ent (

1st to

5th in

star

)2 .

Entir

e la

rval

dev

elop

-m

ent.

10 d

ays d

urin

g 2nd

and

3rd in

star

.

Effe

ct o

f po

llen

from

Bt1

76 c

ultiv

ar.

Eff

ect

of p

olle

n in

com

bina

tion

with

tas-

sel

mat

eria

l fr

om r

e-sp

ectiv

e cu

ltiva

rs.

Incr

ease

d se

arch

ing

toav

oid

Bt i

nges

tion

(no

dire

ct t

oxic

ity

pro-

pose

d).

Eff

ect

of

Cry

1Ac

toxi

n.

Spec

ies-

spec

ific

susc

e-pt

ibili

ty o

f th

e in

sect

sto

the t

oxin

s Red

uced

feed

ing a

nd de

crea

sed

utili

zatio

n of

food

cont

aini

ng a

n en

do-

toxi

n.

Hel

lmic

h et

al.

(200

1)

Hel

lmic

h et

al.

(200

1)

And

erso

n et

al.

(200

4)

Liu

et a

l. (2

005a

)

Dem

l et a

l. (1

999)

Cot

ton

bollw

orm

,H

elic

over

paar

mig

era

(Hüb

ner)

(Noc

tuid

ae)

Toba

cco

horn

wor

m,

Man

duca

sext

a, (L

.)(S

phin

gida

e)

Lepi

dopt

era

spec

ies

Sour

ce o

fB

t exp

osur

eSt

atist

ical

lysig

nific

ant

diff

eren

ces c

ompa

red

to B

t-fre

e co

ntro

l1

durin

g/af

tere

xpos

ure

Expe

rim

enta

l se

t up

wit

h re

gard

to

repo

rted

effe

cts

Dur

atio

n of

Bt e

xpos

ure

Cau

se o

f rep

orte

d di

f-fe

renc

es su

gges

ted/

dis-

cuss

ed b

y th

e au

thor

s

Refe

renc

e


Cry

1Ac

toxi

n pr

o-du

ced

from

Btk

stra

inH

D-7

3;

pu

rifi

edC

ry3A

toxi

n ba

sed

onco

mm

ercia

l for

mul

ation

(Nov

odor

).

Cor

n (B

t176

) po

llen

cont

aini

ng C

ry1A

bto

xin.

Cor

n (B

t176

) po

llen

cont

aini

ng C

ry1A

bto

xin.

Cor

n (B

t176

) po

llen

cont

aini

ng C

ry1A

bto

xin.

Cot

ton

(eve

nt 5

31,

Nuc

otto

n-33

B)

leav

esco

ntai

ning

Cry

1Ac

toxi

n.

Incr

ease

d m

orta

lity,

redu

ced

food

con

-su

mpt

ion

(Cry

3A),

re-

duce

d gr

owth

4 .

Red

uced

feed

ing,

redu

ced

grow

th, a

ndhi

gher

mor

tali

ty i

nca

terp

illa

rs D

egre

ede

pend

s on

amou

nt o

fpo

llen

cons

umed

.

Redu

ced

feed

ing,

redu

ced

grow

th, h

ighe

rm

orta

lity

(and

beh

avi-

oura

l cha

nges

) in

cate

rpill

ars D

egre

ede

pend

s on

amou

nt o

fpo

llen

cons

umed

.

Redu

ced

feed

ing,

redu

ced

grow

th,

and

high

er m

orta

lity

in ca

terp

illar

s Deg

ree

depe

nds

on a

mou

ntof

pol

len

cons

umed

.

Incr

ease

d m

orta

lity,

prol

onge

d de

velo

p-m

ent

tim

e, r

educ

edpr

epup

alw

eigh

t5 .

5day

old

lar

vae

fed

with

toxi

n inc

orpo

rate

din

artif

icia

l die

t in

Petri

dish

es.

Wild

cabb

age (

Bras

sica

oler

acea

) lea

f dis

c ar

-tif

icia

lly d

uste

d w

ithpo

llen

fed

to c

ater

pil-

lars

in p

last

ic b

oxes

.

Wild

cabb

age (

Bras

sica

oler

acea

) lea

f dis

cs ar

-tif

icia

lly d

uste

d w

ithpo

llen

fed

to c

ater

pil-

lars

in p

last

ic b

oxes

.

Wild

cabb

age (

Bras

sica

oler

acea

) lea

f dis

c ar

-tif

icia

lly d

uste

d w

ithpo

llen

fed

to c

ater

pil-

lars

in p

last

ic b

oxes

.

Larv

ae f

ed w

ith c

utle

aves

in P

etri

dish

es.

10 d

ays d

urin

g 2nd

and

3rd in

star

.

24 h

ours

dur

ing

2nd

inst

ar3 .

24 h

ours

dur

ing

2nd

insta

r.

24 h

ours

dur

ing

4th

insta

r.

Entir

e la

rval

dev

elop

-m

ent

until

pre

pupa

lsta

ge.

Spec

ies-

spec

ific

sus-

cept

ibili

ty o

f the

inse

cts

to th

e tox

ins

Redu

ced

feed

ing

and

decr

ease

dut

iliza

tion

of fo

odco

n-ta

inin

g an

endo

-toxi

n.

Toxi

city

of B

t pol

len.

Toxi

city

of B

t pol

len.

Toxi

city

of B

t pol

len.

Effe

ct o

f tr

ansg

enic

cotto

n cu

ltiva

r.

Dem

l et a

l. (1

999)

Felk

e et a

l. (2

002)

Felk

e et a

l. (2

002)

Felk

e et a

l. (2

002)

Bau

r an

d B

oeth

el(2

003)

Euro

pean

cor

n bo

rer,

Ost

rini

a nu

bila

lis

(Hüb

ner)

(Pyr

alid

ae)

Lar

ge w

hite

, P

ieri

sbr

assic

ae L

. (Pi

erid

ae)

Smal

l w

hite

, Pi

eris

rapa

e L.

(Pie

ridae

)

Dia

mon

dbac

k m

oth,

Plut

ella

xyllo

stella

(L.)

(Plu

telli

dae)

Soyb

ean

loop

er,

Pseu

dopl

usia

incl

uden

s (W

alke

r)(N

octu

idae

)

Lepi

dopt

era

spec

ies

Sour

ce o

fB

t exp

osur

eSt

atist

ical

lysig

nific

ant

diff

eren

ces c

ompa

red

to B

t-fre

e co

ntro

l1

durin

g/af

tere

xpos

ure

Expe

rim

enta

l se

t up

wit

h re

gard

to

repo

rted

effe

cts

Dur

atio

n of

Bt e

xpos

ure

Cau

se o

f rep

orte

d di

f-fe

renc

es su

gges

ted/

dis-

cuss

ed b

y th

e au

thor

s

Refe

renc

e


Cor

n (B

t11)

exp

ress

-in

g C

ry1A

b to

xin.

Cor

n(M

on81

0)le

aves

/ste

ms

cont

aini

ng C

ry1A

bto

xin.

Cor

n (B

t11)

exp

ress

-in

g C

ry1A

b to

xin.

Bt s

pray

for

mul

atio

n(D

IPEL

).

Pota

to (N

ewle

afSu

perio

r) le

aves

cont

aini

ngC

ry3A

a to

xin.

Incr

ease

dm

orta

lity,

pro

long

edde

velo

pmen

t tim

e to

2ndin

star.

Incr

ease

d m

orta

lity,

prol

onge

d de

velo

p-m

ent t

imes

, red

uced

larv

al w

eigh

t.

Incr

ease

d m

orta

lity,

incr

ease

d de

velo

p-m

ent t

ime.

Incr

ease

d m

orta

lity,

incr

ease

d de

velo

p-m

ent t

ime.

Red

uced

feed

ing

inca

terp

illar

s, re

duce

dsi

ze b

ody

of p

upae

,re

duce

d fe

cund

ity in

fem

ales

.

Cat

erpi

llars

in ca

ges

clip

ped

on le

aves

.

Cat

erpi

llars

rear

ed o

nm

ixtu

re o

f lea

ves a

ndst

ems (

2-4

wee

k ol

dpl

ants

).

Cat

erpi

llars

rear

ed in

cage

s on

leav

es (1

st

and

2nd in

star

) and

cage

d pl

ants

(fro

m 3

rd

inst

ar o

n).

Cat

erpi

llars

rea

red

inca

ges

on

spra

yed

leav

es (

1st

and

2nd

inst

ar)

and

cage

dsp

raye

d pl

ants

(fr

om3rd

inst

ar o

n).

Larv

ae (r

eare

d on

artif

icia

l die

t unt

il 4th

inst

ar) f

ed w

ith c

utle

aves

.

Dur

ing

1st in

star

.

Entir

e lar

val d

evel

op-

men

t.

Entir

e lar

val d

evel

op-

men

t.

Entir

e lar

val d

evel

op-

men

t.

Dur

ing

5th a

nd 6

th

(last

) ins

tar.

Toxi

city

of p

lant

expr

esse

d ac

tivat

edC

ry1A

b.

Effe

ct o

f Bt c

orn

culti

var.

Effe

ct o

f the

Cry

1Ab

toxi

n.

Effe

ct o

f the

Cry

1Ab

toxi

n.

Red

uced

feed

ing:

use

dC

ry3A

a po

tato

is l

ess

suita

ble

a ho

st p

lant

;re

duce

dbo

dysiz

e:to

xin

coul

d bi

nd t

o m

idgu

tce

lls a

nd re

duce

dig

e-sti

ve fu

nctio

n; re

duce

dfe

cund

ity: u

nkno

wn

effe

ct o

f Cry

3A se

que-

stra

tion

in fe

mal

es.

Dut

ton

et a

l. (2

002)

Vojte

ch et

al.

(200

5)

Dut

ton

et a

l. (2

005)

Dut

ton

et a

l. (2

005)

Hus

sein

et a

l. (2

005)

Hus

sein

et a

l. (2

006)

Egyp

tian

cotto

nle

afw

orm

, Spo

dop-

tera

litto

ralis

(Boi

sduv

al)

(Noc

tuid

ae)

1 If

seve

ral c

ontro

l tre

atm

ents

wer

e in

clud

ed in

the

expe

rimen

ts, th

e sta

tem

ents

refe

r to

the

treat

men

t whi

ch, w

as m

ost s

imila

r to

the

Bt tr

eatm

ent. 2

No

effe

cts

docu

men

ted

whe

n 3rd

thro

ugh

5th in

star l

arva

e w

ere

expo

sed.

3 N

o ef

fect

s doc

umen

ted

whe

n 3rd

insta

r lar

vae

wer

e ex

pose

d. 4 N

o ef

fect

s of t

he p

urifi

ed C

ry3A

toxi

n on

targ

et L

eptin

otar

sade

cem

linea

ta w

ere

repo

rted

in th

is stu

dy. 5

In la

rvae

par

asiti

zed

with

Cop

idos

oma

florid

anum

(Enc

yrtid

ae) a

lso a

n in

crea

sed

cons

umpt

ion

was

obs

erve

d.

Lepi

dopt

era

spec

ies

Sour

ce o

fB

t exp

osur

eSt

atist

ical

lysig

nific

ant

diff

eren

ces c

ompa

red

to B

t-fre

e co

ntro

l1

durin

g/af

tere

xpos

ure

Expe

rim

enta

l se

t up

wit

h re

gard

to

repo

rted

effe

cts

Dur

atio

n of

Bt e

xpos

ure

Cau

se o

f rep

orte

d di

f-fe

renc

es su

gges

ted/

dis-

cuss

ed b

y th

e au

thor

s

Refe

renc

e


Tabl

e 4b

. Su

mm

ary

of f

eedi

ng s

tudi

es c

ondu

cted

with

non

targ

et n

on-L

epid

opte

ra i

nver

tebr

ate

spec

ies

that

rep

orte

d di

ffere

nces

bet

wee

n a

non-

Bt

cont

rol a

nd m

icro

bial

Bt

prot

eins

and

/or

tran

sgen

ic B

t pl

ant

mat

eria

l.

Non

-tar

get

herb

ivor

es:

Aca

ri

Two-

spot

ted

spid

erm

ite, T

etra

nych

usur

ticae

Koc

h(T

etra

nych

idae

)

Hom

opte

ra:

Bla

ck b

ean

aphi

d,Ap

his

faba

e (S

cop.

)(A

phid

idae

)

Pota

to a

phid

,M

acro

siph

umeu

phor

biae

(Th

o-m

as) (

Aph

idid

ae)

Gra

in a

phid

Mac

rosi

phum

aven

ae (F

abr.)

(Aph

idid

ae)

Eggp

lant

expr

essin

gCr

y3Bb

toxi

n.

Cry

1Ac

toxi

npr

oduc

ed f

rom

Btk

stra

in H

D-7

3; p

urifi

edC

ry3A

toxi

n ba

sed

onco

mm

erci

al fo

rmul

a-tio

n (N

ovod

or).

Pota

to (

New

leaf

)pl

antle

ts c

onta

inin

gC

ry3A

toxi

n.

Cry

1Ac

toxi

npr

oduc

ed f

rom

Btk

stra

in H

D-7

3; p

urifi

edC

ry3A

toxi

n ba

sed

onco

mm

erci

al fo

rmul

a-tio

n (N

ovod

or).

Hig

her

host

pre

fer-

ence

for

Bt p

lant

mat

eria

l.

Incr

ease

d (C

ry1A

c) o

rre

duce

d (C

ry3A

)m

orta

lity.

Red

uced

gro

wth

,re

duce

d fe

cund

ity,

high

er fl

ight

inci

denc

eof

you

ng a

lata

eD

iffer

ence

s in

nutri

tiona

l ind

ices

.

Incr

ease

d su

rviv

altim

e.

Two-

choi

ce t

ests

with

adul

ts o

n ha

lf le

ave

disc

s in

Pet

ri di

shes

.

Spec

imen

s fe

d w

ithto

xin

inco

rpor

ated

inar

tific

ial d

iet i

n Pe

tridi

shes

.

0-12

h o

ld a

pter

ous

adul

ts c

aged

on

leav

es.

Spec

imen

s fe

d w

ithto

xin

inco

rpor

ated

inar

tific

ial d

iet i

n Pe

tridi

shes

.

5 da

ys (

+ pr

e-ex

peri-

men

tal e

xpos

ure)

.

3 da

ys

7 da

ys

3 da

ys

Chan

ges i

n pr

imar

y or

seco

ndar

y m

etab

olism

.

Spec

ies-

spec

ific

susc

eptib

ility

of

the

inse

cts

to th

e to

xins

Red

uced

feed

ing

and

decr

ease

d ut

iliza

tion

of fo

od co

ntai

ning

anen

doto

xin.

Mal

nutri

tion

thro

ugh

alte

red

plan

tm

etab

olism

.

Spec

ies-

spec

ific

susc

eptib

ility

of

the

inse

cts

to th

e to

xins

Red

uced

feed

ing

and

decr

ease

d ut

iliza

tion

of fo

od co

ntai

ning

anen

doto

xin.

Zem

ková

Rov

ensk

á et

al. (

2005

)

Dem

l et a

l. 19

99

Ash

ouri

et a

l. (2

001a

)

Dem

l et a

l. 19

99

Taxo

n/sp

ecie

sSo

urce

of

Bt e

xpos

ure

Stat

istic

ally

signi

fican

tdi

ffer

ence

s com

pare

dto

Bt-f

ree

cont

rol1

durin

g/af

tere

xpos

ure

Expe

rim

enta

l se

t up

wit

h re

gard

to

repo

rted

effe

cts

Dur

atio

n of

Bt e

xpos

ure

Cau

se o

f rep

orte

d di

f-fe

renc

es su

gges

ted/

dis-

cuss

ed b

y th

e au

thor

s

Refe

renc

e


Pred

ator

s:A

cari

Pred

ator

y m

ite,

Neos

eiul

uscu

cum

eris

Oud

eman

s(P

hyto

seiid

ae)

Pred

ator

y m

ite,

Phyt

osei

ulus

pers

imili

sA

thia

s-H

enrio

t(P

hyto

seiid

ae)

Het

erop

tera

:

Bige

yed

bug,

Geo

coris

pun

ctip

esSa

y (L

ygei

dae)

Pira

te b

ug, O

rius

tristi

colo

r Whi

te(A

ntho

corid

ae)

Corn

(Bt1

1) p

olle

nco

ntai

ning

Cry

1Ab

toxi

n.

Eggp

lant

expr

essin

gC

ry3B

b to

xin.

Cot

ton

(Nuc

otto

n-33

B) l

eave

s con

tain

-in

g C

ry1A

c tox

in.

Cot

ton

(Nuc

otto

n-33

B) l

eave

s con

tain

-in

g C

ry1A

c tox

in.

Incr

ease

d de

velo

p-m

ent t

ime

in fe

mal

es,

redu

ced

fecu

ndity

.

Low

er p

refe

renc

e for

Bt f

ed p

rey.

Dec

reas

ed lo

ngev

ity.

Dec

reas

ed lo

ngev

ity.

Polle

n fe

d to

prot

onym

phs

insp

ecia

l cag

es.

Cho

ice-

test

s w

itheg

gpla

nt-fe

d (e

ntire

life

span

) non

-targ

etac

arin

e pr

ey(T

etra

nych

us u

rtica

e)in

spe

cial

set

up.

Cot

ton-

fed

(24

hour

s)no

n-ta

rget

lepi

dop-

tera

n pr

ey (S

podo

p-te

ra li

ttora

lis) f

ed to

adul

t bug

s (ad

ditio

n of

cotto

n pl

ant m

ater

ial

in so

me

trial

s).

Cot

ton-

fed

(24

hour

s)no

n-ta

rget

lepi

dop-

tera

n pr

ey (S

podo

p-te

ra li

ttora

lis) f

edto

adu

lt bu

gs (a

dditi

onof

cot

ton

plan

tm

ater

ial i

n so

me

trial

s).

Prot

onym

phal

and

deut

onym

phal

sta

ge.

3 da

ys

Rem

aini

ng li

fe sp

anaf

ter c

olle

ctio

n fr

omfie

ld.

Rem

aini

ng li

fe sp

anaf

ter c

olle

ctio

n fr

omfie

ld.

No

toxi

n ef

fect

3 .U

nint

ende

d ef

fect

of

trans

geni

c pla

nt d

ue to

bree

ding

proc

edur

eaf

ter t

rans

form

atio

n.Po

llen u

nsui

tabl

e foo

d.

Rej

ectio

n of

Bt i

npr

ey2 .

Toxi

city

of B

tpr

otei

ns a

nd p

ossi

bly

met

abol

ites.

Toxi

city

of B

tpr

otei

ns a

nd p

ossi

bly

met

abol

ites.

Obr

ist e

t al.

(200

6b)

Zem

ková

Rov

ensk

á et

al. (

2005

)

Pons

ard

et a

l. (2

002)

Pons

ard

et a

l. (2

002)

Taxo

n/sp

ecie

sSo

urce

of

Bt e

xpos

ure

Stat

istic

ally

signi

fican

tdi

ffer

ence

s com

pare

dto

Bt-f

ree

cont

rol1

durin

g/af

tere

xpos

ure

Expe

rim

enta

l se

t up

wit

h re

gard

to

repo

rted

effe

cts

Dur

atio

n of

Bt e

xpos

ure

Cau

se o

f rep

orte

d di

f-fe

renc

es su

gges

ted/

dis-

cuss

ed b

y th

e au

thor

s

Refe

renc

e


Col

eopt

era:

Two-

spot

lad

ybird

Adal

ia b

ipun

ctat

a L.

(Coc

cine

llida

e)

Car

abid

bee

tle,

Poec

ilus c

upre

us L

.(C

arab

idae

)

Lady

bird

, Pro

pyla

eaja

poni

ca (T

hunb

erg)

Para

sito

ids:

Hym

enop

tera

:

Cote

siam

argi

nive

ntri

s(C

ress

on)

(Bra

coni

dae)

Mic

robi

ally

pro

duce

dtry

psin

-act

ivat

edto

xin

solu

tions

(Cry

1Ab,

Cry

3Bb)

.

Cor

n (M

on81

0)tis

sue

cont

aini

ngC

ry1A

b to

xin.

Ric

e (K

MD

1,K

MD

2) p

olle

nco

ntai

ning

Cry

1Ab

toxi

n.

Cot

ton

(eve

nt 5

31,

Nuc

otto

n-33

B)

leav

esco

ntai

ning

Cry

1Ac

toxi

n.

Cor

n (e

vent

Mon

810)

leav

es/

stem

s con

tain

ing

Cry

1Ab

toxi

n.

Incr

ease

d la

rval

/pup

alm

orta

lity

(Cry

1Ab:

5,

25, 5

0µg/

ml;

Cry

3Bb:

25µg

/ml)

.

Incr

ease

d m

orta

lity4 .

Red

uced

fem

ale

lon-

gevi

ty (o

nly

in K

MD

1co

mpa

red

to n

on-B

tpo

llen)

5 .

Prol

onge

d de

velo

p-m

ent t

ime,

redu

ced

lon-

gevi

ty in

adu

lts, f

ewer

ova

in fe

mal

es.

Incr

ease

d m

orta

lity

until

coc

oon

form

atio

n,pr

olon

ged

deve

lopm

ent

time,

redu

ced

coco

onw

eigh

t; se

x ra

tio s

hif-

ted

tow

ards

fem

ales

.

Toxi

nso

lutio

ns in

dif-

fere

nt c

once

ntra

tions

spra

yed

on

flou

rm

oth

eggs

fed

to la

rvae

in P

etri

dish

es.

Cor

n fe

d no

n-ta

rget

lepi

dopt

eran

pre

y(S

podo

pter

a lit

tora

lis)

fed

to la

rvae

(sta

rting

with

neo

nate

or 1

0 da

yol

d in

divi

dual

s) i

ngl

ass

tube

s.

Ant

herp

owde

r (c

on-

tain

ing

poll

en)

and

aphi

ds (a

dditi

onal

food

with

outt

reat

men

t) fe

dto

larv

ae in

gla

ss tu

bes.

Cot

ton

rear

ed n

on-

targ

etle

pido

pter

anho

sts

(Pse

udop

lusi

ain

clud

ens)

para

sitiz

edat

end

of

2nd in

star

.

Non

-targ

et l

epid

op-

tera

n ho

sts

(Spo

dopt

era

litto

ralis

)re

ared

on

corn

para

sitiz

ed d

urin

g 2nd

insta

r.

Entir

e la

rval

deve

lopm

ent.

40 d

ays

(sta

rting

with

neo

nate

larv

ae);

20 d

ays

(sta

rting

with

10

day

old

larv

ae).

Entir

e la

rval

deve

lopm

ent.

Entir

e im

mat

ure

deve

lopm

ent.

Entir

e im

mat

ure

deve

lopm

ent?

(die

tof

par

asiti

zed

larv

aeno

t des

crib

ed).

Une

xpec

ted

toxi

city

.

“Mos

t lik

ely”

indi

rect

eff

ects

due

topo

or p

rey

qual

ity.

“Dire

ct e

ffec

tsca

nnot

be

excl

uded

”.

Diff

eren

ce n

otdi

scus

sed.

Effe

ct o

f hos

t fee

ding

on u

sed

trans

geni

ccu

ltiva

r.

Indi

rect

eff

ects

due

to l

ow q

ualit

yho

sts;

“dire

ct e

ffec

tsca

nnot

be

excl

uded

alth

ough

ver

yun

likel

y”.

Schm

idt (

2006

);Sc

hmid

t et a

l.(s

ubm

itted

)

Mei

ssle

et a

l. (2

005)

Bai

et a

l. (2

005)

Bau

r and

Boe

thel

(200

3)

Vojte

ch e

t al.

(200

5)

Taxo

n/sp

ecie

sSo

urce

of

Bt e

xpos

ure

Stat

istic

ally

signi

fican

tdi

ffer

ence

s com

pare

dto

Bt-f

ree

cont

rol1

durin

g/af

tere

xpos

ure

Expe

rim

enta

l se

t up

wit

h re

gard

to

repo

rted

effe

cts

Dur

atio

n of

Bt e

xpos

ure

Cau

se o

f rep

orte

d di

f-fe

renc

es su

gges

ted/

dis-

cuss

ed b

y th

e au

thor

s

Refe

renc

e


Copi

doso

ma

florid

anum

(Dal

man

) (En

cyrti

dae)

Cot

esia

flav

ipes

(Cam

eron

)(B

raco

nida

e)

Mic

ropl

itis m

edia

tor

(Hal

iday

)(B

raco

nida

e)

Para

llorh

ogas

pyra

loph

agus

(Mar

sh)

(Bra

coni

dae)

Hyp

erpa

rasit

oids

:H

ymen

opte

ra:

Tetr

astic

hus

how

ardi

(Olli

ff) (

Eulo

phid

ae)

Cot

ton

(eve

nt 5

31,

Nuc

otto

n-33

B)

leav

esco

ntai

ning

Cry

1Ac

toxi

n.

Cor

n (B

t176

) st

empi

ths i

nfus

ed w

ith le

afsu

spen

sion

con

tain

ing

Cry

1Ab

toxi

n.

Cry

1Ac

toxi

n pr

o-du

ced

from

Btk

stra

inH

D-7

3.

Corn

(eve

nt C

BH 3

51)

tiss

ue

cont

aini

ngC

ry9C

toxi

n.

Cor

n (B

t176

) st

empi

ths i

nfus

ed w

ith le

afsu

spen

sion

con

tain

ing

Cry

1Ab

toxi

n.

Red

uced

em

erge

nce

(dep

endi

ng o

n pl

ant

age)

.

Red

uced

num

ber

ofco

coon

s/pu

pae

per

host

, re

duce

d w

eigh

tof

coc

cons

.

Prol

onge

d de

velo

p-m

ent

time,

red

uced

pupa

l wei

ght,

redu

ced

adul

t w

eigh

t, re

duce

dlo

ngev

ity6 .

Incr

ease

d im

mat

ure

mor

talit

y, i

ncre

ased

imm

atur

e de

velo

p-m

ent t

ime,

redu

ced

fe-

mal

e lo

ngev

ity.

Red

uced

par

asiti

sa-

tion,

red

uced

em

er-

genc

e of

adul

ts (

=re-

duce

d nu

mbe

r of s

uc-

cess

fully

par

asiti

zed

coco

ons)

,red

uced

wei

ght o

f fem

ales

.

Cot

ton

rear

ed n

on-ta

r-ge

t lep

idop

tera

n ho

sts

(Pse

udop

lusia

incl

uden

s)pa

rasi

tized

as e

ggs.

Cot

ton

rear

ed n

on-ta

r-ge

t lep

idop

tera

n ho

sts

(Pse

udop

lusia

incl

uden

s)pa

rasi

tized

as e

ggs.

Lep

idop

tera

n ho

st(H

elico

verp

a ar

mig

era)

rear

ed o

n ar

tific

ial d

iet

para

sitiz

ed i

n 2nd

(or

1st ) in

star.

Non

-tar

get

lepi

dop-

tera

n ho

sts(

Eore

uma

lofti

ni)

(28-

32 d

ays

old)

fed

wit

h co

rnst

alk

tissu

e fo

r 48

hpa

rasi

tized

.

Coc

oon

clus

ters

from

host

(pr

imar

y pa

ra-

sito

id C

otes

ia fl

avip

esaf

ter

emer

genc

e fr

omits

her

bivo

rous

hos

tC

hilo

par

tellu

s re

ared

with

in s

tem

pith

s)pa

rasi

tized

.

Entir

e im

mat

ure

deve

l-op

men

t.

Entir

e im

mat

ure

deve

l-op

men

t.

Diff

eren

t (4

-8 d

ays)

depe

ndin

g on

app

lied

conc

entra

tion;

die

t af-

ter

para

sita

tion

not

spec

ified

.

Entir

e im

mat

ure

deve

l-op

men

t (B

t tox

in c

on-

tain

ing

tissu

e in

gut

of

para

lyse

d ho

st).

Expo

sure

to

host

dur

-in

g en

tire

deve

lopm

ent

(see

C. f

lavi

pes a

bove

).

Effe

ct o

f ho

st f

eedi

ngon

use

d tr

ansg

enic

culti

var (

depe

ndin

g on

plan

t age

).

Indi

rect

eff

ects

due

tolo

w q

ualit

y ho

sts

in-

dica

ted.

Indi

rect

eff

ects

due

tolo

w q

ualit

y ho

sts.

Leth

al a

nd s

uble

thal

host

med

iate

d ef

fect

sof

Bt t

oxin

exp

ress

ing

mai

ze ti

ssue

.

Indi

rect

ef

fect

of

trans

geni

c B

t cu

ltiva

r(m

edia

ted

thro

ugh

prim

ary

para

sitoi

d an

dits

her

bivo

rous

hos

t).

Bau

r an

d B

oeth

el(2

003)

Prüt

z an

d D

ettn

er(2

004)

Liu

et a

l. (2

005a

)

Ber

nal e

t al.

(200

2b)

Prüt

z et

al.

(200

4)

Taxo

n/sp

ecie

sSo

urce

of

Bt e

xpos

ure

Stat

istic

ally

signi

fican

tdi

ffer

ence

s com

pare

dto

Bt-f

ree

cont

rol1

durin

g/af

tere

xpos

ure

Expe

rim

enta

l se

t up

wit

h re

gard

to

repo

rted

effe

cts

Dur

atio

n of

Bt e

xpos

ure

Cau

se o

f rep

orte

d di

f-fe

renc

es su

gges

ted/

dis-

cuss

ed b

y th

e au

thor

s

Refe

renc

e


Det

ritiv

ores

:O

ligoc

haet

a:Lu

mbr

icus

ter

rest

ris

L. (L

umbr

icid

ae)

Isop

oda:

Com

mon

roug

hw

oodl

ouse

,Po

rcel

liosc

aber

(Lat

reill

e)(P

orce

llion

idae

)

Cor

n (e

vent

Bt1

1)ex

pres

sing

Cry

1Ab

toxi

n.

Cor

n (e

vent

Bt1

1)ex

pres

sing

Cry

1Ab

toxi

n.

Cor

n (e

vent

s B

t176

,B

t11)

exp

ress

ing

Cry

1Ab

toxi

n.

Red

uced

wei

ght (

200

days

afte

r th

e st

art)7 .

Red

uced

mor

talit

y in

juve

nile

s, re

duce

dw

eigh

t gai

n in

juve

nile

s, fa

ster

wei

ght g

ain

in a

dults

.

Red

uced

con

sum

p-tio

n (D

egre

e de

pend

son

cor

n va

riety

).

Leaf

mat

eria

l fed

toad

ults

in s

oil f

illed

glas

s tu

bes.

Leav

es f

ed to

adu

ltsan

d ne

onat

e ju

veni

les

in e

xper

imen

tal b

oxes

(Con

trol

non-

Bt

mai

ze le

aves

).

Sene

scen

t lea

ves f

edto

adu

lts in

exp

eri-

men

tal b

oxes

.

200

days

120

days

(juve

nile

mor

talit

y);1

31da

ys (j

uven

ilew

eigh

t); 2

2 w

eeks

(adu

lt w

eigh

t).

20 d

ays

Pote

ntia

lly

adve

rse

effe

ct o

f th

e B

ttox

inor

unan

ticip

ated

chan

ges

in th

e pl

ant q

ualit

y or

the

bacte

rialc

omm

unity

on p

lant

s du

e to

the

gene

tic tr

ansf

orm

atio

n.

Bette

r nut

ritio

nal q

ual-

ity o

f Bt f

olia

gesu

ppor

ted

by l

ower

C:N

ratio

, low

er li

gnin

cont

ent a

nd h

ighe

rco

nten

t sol

uble

car

bo-

hydr

ates

mea

sure

d.

Bt e

ffec

ts (d

iffer

ence

sbe

twee

n B

t va

rietie

sac

coun

ted

for d

iffer

ent

toxi

nco

nten

ts), h

ighe

rlig

nin

cont

ent o

rdi

ffere

nt m

icro

bial

colo

nisa

tion.

Zwah

len

et a

l. (2

003)

Esch

er e

t al.

(200

0)

Wan

dele

r et a

l.(2

002)

1 If

sev

eral

con

trol t

reat

men

ts w

ere

incl

uded

in th

e ex

perim

ents

, the

sta

tem

ents

refe

r to

the

treat

men

t whi

ch, w

as m

ost s

imila

r to

the

Bt t

reat

men

t. 2 R

efer

ring

to n

egat

ive

effe

cts

of c

oleo

pter

an-a

ctiv

e B

t tox

ins

on th

e pr

edat

ory

mite

Met

asei

ulus

occ

iden

talis

(Cha

pman

and

Hoy

, 199

1). 3

Sim

ilar e

xper

imen

ts w

ith s

pide

r mite

pre

y co

ntai

ning

high

er to

xin

conc

entra

tions

reve

aled

no

stat

istic

ally

sig

nific

ant e

ffec

ts. 4

Neo

nate

larv

ae w

ere

stro

nger

aff

ecte

d th

an 1

0 da

y ol

d la

rvae

. 5 N

o si

gnifi

cant

diff

eren

ce o

f the

KM

D1-

polle

n+ap

hid

diet

com

pare

d to

the

KM

D2-

polle

n an

d th

e ap

hid-

only

die

t. N

o si

gnifi

cant

diff

eren

ce o

f th

e K

MD

2-po

llen

diet

com

pare

d to

the

non-

Bt-p

olle

ndi

et. 6 E

ffec

ts a

re m

ore

pron

ounc

ed a

t hig

her t

oxin

con

cent

ratio

ns. 7 N

o si

mila

r diff

eren

ces

wer

e ob

serv

ed in

the

field

.

Taxo

n/sp

ecie

sSo

urce

of

Bt e

xpos

ure

Stat

istic

ally

signi

fican

tdi

ffer

ence

s com

pare

dto

Bt-f

ree

cont

rol1

durin

g/af

tere

xpos

ure

Expe

rim

enta

l se

t up

wit

h re

gard

to

repo

rted

effe

cts

Dur

atio

n of

Bt e

xpos

ure

Cau

se o

f rep

orte

d di

f-fe

renc

es su

gges

ted/

dis-

cuss

ed b

y th

e au

thor

s

Refe

renc

e


transgenic plants express Bt toxins in a modifiedform (Hilbeck, 2001) and that plant-expresed Bttoxins can act in concert with other secondarydefense compounds in the plants (Andow andHilbeck, 2004). On the other hand, none of thesestudies confirmed or disproved that the effectsobserved in these non-target species are similar tothose of target species and the mode of action washardly ever studied in adversely affected non-targetspecies. A first exploratory study in this regard wasconducted by Rodrigo-Simon et al. (2006) using Btfed green lacewing larvae (see below) but the studiedpredatory lacewings were not affected and only theprotocols for target herbivores were used.

The Case Example of Bt Toxins and GreenLacewing Larvae

Because the green lacewing is the mostintensively investigated non-lepidopteran, non-targetorganisms to date (Table 2), this case deserves amore detailed analysis. Six studies published on theeffects of Bt toxins are often portrayed as supposedlycontradictory while in reality the differences in theresults can be explained through the differences inthe methodologies used and the underlying researchquestions. In three studies, direct (bi-trophic) effectsof microbially produced Bt toxins were tested(Hilbeck et al., 1998b; Romeis et al., 2004), and infour other studies the effects of prey-mediated (tri-trophic) exposure to Bt toxins from Bt maize (Hilbecket al., 1998a; Dutton et al., 2002) or microbiallyproduced Bt toxins and -protoxins (Hilbeck et al.,1999) were examined (Table 5).

Bi-trophic EffectsDespite the different types of artificial diets used

and parameters measured, a few components of thetwo studies by Hilbeck et al. (1998b) and Romeis etal. (2004) are comparable and yielded indeed similarresults. Hilbeck et al. (1998b) detected a significantdirect lethal effect of Bt toxins that began to manifestitself during the second larval stage but not duringthe first larval stage. Also, Romeis et al. (2004)could not observe adverse effects due to exposure toBt toxin during this larval stage (Table 5, 5: 2.1, 5.2and 5.3). The second instar was not studied.

Hilbeck et al. (1998b) used an artificial dietthat was specifically developed for the commercialmass production of lacewing larvae for biocontrolpurposes and allowed for continuous and completedevelopment of the larvae from egg hatch to adulteclosion. This artificial diet was amended with Bttoxin (100µg/ml) and fed to the lacewing larvaethroughout their entire juvenile feeding stage untilpupation. The authors measured stage-specificmortality and development time. From the secondinstar on, lacewings exhibited a significantly highermortality in the Bt treatment than in the control(Table 5, 5: 2.1) and, additionally, a significantlylonger stage-specific development time. Notably,when only second and third instars were fed with Btdiet, but not the first instars, mortality in the Bt-treatment was still significantly higher than in therespective control but also significantly lower thanin the Bt-treatment that included the first instar.Hence, early instars might be more susceptible thanolder ones, which is consistent with commonknowledge about efficacy of Bt toxins on early larvalstages.

Romeis et al. (2004) used sucrose solution asartificial diet in their trials. This diet does not allow acontinuous and complete development of thesepredaceous larvae. Development of the larvae isarrested, but it allows them to survive periods of lackof prey longer than when sustaining themselves onwater only (Limburg and Rosenheim, 2001). Lacewinglarvae remained in the same larval stage and lived forup to 6 days longer than when being provided withwater only. The only parameter measured was thetime it took until the insects died. Romeis et al. (2004)added Bt toxins to the sucrose solution to seewhether this caused a faster death or not. All testinsects starved to death at the same speed regardlesswhether Bt toxin was added to the sucrose solutionor not (Table 5, 5: 5.2). Also, the exposure to Btsucrose solution during only a part of the first instar– 6 out of 11 days – did not result in a difference,when untreated flour moth eggs were providedafterwards. This high quality food allowed forrecovery of the larvae without sustainedconsequences.

Rodrigo-Simon et al. (2006) provided lacewinglarvae with a total of ca. 3-4 droplets of watercontaining Bt toxin at a low concentration,


Tabl

e 5.

Com

para

tive

over

view

of

six

labo

rato

ry s

tudi

es in

vest

igat

ing

the

effe

cts

of B

t to

xins

on

Gre

en la

cew

ing

(Chr

ysop

erla

car

nea)

larv

ae.

1 (H

ilbec

k et

al.,

Tri-t

roph

ic;

Expe

rimen

t 1.1

:19

98a)

Bt m

aize

(Bt 1

1),

Rep

licat

ions

: 4Fo

od: c

ater

pilla

rsFo

od: c

ater

pilla

rsFo

od: c

ater

pilla

rsiso

geni

c con

trol

Trea

tmen

ts: 2

(non

-targ

et sp

ecie

s)(n

on-ta

rget

spec

ies)

(non

-targ

et sp

ecie

s)m

aize

Larv

ae/tr

t.: 5

0Su

pple

men

ted

with

N =

400

flour

mot

h eg

gs

Dur

atio

n of

Bt e

xpos

ure

Entir

e sta

geEn

tire s

tage

Entir

e sta

ge

Para

met

ers:

Mor

talit

y:B

t: 2

4%B

t: 4

0%B

t: 1

1%B

t: 0

%B

t: 6

0%C

o: 1

0%C

o: 2

1%C

o: 7

%C

o: 2

%C

o: 3

7%

Dev

elop

men

tB

t: 5

.0 d

ays

Bt:

6.5

day

sB

t: 7

.3 d

ays

Bt:

12.

5 da

ysB

t: 3

1 da

ystim

e:C

o: 4

.5 d

ays

Co:

6.5

day

sC

o: 7

.8 d

ays

Co:

12.

5 da

ysC

o: 3

1 da

ys

Expe

rimen

t 1.2

:R

eplic

atio

ns: 4

Food

: cat

erpi

llars

Food

: cat

erpi

llars

Food

: cat

erpi

llars

Trea

tmen

ts: 2

(non

-targ

et sp

ecie

s)(n

on-ta

rget

spec

ies)

(non

-targ

et sp

ecie

s)La

rvae

/trt.:

50

N =

400

Dur

atio

n of

Entir

e sta

geEn

tire s

tage

Entir

e sta

geB

t exp

osur

e

Para

met

ers:

Mor

talit

y:B

t: 2

9%B

t: 4

5%B

t: 1

1%B

t: 8

.0%

Bt:

66%

Co:

10%

Co:

20%

Co:

7%

Co:

2.5

%C

o: 3

8%

Dev

elop

men

tB

t: 5

.8 d

ays

Bt:

7.5

day

sB

t: 7

.5 d

ays

Bt:

12.

5 da

ysB

t: 3

2 da

ystim

e:C

o: 5

.1 d

ays

Co:

5.1

days

Co:

6.5

day

sC

o: 1

2.5

days

Co:

29

days

2 (H

ilbec

k et

al.,

Bi-t

roph

ic; m

icro

-Ex

perim

ent 2

.1:

199

8b)

bial

ly p

rodu

ced

Rep

licat

ions

: 5Fo

od: l

acew

ing

diet

Food

: lac

ewin

g di

etFo

od: l

acew

ing

diet

Bt t

oxin

(Cry

1Ab)

Trea

tmen

ts: 2

[100

µg/m

l],La

rvae

/trt.:

30

Bt-f

ree

cont

rol

N =

300

Stud

ySt

udy

mat

eria

lD

esig

n of

indi

vidu

alex

perim

ents

Firs

t ins

tar (

L1)

Seco

nd in

star

(L2)

Third

inst

ar (L

3)Pu

pa(n

on-fe

edin

g)En

tire j

uven

ile st

age

(L1-

adul

t)


Dur

atio

n of

Entir

e sta

geEn

tire s

tage

Entir

e sta

geB

t exp

osur

e

Para

met

ers:

Mor

talit

y:B

t: 6

%B

t: 2

6%B

t: 2

2%B

t: 3

4%B

t: 5

7%C

o: 6

%C

o: 8

%C

o: 1

2%C

o: 1

4%C

o: 3

0%

Dev

elop

men

tB

t: 7

day

sB

t: 1

1 da

ysB

t: 1

2 da

ysB

t: 1

2 da

ysB

t: 3

7.5

days

time:

Co:

7 d

ays

Co:

10

days

Co:

10

days

Co:

12

days

Co:

37.

5 da

ys

Expe

rimen

t 2.2

:R

eplic

atio

ns: 5

Food

: flo

ur m

oth

Food

: lac

ewin

g di

etFo

od: l

acew

ing

diet

Trea

tmen

ts: 2

eggs

Larv

ae/tr

t.: 3

0N

= 3

00

Dur

atio

n of

Non

eEn

tire s

tage

Entir

e sta

geB

t exp

osur

e

Para

met

ers:

Mor

talit

y:B

t: 2

%B

t: 1

5%B

t: 7

.5%

Bt:

7.5

%B

t: 2

9%C

o: 2

%C

o: 6

%C

o: 5

.0%

Co:

4.0

%C

o: 1

7%

Dev

elop

men

tB

t: 4

.5 d

ays

Bt:

4.3

day

sB

t: 7

.5 d

ays

Bt:

12

days

Bt:

28.

0 da

ystim

e:C

o: 4

.5 d

ays

Co:

4.0

day

sC

o: 7

.5 d

ays

Co:

12

days

Co:

27.

5 da

ys

Expe

rimen

t 2.3

:R

eplic

atio

ns: 5

Food

: flo

ur m

oth

Food

: lac

ewin

g di

etFo

od: l

acew

ing

diet

Trea

tmen

ts: 1

eggs

Larv

ae/tr

t.: 3

0N

= 3

00

Dur

atio

n of

Non

eEn

tire s

tage

Entir

e sta

geB

t exp

osur

e

Para

met

ers:

Mor

talit

y :

ca. 1

%0

%0.

5 %

5%8%

Dev

elop

men

t4.

5 da

ys3.

2 da

ys4.

3 da

ys12

day

s23

day

stim

e:

Stud

ySt

udy

mat

eria

lD

esig

n of

indi

vidu

alex

perim

ents

Firs

t ins

tar (

L1)

Seco

nd in

star

(L2)

Third

inst

ar (L

3)Pu

pa(n

on-fe

edin

g)En

tire j

uven

ile st

age

(L1-

adul

t)


3 (H

ilbec

k et

al.,

Tri-t

roph

ic;

Expe

rimen

t 3:

1999

)m

icro

bial

lyR

eplic

atio

ns: 4

Food

: cat

erpi

llars

Food

: cat

erpi

llars

Food

: cat

erpi

llars

prod

uced

Bt t

oxin

s,Tr

eatm

ents

: 8(n

on-ta

rget

spec

ies)

(non

-targ

et sp

ecie

s)(n

on-ta

rget

spec

ies)

Btt

oxin

s, B

t-fre

eLa

rvae

/trt.:

30

Bt-f

ree

cont

rol

Larv

ae/tr

t. : 3

0N

= 9

60

Dur

atio

n of

Entir

e sta

geEn

tire s

tage

Entir

e sta

geB

t exp

osur

e

Para

met

ers:

Mor

talit

y:

Cry

1Ab

toxi

n[µ

g/m

l]: 10

0, 50

, 25

35, 1

8, 1

0%25

, 12.

5, 1

7.5%

35, 4

6, 3

1%40

, 35,

24%

78, 6

9, 5

5%

Cry

1Ab

prot

oxin

[µg/m

l]: 20

0, 10

0, 50

12.5

, 12,

17.

5%16

, 15,

20%

33, 2

5, 4

1%14

, 9, 2

0%56

, 46,

62%

Cry

2A p

roto

xin

[µg/

ml]:

100

10%

14%

24%

15%

47.5

%C

ontro

l6%

4%13

%10

%26

%

4 (D

utto

n et

al.

Tri-t

roph

ic;

Expe

rimen

t 4.1

:20

02)

Bt m

aize

(Bt 1

1),

Rep

licat

ions

: 2Fo

od: c

ater

pilla

rsFo

od: c

ater

pilla

rsFo

od: c

ater

pilla

rsiso

geni

c con

trol

Trea

tmen

ts: 2

(non

-targ

et sp

ecie

s)(n

on-ta

rget

spec

ies)

(non

-targ

et sp

ecie

s);

mai

zeLa

rvae

/trt.:

30

repl

aced

with

N =

120

flour

mot

h eg

gs

Dur

atio

n of

Entir

e sta

geEn

tire s

tage

2 da

ysB

t exp

osur

e

Para

met

ers:

Surv

ival

Rat

e :B

t: 5

0% (5

0%)

Bt:

40%

(60%

)B

t: 9

0% (1

0%)

=/<

2%B

t: 2

0% (8

0%)

(Mor

talit

y):

Co:

90%

(10%

)C

o: 6

5% (3

5%)

Co:

95%

(5%

)C

o: 6

0% (4

0%)

Dev

elop

men

tB

t: 5

day

sB

t: 8

day

sB

t: 5

day

sB

t: 2

4 da

ystim

e :C

o: 3

day

sC

o: 6

day

sC

o: 5

day

sC

o: 2

1 da

ys

Wei

ght:

–B

t:

< 1m

gB

t: 2

mg

Bt:

9

mg

Co:

ca. 1

mg

Co:

2 m

gC

o: 1

0 m

g

Stud

ySt

udy

mat

eria

lD

esig

n of

indi

vidu

alex

perim

ents

Firs

t ins

tar (

L1)

Seco

nd in

star

(L2)

Third

inst

ar (L

3)Pu

pa(n

on-fe

edin

g)En

tire j

uven

ile st

age

(L1-

adul

t)


Expe

rimen

t 4.2

:R

eplic

atio

ns: 2

Food

: spi

der m

ites

Food

: spi

der m

ites

Food

: spi

der m

ites

Trea

tmen

ts: 2

+ flo

ur m

oth

eggs

Larv

ae/tr

t.: 3

0N

= 1

20D

urat

ion

ofEn

tire s

tage

Entir

e sta

ge2

days

Bt e

xpos

ure

Para

met

ers

Surv

ival

rate

Bt:

95%

(5%

)B

t: 1

00%

(0%

)B

t: 9

5% (5

%)

=/<

2%B

t: 9

5% (5

%)

(Mor

talit

y):

Co:

95%

(5%

)C

o: 9

5% (5

%)

Co:

95%

(5%

)C

o: 9

0% (1

0%)

Dev

elop

men

tC

o: 3

day

sB

t: 3

day

sB

t: 4

day

sB

t: 1

9 da

ystim

e :B

t: 3

day

sC

o: 3

day

sC

o: 4

day

sC

o: 2

0 da

ysW

eigh

t:—

Bt:

ca.

1 m

gB

t: c

a. 3

mg

Bt:

ca.

8 m

gC

o: 9

5% (5

%)

Co:

ca. 1

mg

Co:

ca. 3

mg

Co:

ca. 8

mg

Expe

rimen

t 4.3

:R

eplic

atio

ns: 2

Food

: aph

ids

Food

: aph

ids

Food

: aph

ids

Trea

tmen

ts: 2

Larv

ae/tr

t.: 3

0N

=120

Dur

atio

n of

Non

e1N

one1

Non

e1

Bt e

xpos

ure

Para

met

ers

Surv

ival

rate

Bt:

96%

(4%

)B

t: 9

8% (2

%)

Bt:

98%

(2%

)=/

< 2%

Bt:

95%

(5%

)(M

orta

lity)

:C

o: 9

6% (4

%)

Co:

98%

(2%

)C

o: 1

00%

(0%

)C

o: 9

2% (8

%)

Dev

elop

men

tC

o: 3

day

sB

t: 3

day

sB

t: 4

day

sB

t: 2

0 da

ystim

e :B

t: 3

day

sC

o: 3

day

sC

o: 4

day

sC

o: 2

0 da

ysW

eigh

t:—

Bt:

ca.

1 m

gB

t: c

a. 2

mg

Bt:

ca.

9 m

gC

o: ca

. 1 m

gC

o: ca

. 2 m

gC

o: ca

. 9 m

g

5 (R

omei

s et a

l.B

i-tro

phic

,Ex

perim

ent 5

.1:

2004

)m

icro

bial

lyRe

plic

atio

ns: 1

Food

: sug

ar so

lutio

n—

—pr

oduc

ed B

t tox

inTr

eatm

ents

: 2(C

ry1A

b), B

t-fre

eLa

rvae

/trt.:

40

cont

rol

N=8

0

Stud

ySt

udy

mat

eria

lD

esig

n of

indi

vidu

alex

perim

ents

Firs

t ins

tar (

L1)

Seco

nd in

star

(L2)

Third

inst

ar (L

3)Pu

pa(n

on-fe

edin

g)En

tire j

uven

ile st

age

(L1-

adul

t)


Dur

atio

n of

30 m

inut

es—

—B

t exp

osur

e

Para

met

er:

Upt

ake

rate

(%B

t: 1

5.7%

wei

ght d

iffer

ence

)C

o: 1

4.7%

Expe

rimen

t 5.2

:Re

plic

atio

ns: 6

Food

: sug

ar so

lutio

n—

—Tr

eatm

ents

: 5(4

Bt c

once

n-tra

tions

+ C

o)La

rvae

/trt.:

10

N=3

00

Dur

atio

n of

Prov

ided

unt

il de

ath

——

Bt e

xpos

ure

Para

met

er:

Tim

e to

dea

thB

t (4

conc

.):(la

rval

dev

elop

-9-

10 d

ays

men

t st

oppe

d)C

o: 9

.5 d

ays

Expe

rimen

t 5.3

:Re

plic

atio

ns: 3

Food

: Foo

d: su

gar

Food

:flou

r mot

hFo

od:fl

our m

oth

Trea

tmen

ts: 2

solu

tion

+eg

gs eg

gsLa

rvae

/trt.:

20

flour

mot

h eg

gsN

=120

Dur

atio

n of

6 da

ysN

one

Non

eB

t exp

osur

e

Para

met

ers:

Surv

ival

rate

Bt:

87.

9% (1

2.1%

)B

t: 9

6.1%

(3.9

%)

——

—C

o: 8

4.7%

(15.

3%)

Co:

96.

0% (4

.0%

)—

——

Dev

elop

men

tB

t: 5.

1 da

ys (+

6 B

t)B

t: 3

.4 d

ays

time:

Co:

5.1

day

s (+

6 B

t)C

o: 3

.4 d

ays

Dry

wei

ght L

3—

—B

t: 12

52m

g—

Co:

113

9mg

Stud

ySt

udy

mat

eria

lD

esig

n of

indi

vidu

alex

perim

ents

Firs

t ins

tar (

L1)

Seco

nd in

star

(L2)

Third

inst

ar (L

3)Pu

pa(n

on-fe

edin

g)En

tire j

uven

ile st

age

(L1-

adul

t)


Expe

rimen

t 5.4

:Re

plic

atio

ns: 3

Food

: cat

erpi

llars

Food

:Fo

od:

Trea

tmen

ts: 6

(non

-targ

et sp

ecie

s)B

t- su

gar s

olut

ion

Bt-

suga

r sol

utio

nLa

rvae

/trt.:

10

or fl

our m

oth

eggs

suga

r sol

utio

nsu

gar s

olut

ion

(flo

ur m

oth

eggs

),w

ater

wat

er20

(cat

erpi

llars

)N

= 90

(flo

ur m

oth

eggs

), N

= 18

0(c

ater

pilla

rs)

Dur

atio

n of

Non

eYe

s, al

l lar

vae –

Unc

lear

(brie

f) -

——

Bt e

xpos

ure

dura

tion

uncl

ear

few

larv

aean

d di

ffer

ent

Para

met

er:

Surv

ival

rate

(mor

talit

y) :

Flou

r mot

heg

gs (L

1)C

o: 9

8.9%

(1.1

%)*

——

Cat

erpi

llars

(L1)

Co:

72.

2% (2

7.8%

)—

—

Dev

elop

men

t tim

e:Fl

our m

oth e

ggs (

L1)C

o: 3

.7 da

ys*

——

Cat

erpi

llars

(L1)

Co:

5.7

day

s—

—

Tim

e to

dea

th:

Flou

r mot

h—

46 –

47

days

Not

diff

eren

tiate

deg

gs (L

1)—

46 –

47

days

from

L2

—5.

6 da

ys (w

ater

)

Cat

erpi

llars

(L1)

—20

– 2

1 da

ysN

ot d

iffer

entia

ted

—20

– 2

1 da

ys fr

om L

2—

2.1

days

(wat

er)

Perc

enta

ge to

L3:

Flou

r mot

h egg

s (L1

)—

—37

.9 – 4

6.7%

Cat

erpi

llars

(L1)

——

0-2.

4% (w

ater

)

Stud

ySt

udy

mat

eria

lD

esig

n of

indi

vidu

alex

perim

ents

Firs

t ins

tar (

L1)

Seco

nd in

star

(L2)

Third

inst

ar (L

3)Pu

pa(n

on-fe

edin

g)En

tire j

uven

ile st

age

(L1-

adul

t)


6 (R

odrig

o-Si

mon

Tri-t

roph

ic;

Expe

rimen

t 6.1

:Fo

od: n

oFo

od: M

ixed

Food

: Mix

edet

al.

2006

)m

icro

bial

lyR

eplic

atio

ns: 3

info

rmat

ion?

5 ca

terp

illar

s5

cate

rpill

ars

prod

uced

Bt t

oxin

s:Tr

eatm

ents

: 9(ta

rget

spec

ies,

(targ

et sp

ecie

s,C

ry1A

b, C

ry1A

c,La

rvae

/trt.:

10

stag

e uns

peci

fied)

stag

e uns

peci

fied)

Cry

2Ab

N=2

70pe

r da

y,pe

r da

y,[1

-10µ

g/m

l]flo

ur m

oth

eggs

flour

mot

h eg

gsB

t-fre

e co

ntro

lev

ery

othe

r day

ever

y ot

her d

ay(c

hoic

e).

(cho

ice)

.D

urat

ion

ofPr

esum

ably

non

eEn

tire s

tage

Entir

e sta

geB

t exp

osur

ePa

ram

eter

s:Su

rviv

al ra

teL2

-PL2

-A(M

orta

lity)

:C

ry1A

c—

Not

pro

vide

dN

ot p

rovi

ded

Bt:

ca. 8

5-90

%B

t:ca

.65-

70%

(bot

h co

nc.)

(ca.

10-

15%

)(c

a. 3

0-35

%)

Co:c

a. 8

5%Co

:ca

. 60%

(ca.

15%

)(c

a. 4

0%)

Cry

1Ab

—N

ot p

rovi

ded

Not

pro

vide

dB

t:ca

. 90-

95%

Bt:

ca. 7

5-85

%(b

oth

conc

.)(c

a. 5

-10%

)(c

a. 1

5-25

%)

Co:c

a. 9

0%Co

:ca

. 70%

(ca.

10%

)(c

a. 3

0%)

Cry

2Ab

—N

ot p

rovi

ded

Not

pro

vide

dB

t: 10

0%B

t:ca

. 75-

85%

(bot

h co

nc.)

(0%

)(c

a. 1

5-25

%)

Co:

100

%Co

:ca

. 83%

(0%

)(c

a. 1

7%)

Pupa

L2-A

Dev

elopm

ent ti

me:

Cry

1Ac

—B

t: 2

.1-2

.3 d

Bt:

5.2-

5.8

dB

t: 12

.8-1

3.7

dB

t:20

.2-2

1.8

d(b

oth

conc

.)C

o: 2

dC

o: 5

.5 d

Co:

13.

5 d

Co:

21

dC

ry1A

b—

Bt:

1.8

-1.9

dB

t: 4

.9-5

.0 d

Bt:

12.

3-12

.6 d

Bt:

19.1

-19.

5 d

(bot

h co

nc.)

Co:

2.3

dC

o: 5

.4 d

Co:

12.

8 d

Co:

20.

5 d

Cry

2Ab

—B

t: 1

.8-2

.0 d

Bt:

5.5

-5.7

dB

t: 1

3.8-

14.3

dB

t:21

.1-2

2 d

(bot

h co

nc.)

Co:

1.9

dC

o: 5

.6 d

Co:

14

dC

o: 2

1.6

d

Stud

ySt

udy

mat

eria

lD

esig

n of

indi

vidu

alex

perim

ents

Firs

t ins

tar (

L1)

Seco

nd in

star

(L2)

Third

inst

ar (L

3)Pu

pa(n

on-fe

edin

g)En

tire j

uven

ile st

age

(L1-

adul

t)


Bi-t

roph

ic;

Expe

rimen

t 6.2

:Fo

od: ?

Food

: Mix

edFo

od: M

ixed

micr

obial

ly pr

oduc

edR

eplic

atio

ns: 3

no in

form

atio

n1

drop

wat

er w

/wo

1 dr

op w

ater

w/w

oB

t tox

in (C

ry1A

b)Tr

eatm

ents

: 2B

t 24

h flo

ur m

oth

Bt 2

4 h

flour

mot

h[4

mg/

ml],

Bt-f

ree

Larv

ae/tr

t.: 1

0eg

gs 2

4 h

star

vatio

neg

gs 2

4 h

star

vatio

nco

ntro

lN

=60

Proc

edur

e re

peat

edPr

oced

ure

repe

ated

2-3

times

unt

il2-

3 tim

es u

ntil

pupa

tion.

pupa

tion.

Dur

atio

n of

Bt

Pres

umab

ly n

one

Ever

y 3rd

day

Ever

y 3rd

day

expo

sure

Para

met

ers:

Surv

ival

rate

L2-P

L2-A

(Mor

talit

y):

Cry

1Ac

—

Not

pro

vide

dN

ot p

rovi

ded

Bt:

ca. 9

5%B

t:ca

. 80%

(ca.

5%)

(ca.

20%

)Co

:100

%Co

:ca

. 87%

(0%

)(c

a.13

%)

Dev

elop

men

t tim

e :

Pupa

Cry

1Ac

—B

t: 2

.2 d

Bt:

5.5

dB

t: 1

2.0

dB

t: 2

0.0

dC

o: 2

.1 d

Co:

5.8

dC

o: 1

2.3

dC

o: 1

9.9

d

*Dat

a tha

t wer

e not

incl

uded

in ta

bles

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approximately 1 drop every 2-3 days until pupation.The larvae were raised on flour moth eggsinterrupted by 24 h starvation periods prior to theadministration of the water drop. Exposure beganwith the second larval stage. First instars were notexposed. No effects were observed.

Tri-trophic EffectsBoth Hilbeck et al. (1998a) and Dutton et al.

(2002) fed lacewing larvae with caterpillars that eitherhad fed on Bt maize or isogenic maize. Hilbeck et al.(1998a) continued to feed lacewings with Bt preyuntil pupation, supplemented with meal moth eggsduring the last instar (3 larval stages), while Duttonet al. (2002) stopped feeding Bt prey two days afterlarvae had reached the third instar and reared themexclusively on meal moth eggs until pupation(average 3 days, Table 6). Despite these differences,in both studies significantly more lacewing larvaedied when they were raised with prey caterpillarsthat contained Bt toxin. Dutton et al. (2002) furtherconducted similar feeding studies with other typesof prey, aphids and spider mites. For both prey types,lacewing larvae developed and died at similar ratesregardless whether their prey had fed on Bt orisogenic maize (Table 6). For aphids, this can beexplained, because as strict phloem-feeders they didnot contain Bt toxin. Raps et al. (2001) and Head etal. (2001) did not detect any Bt toxin in the phloemof Bt maize or in aphids feeding on it. By contrast,spider mites did ingest the Bt toxin from the Bt maizebut this did not induce higher mortality in thelacewing larvae. However, no studies on thebiochemical processing of the Bt toxin in the spidermites and its sustained bioactivity were conducted.

Hilbeck et al. (1999) conducted furtherexperiments where they fed lacewing larvae with preycaterpillars that had fed on artificial diet containingdifferent concentrations of microbially produced Bttoxins. Again, significantly higher mortality rates inlacewings were observed that increased as theconcentration of the Bt toxin in the diet for their preyincreased (Table 6). While the prey caterpillars onlyshowed significantly higher mortality of 42% at thehighest Bt toxin concentration, lacewing larvaeexhibited a lethal effect at all concentrationsexceeding 70% when their prey had fed on the

highest concentration diet. At the lowerconcentrations, caterpillars only exhibited sublethaleffects, i.e., reduced weight, when feeding on the dietfor several days. However, when designated as foodfor lacewings in the experiments, caterpillars wereonly allowed to feed for 12-24 h on the Bt diet. Theydid not exhibit noticable adverse effects at that time.

Rodrigo-Simon et al. (2006) raised lacewinglarvae (again, beginning second instar) on five H.armigera larvae per day, supplemented with flourmoth eggs every other day. First instars were notexposed. Also here, untreated flour moth eggsconstituted a significant part of their diet, if not themain part. However, five H. armigera larvae per dayseems rather few. In a study, where lacewing larvaewere offered caterpillars and aphids in a choice andno-choice setup, Meier and Hilbeck (2001) reportedconsumption rates of 4-5 prey caterpillars in only 4hours for second instar lacewing larvae when noother food was available. When a preferred food wasoffered, in that case aphids, second instar lacewinglarvae still ate 2-3 prey larvae in 4 hours. Within the4 hour period, third instar lacewing larvae on averageate only 6-9 prey larvae in a no-choice situation and5-9 prey larvae in a choice situation. Hence, lacewinglarvae in the Rodrigo-Simon et al. study (2006) wereraised, to a substantional degree, on untreated flourmoth egg diet offered every other day. Well-fed larvaecan easily survive one day with a less preferred foodtype in limited supply, possibly allowing for atemporary recovery from the Bt-treatment.

DISCUSSION

Laboratory Studies with Bt Spray FormulationMost laboratory testing on non-target effects

was conducted with spray formulations that arebased on B. thuringiensis subsp. kurstaki (Btk)producing toxins of the Cry1 family and with purifiedCry1 toxins (MacIntosh et al., 1990; Melin and Cozzi,1990), fewer with Bt toxins active against other insectgroups. Studies on non-target effects of Bt sprayformulations showed conflicting results with noeffects in some cases and lethal or sublethal effectsin others. Many of the effects of Bt sprayformulations observed in earlier studies wereattributed to the occurrence of β-exotoxins, which


Table 6. Comparison of the individual similar components of five studies investigating non-target effectsof Bt toxins and Bt-fed prey on Green lacewing (Chrysoperla carnea) larvae (Complementary to Table 5).

Experiment 1.1 4.1 1.1 4.1 5.4 2.3 5.4 2.1 5.2 2.1 5.2Instar Parameter Bt-prey Bt-free prey Food Flour moth Bt artificial Control artificial

as control type eggs diet dietunclear

L1 Mortality 24% 50% 10% 10% 27.8% 1-2% 1% 6% — 6% —Development 5 5 4.5 3 5.7 4.5 3.7 7 — 7 —time days days days days days days days days daysTime to death 9.5 9.5

L2 Mortality 40% 60% 21% 35% — — —Development 6.5 8 6.5 6time days days days days — — —

L1 – A* Mortality 60% 80% 37% 40% — — —Development 31 24 31 21time days days days days — — —

1.1: Hilbeck et al. (1998a) ; 2.1 + 2.3: Hilbeck et al. (1998b); 4.1: Dutton et al. (2002); 5.2 + 5.4: Romeis et al. (2004). *Datathat were not included in tables or in the text were derived from figures of the publications. In these cases, a small inaccuracyin the data presented here is possible..

are known to have a more general toxicity (Sebastaet al., 1981; Melin and Cozzi, 1990), but this wasseldom followed up. The awareness of the acutetoxicity of β-exotoxins on non-target organisms hasled to the proscription of formulations containingthese substances (Lacey and Siegel, 2000). If adverseeffects were observed, both direct (effect of the toxin)and indirect (effects of toxin affected prey/host, whichprovides a less suitable food source) were discussed(Flexner et al., 1986). Despite a considerable numberof studies reporting adverse effects on non-targetinvertebrates, Bt formulations based on δ-endotoxins(Cry proteins) are assumed to be highly specific andhave negligible effects on non-target organismsbecause of their limited bioactivity under fieldconditions (Ignoffo and Garcia, 1978).

Laboratory Studies with Transgenic Bt PlantsNon-target invertebrates and Bt interactions. Overall,the results of the published laboratory studies on non-target effects in the context with transgenic Bt plantsare inconsistent, and no coherent and predictablepattern of the observed Bt effects is emerging yet. Allstudies still represent pieces of a puzzle, the picture ofwhich is not recognizable at this time. The majority ofstudies are isolated experiments following quitedifferent methodologies. Further, the results of thosestudies, which tested a few organisms repeatedly (e.g.

lacewing larvae), did not lead to a scientificconsensus regarding the kind of impact transgenic Btplants might exert and the responsible mode ofaction. In fact, similar to the cases described byCrickmore (2005) (see above), rather differing linesof interpretation complicated the situation further. Assubtle differences in toxin structure could affectbinding and host specificity (Crickmore, 2005), theuncertainty regarding structural differences of Bttoxins expressed by Bt transgenic plants when, inaddition, passing through the digestive tract ofherbivore insects could well explain effects foundwith non-target species. While Crickmore (2005)contemplates that too much research has been put onbinding at the expense of other factors that mighthave an equally important role in determining theefficacy of a toxin, we argue that sublethal effects orlethal effects in non-target organisms at high toxinconcentrations could also be triggered by othermechanisms that are masked in target species by thelethal effects induced through the commonly knownmode of action.Green lacewing larvae and Bt interactions –different interpretations of the same data andremaining gaps of knowledge. From their resultsfrom direct and prey-mediated Bt feeding trials,Romeis et al. (2004), Dutton et al. (2002) andRodrigo-Simon et al. (2006) concluded that the


observed mortality in Bt-fed lacewing larvae is solelydue to lower nutritional quality of the sublethallyaffected prey without the Bt-toxin having a role init. We find this unlikely and a too limitedinterpretation. Firstly, the direct effects of the Bttoxin feeding study clearly document the sensitivityof C. carnea larvae, certainly at higherconcentrations (Hilbeck, et al., 1998b), and cannotbe explained by reduced prey quality as Bt toxinwas fed directly to the predator using a specificlacewing diet. The direct feeding trials by Romeiset al. (2004) and Rodrigo-Simon et al. (2006)complement the findings by Hilbeck et al. (1998b)in as much as they document that short term orintermittent exposure to Bt toxin at mostly lowconcentrations do not lead to measurable adverseeffects, in particular, when lacewing larvae weresubsequently raised on an optimal Bt-free diet. Thetotality of the data on lacewings, but also on othernon-target species and Bt, indeed confirm earlierconclusions (Hilbeck, 2002; Andow and Hilbeck,2004) that complex interactions are involved. Thesecould involve other modes of action of the Bt toxinsor its metabolites, and altered chemistry of Bt toxinswhen, firstly, expressed in a plant and, secondly,passing through the gut of a herbivore preyorganism, including possibly one or all of thefollowing: a) altered nutritional prey quality, b)toxicity of the Bt toxin or its metabolites, c) toxicityof natural plant secondary metabolites interactingwith the Bt toxin/metabolites. To keep theseprocesses apart experimentally is impossible, as toomany possible interactions can be involved (Andowand Hilbeck, 2004).

However, the sustained scientific dissenthighlights another understudied issue concerningthe spread, processing, degradation and re-cyclingof Bt toxins in above- and below-ground ecosystemsand how its bioactivity can be affected by theseprocesses. Only 10 years after large scale commercialproduction of Bt crops in some countries, the firststudies were published that investigated the fateand spread of the expressed novel protein in thefood chain and insect community of the Bt croppingsystem. Harwood et al. (2005) demonstrated thatthe Bt toxins have spread in the food chain and

found surprisingly high concentrations in somehigher trophic level organisms while not in others.Similar results were reported by Obrist et al. (2006a)for different non-target herbivores and predators.In spider mites, Tetranychus urticae, the authorseven documented a concentration that was threetimes higher than in maize leaves the mites had fedupon. Zwahlen and Andow (2005) found the Bttoxin in field collected carabid beetles even if no Btmaize had been planted in the year before. Keyexperiments regarding the molecularcharacterization of Bt toxins – whether or not theyare degraded – and their bioactivity are needed tobetter understand the spread of Bt toxins throughfood webs.

Only one study (Rodrigo-Simon, et al., 2006)has so far considered the mode of action of Bt toxinsin the gut of C. carnea. Based on their results frombinding studies with lacewing midguts by followingthe procotols developed for caterpillars, Rodrigo-Simon et al. (2006) conclude that C. carnea larvaelack specific receptors for Cry1Ab and Cry1Ac. Fromthis, we conclude that Bt toxins do not operate inpredatory lacewing larvae like in herbivorouscaterpillars, but the key experiments on what causedthe significantly higher mortality in Bt-exposedlacewings larvae are still missing to date.

CONCLUSIONSThe reports of adverse effects of Bt toxins and

transgenic Bt plants on arthropod species other thanthe target pest insects went surprisingly unnoticedby the scientific expert community studying Bt modesof action. Some experts simply attributed tri-trophiceffects to poorer prey quality, while the bi-trophicdirect adverse Bt effects were considered as“unlikely”, because it did not fit the commonlyaccepted model of Bt toxin mode of action in targetspecies. While reports of unexplained effects on non-target species and the lack of explanations of theircauses should call for more research, key experimentson alternative modes of action in non-targetinvertebrates are still missing. This is even moreastonishing as the cultivation area of transgenic Btcrop plants increases continuously worldwide (James,2005). Most of the tested species exhibitingunexplained effects are common members of the


insect community occurring in Bt fields and areexposed to Bt toxins (Harwood et al., 2005; Zwahlenand Andow, 2005; Obrist et al., 2006a). Crickmore(2005) emphasises that novel approaches should beapplied in order to provide insights into the complexnature of the toxin-host interactions. A more detailedknowledge of Bt interactions could lead to thedevelopment of improved Bt biopesticides(Crickmore, 2005). We argue that this should not onlybe restricted to target insects but would alsocontribute to a better understanding of unexplainedeffects in non-target organisms. In fact, a recent paperexplores the possibility of alternate modes of actionin target insects (Zhang et al., 2006). Additionally,there are emerging models for the mode of action intarget insects that involve new elements like dualbinding to aminopeptidases and cadherins, lipidtargeting and more (e.g. Bravo et al., 2004). Mostrecently, Broderick, et al (2006) reported that thepresence of certain midgut bacteria is required for Bttoxins to unfold its activity in the investigated targetinsect. Again, we argue that this might also helpexplain some of the peculiar effects observed withnon-target organisms. The currently existing modelof Bt mode of action might have to be revised soon.We believe that including non-target organims intoBt research offers great opportunities to help improveour understanding on what else Bt might do. Withthis review, we hope to stimulate more research andthinking ‘outside of the box’.

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Accepted 15 June 2006

another view on bt proteins – how specific are they and...

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