Managing resistance evolution to transgenic Bt maize in corn borers in Spain.

Since 1998, genetically engineered Bt maize varieties expressing the insecticidal Cry1Ab protein (i.e. event MON 810) have been grown in the European Union (EU), mainly in Spain. These varieties confer resistance against the European and Mediterranean corn borer (ECB and MCB), which are the major lepidopteran maize pests in the EU, particularly in Mediterranean areas. However, widespread, repeated and exclusive use of Bt maize is anticipated to increase the risk of Cry1Ab resistance to evolve in corn borer populations. To delay resistance evolution, typically, refuges of non-Bt maize are planted near or adjacent to, or within Bt maize fields. Moreover, changes in Cry1Ab susceptibility in field populations of corn borers and unexpected damage to maize MON 810, due to corn borers, are monitored on an annual basis. After two decades of Bt maize cultivation in Spain, neither resistant corn borer populations nor farmer complaints on unexpected field damage have been reported. However, whether the resistance monitoring strategy followed in Spain, currently based on discriminating concentration bioassays, is sufficiently sensitive to timely detect early warning signs of resistance in the field remains a point of contention. Moreover, the Cry1Ab resistance allele frequency to Bt maize, which has recently been estimated in MCB populations from north-eastern Spain, might exceed that recommended for successful resistance management. To ensure Bt maize durability in Spain, it is key that adequate resistance management approaches, including monitoring of resistance and farmer compliance with refuge requirements, continue to be implemented and are incorporated in integrated pest management schemes.

Risk Assessment Considerations for Genetically Modified RNAi Plants: EFSA's Activities and Perspective.

Genetically modified plants (GMPs) intended for market release can be designed to induce "gene silencing" through RNA interference (RNAi). The European Food Safety Authority (EFSA) and other international risk assessment bodies/regulatory agencies have taken several actions to determine whether the existing risk assessment approaches for GMPs are appropriate for the risk assessment of RNAi-based GMPs or require complementary or alternative approaches. To our knowledge, at the international level, no dedicated guidelines have been developed for the risk assessment and regulation of RNAi-based GMPs, confirming that existing science-based risk assessment approaches for GMPs are generally considered suitable for RNAi-based GMPs. However, some specificities have been identified for the risk assessment of RNAi-based GMPs. Here, we report on some of these specificities as identified and addressed by the EFSA GMO Panel for the molecular characterisation, food/feed safety assessment and environmental risk assessment of RNAi-based GMPs, using the DvSnf7 dsRNA-expressing maize MON87411 as a case study.

The bean α-amylase inhibitor αAI-1 in genetically modified chickpea seeds does not harm parasitoid wasps.

BACKGROUND: Legumes have been genetically engineered to express α-amylase inhibitor 1 (αAI-1) from common bean in their seeds. Whereas the genetically modified (GM) seeds are immune to multiple bruchid pest species, the cosmopolitan bruchid Acanthoscelides obtectus is tolerant to αAI-1 and their larvae develop normally inside the seeds. Hymenopteran bruchid parasitoids, the most important natural enemies of bruchids, might thus be exposed to αAI-1 when attacking A. obtectus larvae developing inside GM seeds. Exposure might reduce parasitoid fitness, resulting in a decline in the natural control of A. obtectus, and thus promote the spread of this pest. We investigated the impact of the presence of αAI-1 in legume seeds on parasitoid fitness in tritrophic experiments with αAI-1 GM or non-GM chickpea seeds, A. obtectus, and three parasitoid species. Additionally, we investigated the exposure of parasitoids to αAI-1 using a fourth, highly sensitive parasitoid species. RESULTS: Parasitoid fitness was not affected when A. obtectus was used in GM chickpea seeds as hosts, and this lack of effects was probably attributable to the fact that exposure of the parasitoids to αAI-1 was negligible. CONCLUSION: We conclude that the release of GM chickpeas containing αAI-1 should not harm this important group of non-target insects. © 2018 Society of Chemical Industry.

Potential use of an arthropod database to support the non-target risk assessment and monitoring of transgenic plants.

Worldwide, plants obtained through genetic modification are subject to a risk analysis and regulatory approval before they can enter the market. An area of concern addressed in environmental risk assessments is the potential of genetically modified (GM) plants to adversely affect non-target arthropods and the valued ecosystem services they provide. Environmental risk assessments are conducted case-by-case for each GM plant taking into account the plant species, its trait(s), the receiving environments into which the GM plant is to be released and its intended uses, and the combination of these characteristics. To facilitate the non-target risk assessment of GM plants, information on arthropods found in relevant agro-ecosystems in Europe has been compiled in a publicly available database of bio-ecological information during a project commissioned by the European Food Safety Authority (EFSA). Using different hypothetical GM maize case studies, we demonstrate how the information contained in the database can assist in identifying valued species that may be at risk and in selecting suitable species for laboratory testing, higher-tier studies, as well as post-market environmental monitoring.

Impact of αAI-1 expressed in genetically modified cowpea on Zabrotes subfasciatus (Coleoptera: Chrysomelidae) and its parasitoid, Dinarmus basalis (Hymenoptera: Pteromalidae).

Genetically modified (GM) cowpea seeds expressing αAI-1, an α-amylase inhibitor from the common bean, have been shown to be immune against several bruchid species. Effective control of such pests by growing GM cowpea could promote the spread of bruchid species that are αAI-1 tolerant. Consequently, the sustainability of bruchid pest control could be increased by combining GM seeds and hymenopteran parasitoids. However, there are concerns that αAI-1 could interfere with the biological control provided by parasitoids. Here, we assessed the impact of GM cowpea seeds expressing αAI-1 on the αAI-1-tolerant bruchid Zabrotes subfasciatus and its parasitoid Dinarmus basalis. αAI-1 in cowpea seeds did not increase resistance to Z. subfasciatus or affect the mortality rate of Z. subfasciatus larvae. Parasitism of Z. subfasciatus by D. basalis and fitness of D. basalis offspring were not affected by the presence of αAI-1. Thus, αAI-1-expressing cowpeas and parasitoids should be compatible for the control of bruchid pests.

Resistance of αAI-1 transgenic chickpea (Cicer arietinum) and cowpea (Vigna unguiculata) dry grains to bruchid beetles (Coleoptera: Chrysomelidae).

Dry grain legume seeds possessing αAI-1, an α-amylase inhibitor from common bean (Phaseolus vulgaris), under the control of a cotyledon-specific promoter have been shown to be highly resistant to several important bruchid pest species. One transgenic chickpea and four cowpea lines expressing αAI-1, their respective controls, as well as nine conventional chickpea cultivars were assessed for their resistance to the bruchids Acanthoscelides obtectus (Say), Callosobruchus chinensis L. and Callosobruchus maculatus F. All transgenic lines were highly resistant to both Callosobruchus species. A. obtectus, known to be tolerant to αAI-1, was able to develop in all transgenic lines. While the cotyledons of all non-transgenic cultivars were highly susceptible to all bruchids, C. chinensis and C. maculatus larvae suffered from significantly increased mortality rates inside transgenic seeds. The main factor responsible for the partial resistance in the non-transgenic cultivars was deduced to reside in the seed coat. The αAI-1 present in seeds of transgenic chickpea and cowpea lines significantly increases their resistance to two important bruchid pest species (C. chinensis and C. maculatus) essentially to immunity. To control αAI-1 tolerant bruchid species such as A. obtectus and to avoid the development of resistance to αAI-1, varieties carrying this transgene should be protected with additional control measures.

Indirect effect of a transgenic wheat on aphids through enhanced powdery mildew resistance.

In agricultural ecosystems, arthropod herbivores and fungal pathogens are likely to colonise the same plant and may therefore affect each other directly or indirectly. The fungus that causes powdery mildew (Blumeria graminis tritici) and cereal aphids are important pests of wheat but interactions between them have seldom been investigated. We studied the effects of powdery mildew of wheat on two cereal aphid species, Metopolophium dirhodum and Rhopalosiphum padi. We hypothesized that aphid number and size will be smaller on powdery mildew-infected plants than on non-infected plants. In a first experiment we used six commercially available wheat varieties whereas in the second experiment we used a genetically modified (GM) mildew-resistant wheat line and its non-transgenic sister line. Because the two lines differed only in the presence of the transgene and in powdery mildew resistance, experiment 2 avoided the confounding effect of variety. In both experiments, the number of M. dirhodum but not of R. padi was reduced by powdery mildew infection. Transgenic mildew-resistant lines therefore harboured bigger aphid populations than the non-transgenic lines. For both aphid species individual size was mostly influenced by aphid number. Our results indicate that plants that are protected from a particular pest (powdery mildew) became more favourable for another pest (aphids).

Characterization of digestive enzymes of bruchid parasitoids-initial steps for environmental risk assessment of genetically modified legumes.

Genetically modified (GM) legumes expressing the α-amylase inhibitor 1 (αAI-1) from Phaseolus vulgaris L. or cysteine protease inhibitors are resistant to several bruchid pests (Coleoptera: Chrysomelidae). In addition, the combination of plant resistance factors together with hymenopteran parasitoids can substantially increase the bruchid control provided by the resistance alone. If the strategy of combining a bruchid-resistant GM legume and biological control is to be effective, the insecticidal trait must not adversely affect bruchid antagonists. The environmental risk assessment of such GM legumes includes the characterization of the targeted enzymes in the beneficial species and the assessment of the in vitro susceptibility to the resistance factor. The digestive physiology of bruchid parasitoids remain relatively unknown, and their susceptibility to αAI-1 has never been investigated. We have detected α-amylase and serine protease activities in all five bruchid parasitoid species tested. Thus, the deployment of GM legumes expressing cysteine protease inhibitors to control bruchids should be compatible with the use of parasitoids. In vitro inhibition studies showed that sensitivity of α-amylase activity to αAI-1 in the parasitoids was comparable to that in the target species. Direct feeding assays revealed that harmful effects of α-amylase inhibitors on bruchid parasitoids cannot be discounted and need further evaluation.

Development of an early-tier laboratory bioassay for assessing the impact of orally-active insecticidal compounds on larvae of Coccinella septempunctata (Coleoptera: Coccinellidae).

Early-tier studies are the initial step in the environmental risk assessment of genetically engineered plants on nontarget arthropods. They are conducted in the laboratory where surrogate species are exposed to higher concentrations of the arthropod-active compound than those expected to occur in the field. Thus, early-tier tests provide robust data and allow to make general conclusions about the susceptibility of the surrogate to the test substance. We have developed an early-tier test for assessing the toxicity of orally-active insecticidal compounds to larvae of the ladybird beetle Coccinella septempunctata L. (Coleoptera: Coccinellidae). Using potassium arsenate and the protease inhibitor E-64 as model compounds, we validated the bioassay set-up for C. septempunctata. Sucrose solution containing the test compound was offered to larvae for 24 h on the first day of each of its four larval instars. Subsequently, larvae were fed ad libitum with Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) eggs. Both compounds negatively affected C. septempunctata larval survival and development, and adult weight, indicating that the bioassay setup was able to detect dietary effects of insecticidal substances on the ladybird. Power analyses revealed that sample sizes of 20 or 45 per treatment are sufficient to detect 50 or 20% differences between the control and treatment groups, respectively, for the various measurement endpoints.

Infestation of transgenic powdery mildew-resistant wheat by naturally occurring insect herbivores under different environmental conditions.

A concern associated with the growing of genetically modified (GM) crops is that they could adversely affect non-target organisms. We assessed the impact of several transgenic powdery mildew-resistant spring wheat lines on insect herbivores. The GM lines carried either the Pm3b gene from hexaploid wheat, which confers race-specific resistance to powdery mildew, or the less specific anti-fungal barley seed chitinase and ß-1,3-glucanase. In addition to the non-transformed control lines, several conventional spring wheat varieties and barley and triticale were included for comparison. During two consecutive growing seasons, powdery mildew infection and the abundance of and damage by naturally occurring herbivores were estimated under semi-field conditions in a convertible glasshouse and in the field. Mildew was reduced on the Pm3b-transgenic lines but not on the chitinase/glucanase-expressing lines. Abundance of aphids was negatively correlated with powdery mildew in the convertible glasshouse, with Pm3b wheat plants hosting significantly more aphids than their mildew-susceptible controls. In contrast, aphid densities did not differ between GM plants and their non-transformed controls in the field, probably because of low mildew and aphid pressure at this location. Likewise, the GM wheat lines did not affect the abundance of or damage by the herbivores Oulema melanopus (L.) and Chlorops pumilionis Bjerk. Although a previous study has revealed that some of the GM wheat lines show pleiotropic effects under field conditions, their effect on herbivorous insects appears to be low.

Potential use of a serpin from Arabidopsis for pest control.

Although genetically modified (GM) plants expressing toxins from Bacillus thuringiensis (Bt) protect agricultural crops against lepidopteran and coleopteran pests, field-evolved resistance to Bt toxins has been reported for populations of several lepidopteran species. Moreover, some important agricultural pests, like phloem-feeding insects, are not susceptible to Bt crops. Complementary pest control strategies are therefore necessary to assure that the benefits provided by those insect-resistant transgenic plants are not compromised and to target those pests that are not susceptible. Experimental GM plants producing plant protease inhibitors have been shown to confer resistance against a wide range of agricultural pests. In this study we assessed the potential of AtSerpin1, a serpin from Arabidopsis thaliana (L). Heynh., for pest control. In vitro assays were conducted with a wide range of pests that rely mainly on either serine or cysteine proteases for digestion and also with three non-target organisms occurring in agricultural crops. AtSerpin1 inhibited proteases from all pest and non-target species assayed. Subsequently, the cotton leafworm Spodoptera littoralis Boisduval and the pea aphid Acyrthosiphon pisum (Harris) were fed on artificial diets containing AtSerpin1, and S. littoralis was also fed on transgenic Arabidopsis plants overproducing AtSerpin1. AtSerpin1 supplied in the artificial diet or by transgenic plants reduced the growth of S. littoralis larvae by 65% and 38%, respectively, relative to controls. Nymphs of A. pisum exposed to diets containing AtSerpin1 suffered high mortality levels (LC(50) = 637 µg ml(-1)). The results indicate that AtSerpin1 is a good candidate for exploitation in pest control.

Aphid-parasitoid community structure on genetically modified wheat.

Since the introduction of genetically modified (GM) plants, one of the main concerns has been their potential effect on non-target insects. Many studies have looked at GM plant effects on single non-target herbivore species or on simple herbivore-natural enemy food chains. Agro-ecosystems, however, are characterized by numerous insect species which are involved in complex interactions, forming food webs. In this study, we looked at transgenic disease-resistant wheat (Triticum aestivum) and its effect on aphid-parasitoid food webs. We hypothesized that the GM of the wheat lines directly or indirectly affect aphids and that these effects cascade up to change the structure of the associated food webs. Over 2 years, we studied different experimental wheat lines under semi-field conditions. We constructed quantitative food webs to compare their properties on GM lines with the properties on corresponding non-transgenic controls. We found significant effects of the different wheat lines on insect community structure up to the fourth trophic level. However, the observed effects were inconsistent between study years and the variation between wheat varieties was as big as between GM plants and their controls. This suggests that the impact of our powdery mildew-resistant GM wheat plants on food web structure may be negligible and potential ecological effects on non-target insects limited.

Laboratory toxicity studies demonstrate no adverse effects of Cry1Ab and Cry3Bb1 to larvae of Adalia bipunctata (Coleoptera: Coccinellidae): the importance of study design.

Scientific studies are frequently used to support policy decisions related to transgenic crops. Schmidt et al., Arch Environ Contam Toxicol 56:221-228 (2009) recently reported that Cry1Ab and Cry3Bb were toxic to larvae of Adalia bipunctata in direct feeding studies. This study was quoted, among others, to justify the ban of Bt maize (MON 810) in Germany. The study has subsequently been criticized because of methodological shortcomings that make it questionable whether the observed effects were due to direct toxicity of the two Cry proteins. We therefore conducted tritrophic studies assessing whether an effect of the two proteins on A. bipunctata could be detected under more realistic routes of exposure. Spider mites that had fed on Bt maize (events MON810 and MON88017) were used as carriers to expose young A. bipunctata larvae to high doses of biologically active Cry1Ab and Cry3Bb1. Ingestion of the two Cry proteins by A. bipunctata did not affect larval mortality, weight, or development time. These results were confirmed in a subsequent experiment in which A. bipunctata were directly fed with a sucrose solution containing dissolved purified proteins at concentrations approximately 10 times higher than measured in Bt maize-fed spider mites. Hence, our study does not provide any evidence that larvae of A. bipunctata are sensitive to Cry1Ab and Cry3Bb1 or that Bt maize expressing these proteins would adversely affect this predator. The results suggest that the apparent harmful effects of Cry1Ab and Cry3Bb1 reported by Schmidt et al., Arch Environ Contam Toxicol 56:221-228 (2009) were artifacts of poor study design and procedures. It is thus important that decision-makers evaluate the quality of individual scientific studies and do not view all as equally rigorous and relevant.

A barley cysteine-proteinase inhibitor reduces the performance of two aphid species in artificial diets and transgenic Arabidopsis plants.

Cystatins from plants have been implicated in plant defense towards insects, based on their role as inhibitors of heterologous cysteine-proteinases. We have previously characterized thirteen genes encoding cystatins (HvCPI-1 to HvCPI-13) from barley (Hordeum vulgare), but only HvCPI-1 C68 → G, a variant generated by direct-mutagenesis, has been tested against insects. The aim of this study was to analyze the effects of the whole gene family members of barley cystatins against two aphids, Myzus persicae and Acyrthosiphon pisum. All the cystatins, except HvCPI-7, HvCPI-10 and HvCPI-12, inhibited in vitro the activity of cathepsin L- and/or B-like proteinases, with HvCPI-6 being the most effective inhibitor for both aphid species. When administered in artificial diets, HvCPI-6 was toxic to A. pisum nymphs (LC(50) = 150 µg/ml), whereas no significant mortality was observed on M. persicae nymphs up to 1000 µg/ml. The effects of HvCPI-6 ingestion on A. pisum were correlated with a decrease of cathepsin B- and L-like proteinase activities. In the case of M. persicae, there was an increase of these proteolytic activities, but also of the aminopeptidase-like activity, suggesting that this species is regulating both target and insensitive enzymes to overcome the effects of the cystatin. To further analyze the potential of barley cystatins as insecticidal proteins against aphids, Arabidopsis plants expressing HvCPI-6 were tested against M. persicae. For A. pisum, which does not feed on Arabidopsis, a combined diet-Vicia faba plant bioassay was performed. A significant delay in the development time to reach the adult stage was observed in both species. The present study demonstrates the potential of barley cystatins to interfere with the performance of two aphid species.

Bt maize fed-prey mediated effect on fitness and digestive physiology of the ground predator Poecilus cupreus L. (Coleoptera: Carabidae).

We investigated the effects of a Bt maize hybrid on fitness and digestive physiology of the ground-dwelling predator Poecilus cupreus L., as compared with the near-isogenic hybrid. A tritrophic assay revealed that there was a great decline in the detection of Cry1Ab toxin through the trophic chain, the concentration of the toxin being 945, 349 and 37 ng g(-1) of fresh weight in Bt maize leaves, Spodoptera littoralis (Boisduval) larvae and P. cupreus larvae, respectively. Moreover, the toxin was only detected in 8% of the P. cupreus adults collected from fields growing Bt maize. Developmental time of both larvae and pupae of P. cupreus was not adversely affected by the Cry1Ab toxin via fed-prey. To elucidate potential detrimental effects due to a reduction in the quality of the prey, we assessed the digestive proteolytic activities of P. cupreus adults from a laboratory culture and insects collected in commercial Bt and non-Bt maize fields. Field-collected P. cupreus adults had higher proteolytic activities than those reared in the laboratory, whereas no significant differences were found between P. cupreus adults reared on Bt and non-Bt maize fed-S. littoralis or between P. cupreus adults collected in commercial Bt and non-Bt maize fields.

Prey mediated effects of Bt maize on fitness and digestive physiology of the red spider mite predator Stethorus punctillum Weise (Coleoptera: Coccinellidae).

The present study investigated prey-mediated effects of two maize varieties expressing a truncated Cry1Ab, Compa CB (event Bt176) and DKC7565 (event MON810), on the biology of the ladybird Stethorus punctillum. Although immuno-assays demonstrated the presence of Cry1Ab in both prey and predator collected from commercial maize-growing fields, neither transgenic variety had any negative effects on survival of the predator, nor on the developmental time through to adulthood. Furthermore, no subsequent effects on ladybird fecundity were observed. As a prerequisite to studying the interaction of ladybird proteases with Cry1Ab, proteases were characterised using a range of natural and synthetic substrates with diagnostic inhibitors. These results demonstrated that this predator utilises both serine and cysteine proteases for digestion. In vitro studies demonstrated that T. urticae were not able to process or hydrolyze Cry1Ab, suggesting that the toxin passes through the prey to the third trophic level undegraded, thus presumably retaining its insecticidal properties. In contrast, S. punctillum was able to activate the 130 kDa protoxin into the 65 kDa fragment; a fragment of similar size was also obtained with bovine trypsin, which is known to cleave the protoxin to the active form. Thus, despite a potential hazard to the ladybird of Bt-expressing maize (since the predator was both exposed to, and able to proteolytically cleave the toxin, at least in vitro), no deleterious effects were observed.

Reproduction, longevity and life table parameters of Tyrophagus neiswanderi (Acari: Acaridae) at constant temperatures.

The astigmatid mite Tyrophagus neiswanderi Johnston and Bruce is mainly considered a pest of ornamental and horticultural crops. However, this mite has been found infesting Cabrales cheese in Spain, though its population density is low compared to Acarus farris, the prevalent species of astigmatid mite encountered in Cabrales cheese maturing caves. One of the factors that might be influencing this differential abundance is temperature. In the present study the effect of temperature on reproductive parameters and longevity of T. neiswanderi was examined at six constant temperatures, ranging from 10 to 31 degrees C, and a relative humidity of 90 +/- 5%. Preoviposition period, fecundity and daily fecundity were adversely affected by extreme temperatures while the oviposition period increased as temperature was reduced. Male and female longevity increased as temperature decreased, but males showed significantly greater longevity than females. Additionally, this difference was greater as temperature decreased. The effect of temperature on the intrinsic rate of natural increase of T. neiswanderi populations was described by the non-linear Lactin model. The optimum temperature for development was predicted at 26.6 degrees C. At this temperature, the population doubling time is 2.8 days. The lower and upper thresholds for T. neiswanderi populations were calculated at 7.4 and 31.7 degrees C, respectively. According to these results, the influence of temperature on the low population density of this mite found in Cabrales maturing caves compared with A. farris is discussed.

Effects of relative humidity on development, fecundity and survival of three storage mites.

The developmental rate of immature stages and the reproduction of adults of Tyrophagus putrescentiae (Schrank), T. neiswanderi Johnston and Bruce and Acarus farris (Oudemans) were examined at 70, 80 and 90% r.h. and a constant temperature of 25 degrees C. At 70% r.h., T. putrescentiae and A. farris immature stages failed to reach the protonymph stage as 100% of the larvae died, whereas T. neiswanderi was able to complete development. The developmental time of all immature stages for the three species was significantly increased as relative humidity was reduced. The mobile stages were particularly susceptible, as the time needed to complete their development at lower relative humidities suffered greater increases than the egg stage. At 70% r.h., T. putrescentiae and A. farris were not able to lay eggs and only 24% of T. neiswanderi pairs were fertile. The reproductive parameters of the three species at the relative humidities at which they were able to lay eggs showed significant differences, except for the percentage of fertile mating at 80 and 90% r.h. As relative humidity increased, preoviposition period was reduced and fecundity and daily fecundity was increased, whereas the oviposition period showed different patterns for the three species. The intrinsic rate of increase (r ( m )) of T. neiswanderi at 70% r.h. was negative indicating that, at these conditions, mite populations of this species will diminish until they disappear. As relative humidity increased from 80 to 90% r.h. this parameter was almost twofold for both Tyrophagus species. The r ( m ) obtained for A. farris at 90% r.h. was similar to that of T. neiswanderi at the same humidity while at 80% r.h. it was very small so that the population doubling time was more than 84 days. The influence of relative humidity on biology of these mites and its practical application as control measure are discussed.

Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant.

The aim of this study was to assess the effects of potato plants expressing a barley cystatin on a potentially cystatin-susceptible natural enemy by predation on susceptible and non-susceptible preys feeding on the plant. We have focussed on the impact of the variant HvCPI-1 C68 --> G, in which the only cysteine residue was changed by a glycine, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the Egyptian cotton leafworm (ECW), Spodoptera littoralis. Moreover, we have studied the prey-mediated effects of the barley cystatin at the third trophic level, using the predatory spined soldier bug (SSB), Podisus maculiventris, as a model. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing the C68 --> G variant resulted in significantly lower weight gains compared to those fed on non-transformed (NT) plants. On the contrary, larger weight gains were obtained when ECW larvae, that lack digestive cysteine proteases, were reared on transgenic potato expressing the cystatin, as compared to larvae fed on NT plants. No negative effects on survival and growth were observed when SSB nymphs were exposed to HvCPI-1 C68 --> G by predation on either CPB or ECW larvae reared on transgenic potato plants expressing the barley cystatin, despite the fact that the inhibitor suppressed in vitro gut proteolysis of the predatory bug. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays.