Fernando Álvarez-Alfageme

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.

Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in greek and spanish population of Sesamia nonagrioides (Lepidoptera: Noctuidae)

Abstract The high-dose/refuge strategy is considered as the main strategy for delaying resistance in target pests to genetically modified crops that produce insecticidal proteins derived from Bacillus thuringiensis Berliner. This strategy is based on a key assumption that resistance alleles are initially rare (<10−3). To test this assumption, we used an F2 screen on natural populations of Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) from Greece and Spain. In total, 75 lines from Greece and 85 lines from Spain were screened for survival of F2 larvae on Cry1Ab corn, Zea mays L., leaves. No major resistance alleles were found. The frequency of resistance alleles in the Greek population was <9.7 × 10−3 with 95% probability, which was very similar to that of the Spanish population (<8.6 × 10−3 with 95% probability), and the expected frequencies were 3.2 × 10−3 (0–0.0097) and 2.9 × 10−3 (0–0.0086) in Greece and Spain (pooled 1.5 × 10−3). The experiment-wise detection probability of resistance was 94.0 and 97.5% for the Greek and the Spanish population, respectively. Evidence of alleles conferring partial resistance to Cry1Ab was found only for the Greek population. The frequency of alleles for partial resistance was estimated as 6.5 × 10−3 with a 95% credibility interval between 8 × 10−4 and 17.8 × 10−3 and a detection probability of 94%. Our results suggest that the frequency of alleles conferring resistance to Cry1Ab, regarding the population of S. nonagrioides, may be rare enough so that the high-dose/refuge strategy could be applied with success for resistance management.

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

Abstract 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.

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.

Development of an Early-Tier Laboratory Bioassay for Assessing the Impact of Orally-Active Insecticidal Compounds on Larvae of Coccinella septempunctata (Coleoptera: Coccinellidae)

ABSTRACT 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.

The effects of different prey regimes on the proteolytic digestion of nymphs of the spined soldier bug, Podisus maculiventris (Hemiptera: Pentatomidae).

The effects of different prey regimes on the performance and digestive physiology of the spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae), were assessed. Specifically, P. maculiventris nymphs were fed on Colorado potato beetle (CPB), Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), larvae; Egyptian cotton leafworm (ECW); Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae); larvae; Calliphora spp. (CAL) (Diptera: Calliphoridae) pupae or a mixture of the three prey. No differences in development and weight gain were observed when P. maculiventris nymphs were fed different prey species (CPB, ECW or CAL). However, an increase in weight gain and a reduction in the duration of the stadia were observed for nymphs fed with a mixture of the three prey. To investigate the physiological background, biochemical analysis were carried out on insects dissected at the end of the feeding assay. We have found that the proteolytic activity in the salivary glands of P. maculiventris nymphs was not affected by prey species, whereas the relative activity of these proteases in the midgut depends on the prey. Moreover, gel assays proved that the proteolytic profiles of midguts from P. maculiventris nymphs feeding on CPB, ECW and CPB closely resembled those of their prey. All together, these results suggest that P. maculiventris may utilize enzymes from the prey they consume that may facilitate the process of digestion.

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.

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.

Putative effects of Cry1Ab to larvae of Adalia bipunctata - reply to Hilbeck et al. (2012)

In their recent study, Hilbeck et al. (2012) report that Cry1Ab causes lethal effects on larvae of the ladybird beetle Adalia bipunctata when fed directly to the predator. Such toxic effects were not previously observed in a direct feeding study conducted by us (Álvarez-Alfageme et al. 2011). Because Hilbeck et al. (2012) claim that our study design did not allow us to detect any adverse effects we provide arguments for the value and relevance of our study in this commentary. Furthermore we discuss two additional published studies that have not revealed any direct effects of Cry1Ab on larvae of A. bipunctata and are not mentioned by Hilbeck et al. (2012). One of the studies was conducted in our laboratory under more realistic exposure conditions (Álvarez-Alfageme et al. 2011). Feeding A. bipunctata larvae with spider mites reared on Bt maize did not reveal any adverse effects on lethal and sublethal parameters of the predator. This was despite the fact that the larvae had ingested high amounts of biologically-active Cry1Ab protein. Thus, we do not see verified evidence that A. bipunctata larvae are sensitive to Cry1Ab at realistic worst-case exposure concentrations. This, together with the fact that A. bipunctata will be little exposed to Cry1Ab under field conditions, allows us to conclude that the risk of Bt maize to this predator is negligible. Support for this comes from the results of many Bt maize field studies that have not revealed evidence for direct Cry1Ab-effects on non-Lepidoptera species.

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).