The effect of Xenorhabdus bacteria metabolites on Colorado potato beetle (Leptinotarsa decemlineata) adult feeding and larval survival.

Colorado Potato Beetle (CPB) is one of the most destructive potato pests that can quickly develop resistance to insecticides. Therefore, new safe and effective control strategies that are less susceptible to the development of resistance by CPB are urgently needed. Due to their complex mode of action, the likelihood of resistance development by target pests is generally low with antifeedants. In the present study, we assessed the effect of secondary metabolites of various Xenorhabdus bacteria species and strains on CPB adult feeding and on larval development. The metabolites were applied in the form of cell free supernatants (CFSs) from Xenorhabdus cultures. In bioassay 1, leaves treated with ten Xenorhabdus cultures were fed to CPB adults, and their feeding was assessed daily for one week. In bioassay 2, CPB egg masses were placed on the leaves treated with five bacterial cultures, and larval development to pupae was monitored. Out of the ten Xenorhabdus cultures tested, two strains exhibited a significant reduction in the feeding behavior of Colorado Potato Beetle adults, with reductions of up to 70% compared to the control. The effect of CFSs on larval development was variable, and when treated with X. khoisanae SGI 197, over 90% of larvae died in the first few days before reaching the 2nd instar, and complete mortality was achieved on the 8th day of the experiment. Our study is the first study to demonstrate the antifeedant effect of Xenorhabdus cultures towards herbivorous beetles, and the metabolites of these bacteria may have potential for CPB control. Clearly, the metabolites produced by X. khoisanae SGI-197 may be a promising tool for CPB larvae control with the potential to significantly decrease damage to potato plants.

Different maize varieties have greater impact on Tetranychus urticae (Acari: Tetranychidae) than GE maize expressing Cry3Bb1 insecticidal protein.

Genetically engineered (GE) maize expressing Bacillus thuringiensis Cry3Bb1 protein was developed to control Diabrotica spp. (Coleoptera: Chrysomelidae). However, Cry proteins have been reported to have effects on non-target arthropods. We therefore investigated whether the non-target pest Tetranychus urticae (Acari: Tetranychidae) was negatively affected by GE maize expressing the insecticidal Cry3Bb1 protein. Five treatments were used in the laboratory tests to determine the life history parameters of T. urticae on leaves of field-grown maize varieties: (1) GE maize MON 88017, (2) isogenic maize, (3) isogenic maize protected with the soil applied insecticide chlorpyrifos (Dursban 10G), and two unrelated varieties (4) Kipous and (5) PR38N86. Newly emerged T. urticae larvae were individually released on the upper surface of leaf discs placed on water saturated cotton wool. Immatures and adults survival, duration of developmental stages and female fecundity were recorded daily until T. urticae died. Age-stage, two-sex life table method and test for trends, revealed no significant differences in 13 of 18 studied parameters. The unrelated varieties Kipous and PR38N86 on one side and maize with the same genetic background, namely GE maize and isogenic maize with or without insecticide protection, on the other side, showed significant variations in male longevity, larval survival rate, preoviposition period, and fecundity. In addition to the differences between varieties, GE maize and insecticide-protected isogenic maize showed a substantial difference in age-specific fecundity, but not in the mean number of eggs laid by females. The obtained results do not indicate that consumption of Cry3Bb1 has negative effect on T. urticae and suggest that GE maize does not pose a risk with respect to the non-target mite pest T. urticae. The results may have implications for the approval and renewal of import and cultivation for GE crop in the European Union.

No bioaccumulation of Cry protein in the aphidophagous predator Harmonia axyridis.

The uptake of insecticidal Cry1Ab from genetically engineered (GE) maize, via herbivore Rhopalosiphum padi, to a predator Harmonia axyridis and its potential intergenerational transfer were investigated. Cry1Ab concentration was found to be 400-fold lower in R. padi compared to GE maize, and more than two-fold lower in H. axyridis. For 62% of H. axyridis samples, Cry1Ab was under the limit of detection (LOD), for another 13% were under the limit of quantification (LOQ). The concentration of Cry1Ab was similar between H. axyridis exposed short-term and long-term with the exception of adults after long-term. There was no correlation between Cry1Ab in females and eggs and neonates. The performance of H. axyridis was comparable between Cry1Ab and control. Histological investigation did not show any pathological changes in the digestive and reproductive systems. The detected route of exposure is unlikely to be important for functional biological control by H. axyridis in agroecosystem.

Essential Oils: Useful Tools in Storage-Pest Management.

This study aimed to verify the level of repellent and mortality effect of two chemical substances (DEET and 2-undecanone) and seven essential oils (EOs), Allium sativum, Artemisia annua, Ocimum basilicum, Lavandula angustifolia, Eucalyptus globulus, Pinus sylvestris, and Curcuma longa. The storage pests Tribolium confusum, Tenebrio molitor, and Acanthoscelides obtectus were exposed to various concentrations in an olfactometer-and-mortality test. The effects were recorded 24-48-72 h after the treatments were applied. A. sativum, E. globulus, and L. augustifolia were found to have significant repellence effects. A substantial lethal effect was observed for A. sativum, E. globulus, and O. basilicum. We also found that even if the most efficient EOs were diluted to low concentrations, they still produced repellent and mortality effects. The presented results indicate that A. sativum and O. basilicum were the most effective against T. confusum and T. molitor; simultaneously, L. angustifolia and C. longa showed high activity against A. obtectus. All of these efficient EOs could be applied as effective bio-control agents in various stored conditions.

Role of adipokinetic hormone in the Colorado potato beetle, Leptinotarsa decemlineata infected with the entomopathogenic nematode Steinernema carpocapsae.

The effects of the entomopathogenic nematode Steinernema carpocapsae on the Colorado potato beetle (CPB) Leptinotarsa decemlineata and the involvement of adipokinetic hormone (AKH) in the responsive reactions were examined in this study. It was observed that nematode application doubled the amount of AKH (Peram-CAH-I and Peram-CAH-II) in the central nervous system of L. decemlineata, indicating mobilization of anti-stress reactions in the body. Furthermore, the external co-application of Peram-CAH-II with the nematode significantly increased beetle mortality (5.6 and 1.8 times, 1 and 2 days after application, respectively). The mechanism underlying this phenomenon was investigated. As the effect on gut characteristics was equivocal, it was assumed that the nematodes profited from the observed mobilization of metabolites from the fat body into the Peram-CAH-II-induced hemolymph. This phenomenon supplied nematodes with a more nutrient-dense substrate on which they propagated. Furthermore, Peram-CAH-II lowered vitellogenin expression in the fat body, particularly in males, thus limiting the anti-pathogen defense capacity of the protein. However, there could be other possible mechanisms underpinning this chain of events. The findings could be theoretically intriguing but could also aid in developing real insect pest control methods in the future.

Could the Presence of Thrips AFFECT the Yield Potential of Genetically Modified and Conventional Maize?

Maize pests like Ostrinia nubilalis and Diabrotica virgifera virgifera are eradicated using genetically modified maize. This study's goal was to see if the genetically modified maize MON810 is also toxic to thrips communities on maize. The impact of Bt maize on thrips diversity and abundance, as well as yield losses, was studied in the field in Borovce for three years (Slovakia). The study used 10 Bt and 10 non-Bt maize cultivars. Thrips were monitored every two weeks during the season using transparent sticky traps installed on the experimental plots (one per plot, 20 per year). In total, 3426 thrips were caught. Thrips populations usually peak around the end of July at BBCH55. Among the species identified were Limothrips denticornis, Limothrips cerealium, Haplothrips aculeatus, Frankliniella schultzei, Frankliniella occidentalis, Thrips tabaci, Aeolothrips fasciatus, Frankliniella tenuicornis, and Chirothrips spp. We found that MON810 maize had no effect on the occurrence or composition of thrips. Their presence was affected by the maize growth phase and growing seasons and partially by the weather. The direct effect on the grain yield was not confirmed. Our research contributed to scientific knowledge of thrips communities found on maize plants in Central Europe, including Bt maize.

Cry3Aa Toxin Is Not Suitable to Control Lepidopteran Pest Spodoptera littoralis (Boisd.).

The toxicity of the Bacillus thuringiensis (Bt) toxin Cry3Aa-originally used against the main potato pest, the Colorado potato beetle, Leptinotarsa decemlineata-was verified on this species and then evaluated against the Egyptian armyworm, Spodoptera littoralis, which is a pest of several economically important plants. Larvae of S. littoralis were fed a semi-artificial diet supplemented either with a recombinant or with a natural Bt toxin Cry3Aa and with the genetically engineered (GE) potato of variety Superior NewLeaf (SNL) expressing Cry3Aa. Cry3Aa concentration in the diet and the content in the leaves were verified via ELISA (enzyme-linked immunosorbent assay) and HPLC (high-performance liquid chromatography) during and at the end of the experiments. The biological effectiveness of the coleopteran-specific Cry3Aa with previous reports of activity against S. littoralis was tested on five different populations of S. littoralis larvae by monitoring 13 parameters involving development from penultimate instar, weight, the efficiency of food conversion to biomass, ability to reproduce, and mortality. Although some occasional differences occurred between the Cry3Aa treatments and control, any key deleterious effects on S. littoralis in this study were not confirmed. We concluded that the Cry3Aa toxin appears to be non-toxic to S. littoralis, and its practical application against this pest is unsuitable.

Virulence of Beauveria bassiana Strains Isolated from Cadavers of Colorado Potato Beetle, Leptinotarsa decemlineata.

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a serious, widely distributed pest of potato and other crops. This pest is able to defoliate the host plant and cause severe yield loss. Moreover, the pest quickly becomes resistant to many chemical pesticides. Therefore, the development of novel biopesticides targeting this pest is urgently needed. The purpose of this study was to obtain new strains of the entomopathogenic fungus Beauveria bassiana and assess their efficacy against L. decemlineata adults under laboratory conditions. Twelve strains were isolated from cadavers of Colorado potato beetles collected in potato fields in the Czech Republic. Test beetles were treated by suspensions of conidia at the concentration of 1 × 107 spores per milliliter and their survival was recorded daily for three weeks. The results of the bioassays revealed that all new native strains were pathogenic to L. decemlineata adults and caused mortality up to 100% at the end of the trial period with an LT50 of about 7 days. These strains were more virulent than a reference strain GHA and some of them can be recommended for the development of a new mycoinsecticide against L. decemlineata. Our findings also highlight the importance of searching for perspective strains of entomopathogenic fungi among naturally infected hosts.

Efficacy of the Applied Natural Enemies on the Survival of Colorado Potato Beetle Adults.

Colorado potato beetle Leptinotarsa decemlineata is among the most destructive pests of potatoes quickly developing resistance to traditional insecticides. In the present study, we tested the effect of various species and strains of entomopathogenic nematodes on CPB adults, and subsequently, the most effective nematodes were applied alone and in combination with entomopathogenic fungus B. bassiana in pots with potato plants and in the field and their effect on the number of emerging adults was evaluated. In the experimental infections, both the nematode invasion and pathogenicity were variable, and, in several strains, the mortality reached 100%. In pot experiments, soil application of nematodes S. carpocapsae 1343 and S. feltiae Jakub and fungus significantly decreased numbers of emerging CPB adults, while, after the application on leaves, only fungal treatment was effective. The field application of fungus B. bassiana significantly decreased the number of emerging CPB adults in comparison to control sites by ca. 30% while the effect of nematodes and the nematodes-fungus combination was not significant. In conclusion, we demonstrate the necessity of thorough bioassays to select the most effective nematode strains. Entomopathogenic nematodes have the potential to effectively decrease the emergence of CPB adults, but further research is needed to improve the effectiveness in the field.

Importance of functional classification in the use of carabids for the environmental risk assessment of the GE crops and other agricultural practices.

Carabids (Coleoptera: Carabidae) seem to be suitable bioindicators of the environmental impacts of novel agrotechnologies, including deployment of the genetically engineered (GE) crops. In this article, we describe our effort to employ carabids in the environmental risk assessment (ERA). GE maize MON88017, its near-isogenic hybrid nontreated or treated with the soil insecticide chlorpyrifos, and two reference hybrids were used to compare three different ways how to utilize carabids in ERA. The analysis of abundance of all captured carabids or of the most abundant carabid species did not disclose any differences between the treatments. The analysis based on the categories of functional traits revealed distinct features of some treatments and proved suitable for ERA because it permitted field data transportability in spite of different species compositions. Our results indicate that GE maize has no detrimental environmental effect. On the other hand, we found significant trends toward lower abundance and lower species number (including analysis of all carabid species together) in plots treated with the insecticide, and some tendencies to higher abundance and higher species number in plots sown with the reference hybrid PR38N86. Using functional group indicators allows identification of unintended changes in ecological functions of agroecosystem and comparability across geographies. We recommend data evaluation at the level of the categories of functional traits in ERA of GE crops and other agricultural practices.

Stacked Bt maize and arthropod predators: exposure to insecticidal Cry proteins and potential hazards.

Genetically engineered (GE) crops with stacked insecticidal traits expose arthropods to multiple Cry proteins from Bacillus thuringiensis (Bt). One concern is that the different Cry proteins may interact and lead to unexpected adverse effects on non-target species. Bi- and tri-trophic experiments with SmartStax maize, herbivorous spider mites (Tetranychus urticae), aphids (Rhopalosiphum padi), predatory spiders (Phylloneta impressa), ladybeetles (Harmonia axyridis) and lacewings (Chrysoperla carnea) were conducted. Cry1A.105, Cry1F, Cry3Bb1 and Cry34Ab1 moved in a similar pattern through the arthropod food chain. By contrast, Cry2Ab2 had highest concentrations in maize leaves, but lowest in pollen, and lowest acquisition rates by herbivores and predators. While spider mites contained Cry protein concentrations exceeding the values in leaves (except Cry2Ab2), aphids contained only traces of some Cry protein. Predators contained lower concentrations than their food. Among the different predators, ladybeetle larvae showed higher concentrations than lacewing larvae and juvenile spiders. Acute effects of SmartStax maize on predator survival, development and weight were not observed. The study thus provides evidence that the different Cry proteins do not interact in a way that poses a risk to the investigated non-target species under controlled laboratory conditions.

Use of Carabids for the Post-Market Environmental Monitoring of Genetically Modified Crops.

Post-market environmental monitoring (PMEM) of genetically modified (GM) crops is required by EU legislation and has been a subject of debate for many years; however, no consensus on the methodology to be used has been reached. We explored the suitability of carabid beetles as surrogates for the detection of unintended effects of GM crops in general PMEM surveillance. Our study combines data on carabid communities from five maize field trials in Central Europe. Altogether, 86 species and 58,304 individuals were collected. Modeling based on the gradual elimination of the least abundant species, or of the fewest categories of functional traits, showed that a trait-based analysis of the most common species may be suitable for PMEM. Species represented by fewer than 230 individuals (all localities combined) should be excluded and species with an abundance higher than 600 should be preserved for statistical analyses. Sixteen species, representing 15 categories of functional traits fulfill these criteria, are typical dominant inhabitants of agroecocoenoses in Central Europe, are easy to determine, and their functional classification is well known. The effect of sampling year is negligible when at least four samples are collected during maize development beginning from 1 April. The recommended methodology fulfills PMEM requirements, including applicability to large-scale use. However, suggested thresholds of carabid comparability should be verified before definitive conclusions are drawn.

Laboratory Evaluation of Isaria fumosorosea CCM 8367 and Steinernema feltiae Ustinov against Immature Stages of the Colorado Potato Beetle.

The Colorado potato beetle, Leptinotarsa decemlineata, has developed resistance to most registered pesticides and has become one of the most difficult insect pests to control. Development of new biopesticides targeting this pest might solve the resistance problem and contribute to sustainable crop production. Laboratory experiments were conducted to assess the efficacy of Isaria fumosorosea (syn. Paecilomyces fumosoroseus) strain CCM 8367 against L. decemlineata when applied alone or combined with the entomopathogenic nematode Steinernema feltiae. The last-instar larvae of the Colorado potato beetle showed the highest susceptibility to I. fumosorosea followed by pre-pupae and pupae. The median lethal concentration (LC50) was estimated to be 1.03×106 blastospores/ml. The strain CCM 8367 was more virulent, causing 92.6% mortality of larvae (LT50 = 5.0 days) compared to the reference strain Apopka 97, which caused 54.5% mortality (LT50 = 7.0 days). The combined application of the fungus with the nematodes increased the mortality up to 98.0%. The best results were obtained when S. feltiae was applied simultaneously with I. fumosorosea (LT50 = 2.0 days); later application negatively affected both the penetration rate and the development of the nematodes. We can conclude that the strain CCM 8367 of I. fumosorosea is a prospective biocontrol agent against immature stages of L. decemlineata. For higher efficacy, application together with an entomopathogenic nematode is recommended.

Risk Assessment of Genetically Engineered Maize Resistant to Diabrotica spp.: Influence on Above-Ground Arthropods in the Czech Republic.

Transgenic maize MON88017, expressing the Cry3Bb1 toxin from Bacillus thuringiensis (Bt maize), confers resistance to corn rootworms (Diabrotica spp.) and provides tolerance to the herbicide glyphosate. However, prior to commercialization, substantial assessment of potential effects on non-target organisms within agroecosystems is required. The MON88017 event was therefore evaluated under field conditions in Southern Bohemia in 2009-2011, to detect possible impacts on the above-ground arthropod species. The study compared MON88017, its near-isogenic non-Bt hybrid DK315 (treated or not treated with the soil insecticide Dursban 10G) and two non-Bt reference hybrids (KIPOUS and PR38N86). Each hybrid was grown on five 0.5 ha plots distributed in a 14-ha field with a Latin square design. Semiquantitative ELISA was used to verify Cry3Bb1 toxin levels in the Bt maize. The species spectrum of non-target invertebrates changed during seasons and was affected by weather conditions. The thrips Frankliniella occidentalis was the most abundant species in all three successive years. The next most common species were aphids Rhopalosiphum padi and Metopolophium dirhodum. Frequently observed predators included Orius spp. and several species within the Coccinellidae. Throughout the three-year study, analysis of variance indicated some significant differences (P<0.05). Multivariate analysis showed that the abundance and diversity of plant dwelling insects was similar in maize with the same genetic background, for both Bt (MON88017) and non-Bt (DK315) untreated or insecticide treated. KIPOUS and PR38N86 showed some differences in species abundance relative to the Bt maize and its near-isogenic hybrid. However, the effect of management regime on arthropod community was insignificant and accounted only for a negligible portion of the variability.

Protease inhibitor from insect silk-activities of derivatives expressed in vitro and in transgenic potato.

Several recombinant derivatives of serine protease inhibitor called silk protease inhibitor 2 (SPI2), which is a silk component in Galleria mellonella (Lepidoptera, Insecta), were prepared in the expression vector Pichia pastoris. Both the native and the recombinant protease inhibitors were highly active against subtilisin and proteinase K. The synthetic SPI2 gene with Ala codon in the P1 position was fused with mGFP-5 to facilitate detection of the transgene and its protein product. A construct of the fusion gene with plant regulatory elements (promoter 35S and terminator OCS) was inserted into the binary vector pRD400. The final construct was introduced into Agrobacterium tumefaciens that was then used for genetic transformation of the potato variety Velox. The transgene expression was monitored with the aid of ELISA employing polyclonal antibody against natural SPI2. In vitro tests showed increased resistance to the late blight Phytophthora infestans in several transformed lines. No effect was seen on the growth, mortality, life span or reproduction of Spodoptera littoralis (Lepidoptera, Insecta) caterpillars, while feeding on transformed potato plants expressing the fusion protein, indicating that the transformed potatoes may be harmless to non-target organisms.