Hany M. Hussein

Impact of Cry1Ab toxin expression on the non-target insects dwelling on maize plants

The effect of transgenic maize MON810 (Bt maize) on the diversity and abundance of plant‐dwelling insects was tested under field conditions in southern Bohemia (coordinates 48°N, 14ºE, 384 m a.s.l.) for three successive years. The experiment was carried out on 10 0.5‐ha plots of which five were seeded with the Bt maize and five with the non–Bt parental cultivar. The content of Bt toxin (Cry1Ab) was measured in plant tissues with a commercial ELISA kit. Randomly chosen plants (10 per plot) were taken from the field during the vegetation period in about 2‐week intervals and thoroughly examined in the laboratory. Collected insects were identified and their counts were statistically analysed with CANOCO with respect to the Bt toxin, developmental stage of maize and the year of cultivation. No significant effect of Bt maize on the plant‐dwelling non‐target insects was detected. Correlation between the number of plants and detected insect diversity revealed that inspection of 20 plants (four per each of five plots) provided data reliable at 95% probability level; six plants per plot were sufficient for the analysis of aphids, thrips and Orius bugs.

Potato Expressing Beetle-Specific Bacillus thuringiensis Cry3Aa Toxin Reduces Performance of a Moth

Expression of the Bacillus thuringiensis beetle-specific toxin Cry3Aa, which renders a genetically modified potato cultivar resistant to the Colorado potato beetle Leptinotarsa decemlineata, exerts a deleterious effect on the polyphagous moth Spodoptera littoralis. The caterpillars of S. littoralis feed less and produce smaller pupae on the genetically modified cultivar (NewLeaf Superior) than on the parental nontransgenic cultivar (Superior). The conversion efficiencies of total dry matter, combustion heat, carbon, and nitrogen from leaves to insect biomass are similar on both cultivars. In spite of similar food utilization and a relatively small difference in the body mass at pupation, female adults that developed from caterpillars fed on NewLeaf Superior lay a mean of 309 eggs compared to a mean of 713 eggs deposited by females that developed from caterpillars fed on Superior. Because of this difference and a simultaneous reduction in fertility (egg hatchability) from 78 to 48%, a pair of adults that fed as larvae on NewLeaf Superior produces only 148 larvae, whereas a pair of adults that fed as larvae on Superior produces 556 larvae. We suggest that small amounts of Cry3Aa that accumulate in insect tissue and persist until the adult stage are responsible for the decline in reproduction.

Epigeic spiders are not affected by the genetically modified maize MON 88017

The genetically modified (GM) maize MON 88017 facilitates weed management owing to its tolerance to glyphosate, and resists western corn rootworm (WCR), Diabrotica virgifera virgifera, owing to the expression of Cry3Bb1 toxin. MON 88017 could therefore contribute to the solution of two major problems of European agriculture: continuous WCR spreading and high use of herbicides. To assess possible unwanted environmental impacts of MON 88017, we compared communities of spiders in plots planted in three successive years with this maize, its near isogenic non‐GM cultivar treated or not treated with an insecticide and two unrelated maize cultivars. Each of the five treatments was applied on five 0.5 ha plots in a 14 ha field. Spiders were collected in five pitfall traps per plot five times per year. Upon reaching the waxy ripening stage, all plants of first‐year cultivation were shredded to small pieces and ploughed into the soil in the respective plot, whereas in the 2nd and 3rd year the harvest was used for biogas production and only digestate was returned to the field. Out of 79 spider species, Pardosa agrestis, Pachygnatha degeeri and Oedothorax apicatus made up 28%, 25% and 23% of the total spider count in the 1st year of study; 2%, 8% and 84% in the 2nd; and 40%, 8% and 35% in the 3rd year. Statistical analysis did not reveal any influence of GM maize on the spider abundance and biodiversity. The abundance, and in two years also the species diversity, was insignificantly higher on the plots with GM maize than on plots with the insecticide‐treated non‐GM maize. The composition and size of spider community varied year to year, probably reflecting weather conditions and differences in field fertilization with organic matter.

Communities of ground‐dwelling arthropods in conventional and transgenic maize: background data for the post‐market environmental monitoring

To verify the validity of concerns about environmental safety of maize expressing insecticidal Cry toxins (referred to as Bt maize), we compared communities of ground beetles (Carabidae), rove beetles (Staphylinidae) and spiders (Araneae) in plots planted either with Bt maize cultivar YieldGard® or with the non‐transgenic parental cultivar Monumental. Each cultivar was grown on 5 plots of 0.5 ha for three consecutive years. To increase the field load of Cry toxin, the fully grown maize of the first study year was shredded to small pieces that were ploughed into the soil. Arthropods were collected in pitfall traps and determined to the species level. The abundance and species richness of all studied groups greatly varied over the season and between the seasons but without statistically significant differences between the Bt and non‐Bt plots. A single spider species and three ground beetle species dominated in the catches every year, whereas a set of 1–4 most abundant rove beetle species changed every year. Frequently occurring species were typical for most of Europe. The total counts of ground beetles, rove beetles and spiders collected once or twice per season are proposed to serve as bioindicators in the post‐market environmental monitoring (PMEM).

Effects of nonsteroidal ecdysone agonist RH-5992 and chitin biosynthesis inhibitor lufenuron on Spodoptera littoralis (Boisduval, 1833)

Comparative studies of the effects of two compounds, tebufenozide (an ecdysone agonist) and lufenuron (an insect growth regulator inhibiting chitin synthesis), were conducted on Spodoptera littoralis (Boisduval, 1833). The compounds, orally administered, caused larval mortality proportional to the concentrations in the food source. Tebufenozide initiated precocious molting, and lufenuron, and inhibited chitin synthesis. In both cases, larvae were unable to complete the molting process and died in the old larval cuticle. Larvae contaminated by sublethal doses completed their development to adulthood. Lufenuron is more active than is tebufenozide. LD-50 for lufenuron is 0.0001ppm and for tebufenozide 0.001ppm. Topical application of the test compounds to eggs caused dose- and agedependent inhibition of embryonic development. Application of tebufenozide in the second half of embryogenesis caused precocious molting of eclosed larvae of the 1st instar. Some morphological changes in the process of larval-pupal transformation were also observed. Tested compounds also reduced reproduction in adult individuals that had been treated by the tested compounds in the larval stage.

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.

Laboratory evaluation of a new strain CCM 8367 of Isaria fumosorosea (syn. Paecilomyces fumosoroseus) on Spodoptera littoralis (Boisd.)

Laboratory experiments were conducted to assess the efficiency of the new strain CCM 8367 of Isaria fumosorosea on different stages of the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.), including soil treatments with pre-pupal and pupal stages. Treatments of larval instars showed a high susceptibility with 3rd, 5th and last instars to suspension of this fungus with concentration 5 × 107 spores/ml. Larval mortality was over 90%. There were no significant differences (P = 0.7929, F = 0.2346) between instar treatments. The commercial fungus, PreFeRal® strain Apopka 97 of I. fumosorosea, which was used in comparison with this new strain caused mortality rates of between 63.33 and 90%. Statistically, differences between the effects of CCM 8367 strain and Apopka 97 were highly significant on the last instar (P = 0.0064, F = 6.479) and extremely significant on the 3rd instar (P < 0.0001, F = 13.29). No significant differences were recorded between the two strains on the 5th instar (P = 0.0597, F = 3.233). Fungal treatments with the late stage insects (end of the last instar or pupal stage) in soil yielded interesting results: the mortality rate on end of final instar larvae was 16.66% when treated with Apopka 97 and 83.33% when treated with CCM 8367. Soil containing pupae of S. littoralis that were inoculated with CCM8367 resulted in a high number of malformed adults, and the mortality rate was 64.52% (32.27% of malformed adults died and 32.25% of pupae fully infected by fungus). Only 3.23% of samples produced morphologically normal adults in this test. The results conclude that the strain of I. fumosorosea CCM 8367 has strong insecticidal effects on S. littoralis and has the potential to be implemented as a novel biocontrol agent.

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.

Effectiveness of the entomopathogenic nematode Steinernema feltiae in agar gel formulations against larvae of the Colarado potato beetle, Leptinotarsa decemlineata (Say.) (Coleoptera: Chrysomelidae)

The entomopathogenic nematode Steinernema feltiae strain Ustinov Russia was used on potato foliage to control larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say.) (Coleoptera: Chrysomelidae). Nematodes were applied in formulations of agar at 4%, 2%, 1% and 0.5% concentrations and compared to a control application of nematodes in water for nematode survival. Agar formulation significantly improved efficacy by increasing nematode survival through reduction in desiccation when compared to water-based formulation. More than 70% of infective juvenile nematodes (IJs) died after being incubated in the highest concentration of agar for 12 h, while only 8% mortality was recorded at the 1% concentration. Suspension of nematodes in 1% agar gel was shown to be efficacious in both laboratory and greenhouse tests for extension of the nematodes’ survival. Agar formulation droplets dried slower than control droplets by 127.8 min. Leptinotarsa decemlineata mortality significantly increased when insects were exposed to infective juvenile nematodes for four hours after application. In conclusion, the agar formulation enhanced nematode survival by providing a suitable environment thereby delaying dryingand increasing the possibility for nematodes to invade their host on the foliage.

Effectiveness of entomopathogenic nematodes Steinernema feltiae and Heterorhabditis bacteriophora on the Colorado potato beetle Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) under laboratory and greenhouse conditions

In laboratory and greenhouse studies, the invading ability, virulence, and mortality caused by Stinernema feltiae and Heterorhabditis bacteriophora were compared. After one and two days of exposure to either nematode species, the mortality of Colordo potato beetle (CPB) Leptinotarsa decemlineata larvae at different instars, third and fourth, was recorded and the number of nematodes invading cadavers was more than the number of nematodes inside the larvae at the late last instar (one day before pre-pupa). Two concentrations, 250 and 500 IJs/dish, infective juvenile nematodes/0.5 ml were tested on different CPB larval instar. S. feltiae was more effective, with fourth instar rather than third and late last instar. On the other hand, H. bacteriophora showed a very weak effect with L. decemlineata. Also it was clear that S. feltiae was more effective and faster than H. bacteriophora: more than 70% of larvae were killed within 24 hours compared with H. bacteriophora which killed 40% of larvae within 48–72 hours. A significant difference in invading efficiency was observed with concentration 2500 IJs/pot in the greenhouse test. The number of adult females found in the cadavers of L. decemlineata larvae was always higher than the number of males. Foliage application of S. feltiae and H. bacteriophora resulted in a significant reduction of the number of damaged leaves and a lower index of damage compared with that in the control. We conclude that S. feltiae has significant potential and can help in the management of the Colorado potato beetle.