Farmers, industry, governments and environmental groups agree that it would be useful to manage transgenic crops producing insecticidal proteins to delay the evolution of resistance in target pests. The main strategy proposed for delaying resistance to Bacillus thuringiensis ( Bt) toxins in transgenic crops is the high-dose/refuge strategy. This strategy is based on the unverified assumption that resistance alleles are initially rare (<10(-3)). We used an F(2) screen on >1,200 isofemale lines of Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) collected in France and the US corn belt during 1999-2001. In none of the isofemale lines did we detect alleles conferring resistance to Bt maize producing the Cry1Ab toxin. A Bayesian analysis of the data indicates that the frequency of resistance alleles in France was <9.20 x 10(-4) with 95% probability, and a detection probability of >80%. In the northern US corn belt, the frequency of resistance to Bt maize was <4.23 x 10(-4) with 95% probability, and a detection probability of >90%. Only 95 lines have been screened from the southern US corn belt, so these data are still inconclusive. These results suggest that resistance is probably rare enough in France and the northern US corn belt for the high-dose plus refuge strategy to delay resistance to Bt maize.

-In a comparative approach, we evaluated the effects of Cry1Ab protoxin, deltamethrin and imidacloprid insecticides on mortality, syrup consumption, foraging activity and olfactory learning capacities of free-flying honeybees. In an indoor flight cage we exposed bee colonies to different syrups containing Cry1Ab protoxin, deltamethrin or imidacloprid at 1000 µg/kg, 500 µg/kg and 48 µg/kg, respectively. Cry1Ab did not affect mortality, syrup consumption or learning capacities. However, foraging activity was reduced during and after the treatment. Deltamethrin and imidacloprid both affected syrup consumption and foraging activity. Deltamethrin also induced a reduction in learning capacities. With the tested concentrations, our study suggests that for honeybees, synthetic insecticides such as deltamethrin may induce a greater hazard than Cry1Ab protein, potentially expressed in Bt corn pollen at concentrations lower than 1000 µg/kg.Apis mellifera / Cry1Ab protoxin / insecticide / behaviour / risk assessment

The spo0A gene of Bacillus subtilis encodes the key factor involved in the initiation of sporulation. It was previously shown that the B. thuringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopteran larvae, is overexpressed in an spo0A mutant of B. subtilis. In this paper we describe the construction of a Bt spo0A mutant strain and its use to produce insecticidal crystal proteins. The spo0A gene of Bt was cloned and identified by its ability to transform a B. subtilis spo0A mutant to prototrophy. Its nucleotide sequence is homologous to the B. subtilis gene. The spo0A gene was replaced in the Bt genome with a disrupted copy to give an Spo- strain unable to initiate sporulation. When the cryIIIA gene was cloned in the Bt spo0A mutant, large amounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell. These encapsulated toxins were highly active against coleopteran larvae. We anticipate that the cryIIIA expression system and the Bt spo0A mutant will provide a convenient process to generate novel formulations of stabilized and environmentally safe Bt-based biopesticides.

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Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis are closely related species belonging to the Bacillus cereus group. B. thuringiensis and B. cereus generally produce extracellular proteins, including phospholipases and hemolysins. Transcription of the genes encoding these factors is controlled by the pleiotropic regulator PlcR. Disruption of plcR in B. cereus and B. thuringiensis drastically reduces the hemolytic, lecithinase, and cytotoxic properties of these organisms. B. anthracis does not produce these proteins due to a nonsense mutation in the plcR gene. We screened 400 B. thuringiensis and B. cereus strains for their hemolytic and lecithinase properties. Eight Hly- Lec- strains were selected and analyzed to determine whether this unusual phenotype was due to a mutation similar to that found in B. anthracis. Sequence analysis of the DNA region including the plcR and papR genes of these strains and genetic complementation of the strains with functional copies of plcR and papR indicated that different types of mutations were responsible for these phenotypes. We also found that the plcR genes of three B. anthracis strains belonging to different phylogenetic groups contained the same nonsense mutation, suggesting that this mutation is a distinctive trait of this species.

In this study, we evaluated in field trials the effects on non-target species, of transgenic corn producing the Cry1Ab toxin of Bacillus thuringiensis (Bt). In 1998, we collected Ostrinia nubilalis (Hübner) larvae from transgenic Bt corn (Novartis Hybrid 176) and non-Bt corn at four geographical sites. We found a significant variation in parasitism by the tachinids Lydella thompsoni (Herting) and Pseudoperichaeta nigrolineata (Walker) among sites, and more parasitism in non-Bt than in Bt fields. The Bt effect did not vary significantly among fields. In 1999, we performed a field experiment at two sites, comparing the temporal abundance of non-target arthropods in Bt corn (Monsanto Hybrid MON810) and non-Bt corn. The non-target insects studied included the aphids Metopolophium dirhodum (Walker), Rhopalosiphum padi (L.) and Sitobion avenae (F.), the bug Orius insidiosus (Say), the syrphid Syrphus corollae (Meigen), the ladybird Coccinella septempunctata (L.), the lacewing Chrysoperla carnea (Stephens), thrips and hymenopteran parasitoids. For all species but one, the number of individuals varied greatly over the season but did not differ between the types of corn. The only exception was thrips which, at one site, was significantly more abundant in Bt corn than in non-Bt corn. However this difference did not remain significant when we took the multiple tests into account. Implications for pest resistance management, population dynamics and risk assessment are discussed.

We describe a novel approach for the insertion of an insecticidal toxin gene into a resident plasmid in Bacillus thuringiensis (Bt). A gene encoding a coleopteran-specific toxin was cloned within a fragment of IS232 and inserted into a plasmid thermosensitive for replication in Bt. The plasmid was used to transform a Bt strain toxic to lepidoptera, and the transformants were then selected at non-permissive temperature for clones in which the vector had integrated into a copy of IS232 present on a resident plasmid. A second recombination event was selected such that the vector was eliminated and the newly introduced toxin gene was conserved. The resulting strain contained only DNA of Bt origin, and displayed insecticidal activity against both lepidoptera and coleoptera.

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