41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

The use of transgenic Bt-maize is increasing yearly (last year accounting for about 19% of the total maize planted area in the world) because of the efficient control of the corn borers, in especial Ostrinia nubilalis . Resistance to Bacillus thuringiensis (Bt) insecticidal toxins has been linked to the 12-domain cadherin locus in 3 lepidopteran species. The O. nubilalis cadherin gene has been revealed as a complex gene of about 20 kbp in length, with 34 introns. In the present work, we have studied the size polymorphism of the gene in a Spanish population, by amplifying the genomic sequence of the gene in 16 overlapping regions. The variability observed was not uniformly distributed, with a maximum in region 14 and a minimum (no polymorphism) in region 4. All this size variability must be due to changes in the intronic regions because we found no detectable size differences in mRNA. This variability can be useful to select appropriate polymorphic regions to be used as markers of this gene in experiments such us to determine the genetic linkage of the cadherin to Bt resistance traits.

Increasing evidence implicates a role for the innate immune system of Trichoplusia ni in mitigating Bacillus thuringiensis kurstaki (Btk) toxicity. We studied the immune response to Btk in susceptible and resistant T. ni by measuring the resistance levels, the expression of hemolymph antimicrobial proteins (AMP), and the differential number of circulating hemocytes in individual larvae. The immune response was evaluated after injections of a cocktail of gram negative (Escherichia coli) and gram positive (Staphylococcus epidermidis) bacteria, and after per os exposure to a commercial formulation of Btk endotoxins. The differential expression of genes encoding several AMPs were assessed in fat body and midgut tissues by quantitative real-time PCR 8, 24, and 48 hours after treatments. The protein-level changes in the hemolymph were determined by reverse-phase HPLC analysis, and antimicrobial activity assays. Exposure to treatments caused significant increases in the number of circulating hemocytes, the levels of AMP gene expression, as well as changes in hemolymph protein composition. The overall increase in cellular and humoral immune factors after exposure to Btk suggests that multiple systems are stimulated by exposure, and may contribute to reducing the toxicity of Btk.

The response of insects to pathogens involves changes in gene expression, which may help the insect to overcome the infection by the pathogens or the effect of their toxic compounds. Studying the response of Spodoptera exigua to its pathogens we detected a novel family of genes that were up-regulated after larval exposure to different B. thuringiensis toxins and also during the infection with the baculovirus Autographa californica (Ac)MNPV. These genes, due to their expression in response to pathogen, were called Repat genes. So far, we have detected 8 members of this family, all coding for proteins with a predicted molecular weight of approximately 15-20 KDa. Characterization of the genomic structure of 2 of the most distant members of the Repat family has revealed a similar organization, suggesting a common origin for the different members. In the present work we summarize our recent advances in the determination of the molecular function of REPAT proteins. We also report here current evidences supporting the role of REPAT proteins in attenuating the pathological effects of B. thuringiensis and baculovirus

The use of transgenic crops expressing lepidopteran-specific Cry proteins derived from the soil bacterium Bacillus thuringiensis (Bt) is a useful method to control the polyphagous pest Helicoverpa armigera. However, as H. armigera potentially develops resistance to Cry proteins, the combination of Bt and natural enemies such as the entomopathogenic fungus Metarhizium anisopliae might be an effective control method. Studies were conducted using a Cry2Aa-expressing chickpea line and a susceptible and Cry2A-resistant H. armigera strain. In a concentration-response assay, Cry2A-resistant larvae were more tolerant to M. anisopliae than susceptible larvae. In a second bioassay, however, similar mortality levels among the two strains were observed when fed on M. anisopliae treated control chickpea leaves. Thus, resistance to Cry2A did not cause any fitness costs that would become visible in an increased susceptibility to the fungus. On Bt chickpea leaves, in contrast, susceptible H. armigera larvae were more sensitive to M. anisopliae than on control leaves. It appeared that sublethal Bt damage enhanced the effectiveness of M. anisopliae. For Cry2A-resistant larvae the mortality caused by the fungus was similar independent from the food source. It is concluded that Bt chickpea plants and M. anisopliae are compatible for the control of H. armigera larvae.