Vitellogenin knockdown strongly affects cotton boll weevil egg viability but not the number of eggs laid by females.

Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development in this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis, but hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer the resistance against this devastating cotton pest.

Molecular approaches to improve the insecticidal activity of Bacillus thuringiensis Cry toxins.

Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity.

Variant Cry1Ia toxins generated by DNA shuffling are active against sugarcane giant borer.

Sugarcane giant borer (Telchin licus licus) is a serious sugarcane pest in Americas whose endophytic lifestyle hampers effective chemical and biological controls. Therefore, development of alternative control methods is extremely important. Envisaging development of transgenic plants resistant to this pest, we investigated the effect of the Bacillus thuringiensis Cry protein Cry1Ia12synth (truncated protein lacking C-terminus with plant codon usage) and variants against T. l. licus. cry1Ia12synth gene was used to generate mutated variants, which were screened for toxicity toward T. l. licus. For that purpose, an innovative technique combining cry gene shuffling with phage-display was used to build a combinatorial library comprising 1.97x10(5) Cry1Ia12synth variants. Screening of this library for variants binding to T. l. licus Brush Border Midgut Vesicles led to the identification of hundreds of clones, out of which 30 were randomly chosen for toxicity testing. Bioassays revealed four variants exhibiting activity against T. l. licus as compared to the non-toxic Cry1Ia12synth. Eight single substitutions sites were found in these active variants. Based on theoretical molecular modelling, the probable implications of these mutations are discussed. Therefore, we have four genes encoding Cry1Ia12synth variants active against T. l. licus promising for future development of resistant transgenic sugarcane lines.