The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented. Characterization was based on their insecticidal activity against Aedes aegypti , Culex quinquefasciatus , and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for cry and cyt genes encoding proteins active against mosquitoes ( cyt1 , cyt2 , cry2 , cry4A , cry4B , cry10 , cry11 , cry17 , cry19 , cry24 , cry25 , cry27 , cry29 , cry30 , cry32 , cry39 , and cry40 ). Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A . aegypti and C . quinquefasciatus larvae than B . thuringiensis subsp. israelensis and displayed high similarities with the B . thuringiensis subsp. israelensis used in this study with regard to protein and plasmid profiles and the presence of cry genes. Strain 147-8906 has activity against A . aegypti similar to that of B . thuringiensis subsp. israelensis but has different protein and plasmid profiles. This strain, harboring cry11 , cry30 , cyt1 , and cyt2 genes, could be relevant for future resistance management interventions. Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene.

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti . The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus , Bacillus sphaericus , B. anthracis , B. licheniformis , and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.

The sugarcane aphid, Melanaphis sacchari Zehntner (Hemiptera: Aphididae), is recognized as an important pest of sorghum cultivation. The use of natural products in the form of botanical extracts represents an alternative for its control. In this investigation, we evaluated the insecticidal activity of hexanic, acetonic, and methanolic extracts of leaves, fruits, and roots of Ricinus communis L. (Euphorbiaceae). These were applied in contact bioassays at different concentrations to control apterous adults of M. sacchari. We found that the chemical components of lower polarity contained in the hexane extract of leaves (RcLH) produced the best biological effect, with 96% mortality at 72 h. Thin layer chromatography allowed fractions of this extract to be grouped into 7 categories (F1–F7) based on their chemical content. The F3 category produced 90% mortality at 10,000 ppm at 72 h in contact bioassays. The 1H and 13C nuclear magnetic resonance analysis in addition to the gas chromatography-mass spectrometry of F3 revealed the presence of myristic and stearic acid. Our results showed that the hexanic extracts of R. communis and their fatty acids may be an alternative for the development of new insecticides, constituting a better option in terms of effectiveness and lower toxicity compared with the synthetic products currently on the market used for their control.

Bacillus thuringiensis Cry1Ab and Cry1Fa toxins are environmentally safe insecticides that control important insect pests. Spodoptera frugiperda is an important maize pest that shows low susceptibility to Cry1A toxins, in contrast to Cry1Fa, which is highly active against this pest and is used in transgenic maize for S. frugiperda control. The β16 region from domain III of Cry1Ab has been shown to be involved in interactions with receptors such as alkaline phosphatase (ALP) or aminopeptidase (APN) in different lepidopteran insects. Alanine-scanning mutagenesis of amino acids of Cry1Ab β16 (⁵⁰⁹STLRVN⁵¹⁴) revealed that certain β16 mutations, such as N514A, resulted in increased toxicity of Cry1Ab for S. frugiperda without affecting the toxicity for other lepidopteran larvae, such as Manduca sexta larvae. Exhaustive mutagenesis of N514 was performed, showing that the Cry1Ab N514F, N514H, N514K, N514L, N514Q, and N514S mutations increased the toxicity toward S. frugiperda. A corresponding mutation was constructed in Cry1Fa (N507A). Toxicity assays of wild-type and mutant toxins (Cry1Ab, Cry1AbN514A, Cry1AbN514F, Cry1Fa, and Cry1FaN507A) against four S. frugiperda populations from Mexico and one from Brazil revealed that Cry1AbN514A and Cry1FaN507A consistently showed 3- to 18-fold increased toxicity against four of five S. frugiperda populations. In contrast, Cry1AbN514F showed increased toxicity in only two of the S. frugiperda populations analyzed. The mutants Cry1AbN514A and Cry1AbN514F showed greater stability to midgut protease treatment. In addition, binding analysis of the Cry1Ab mutants showed that the increased toxicity correlated with increased binding to brush border membrane vesicles and increased binding affinity for S. frugiperda ALP, APN, and cadherin receptors. IMPORTANCESpodoptera frugiperda is the main maize pest in South and North America and also is an invasive pest in different African countries. However, it is poorly controlled by Bacillus thuringiensis Cry1A toxins expressed in transgenic crops, which effectively control other lepidopteran pests. In contrast, maize expressing Cry1Fa is effective in the control of S. frugiperda, although its effectiveness is being lost due to resistance evolution. Some of the Cry1Ab domain III mutants characterized here show enhanced toxicity for S. frugiperda without loss of toxicity to Manduca sexta. Thus, these Cry1Ab mutants could provide useful engineered toxins that, along with other Cry toxins, would be useful for developing transgenic maize expressing stacked proteins for the effective control of S. frugiperda and other lepidopteran pests in the field.