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The fall armyworm, Spodoptera frugiperda , is an invasive maize pest that has spread from the Americas into Africa and Asia and causes severe crop damage worldwide. Most populations of S. frugiperda show low susceptibility to Bacillus thuringiensis (Bt) Cry1Ab or Cry1Ac toxins, which have been proved to be effective against several other lepidopteran pests. In addition, S. frugiperda has evolved resistance to transgenic maize expressing Cry1Fa toxin. The specificity and toxicity of Cry toxins are determined by their binding to different larval midgut proteins, such as aminopeptidase N (APN), alkaline phosphatase (ALP), and cadherin (CAD), among other proteins, by means of exposed domain II loop regions and also by the domain III β-sheets β-16 and β-22. Here, we analyzed different Cry1Ab mutants with mutations in the domain III β-22 region. Alanine-scanning mutagenesis of this region revealed that all mutants showed increased toxicity against a nonsusceptible Cry1Ab S. frugiperda population. Further analysis of the mutant toxin Cry1AbS587A (bearing a mutation of S to A at position 587) revealed that, compared to Cry1Ab, it showed significantly increased toxicity to three other S. frugiperda populations from Mexico but retained similar toxicity to Manduca sexta larvae. Cry1AbS587A bound to brush border membrane vesicles (BBMV), and its higher toxicity correlated with higher binding affinities to APN, ALP, and CAD recombinant proteins. Furthermore, silencing the expression of APN1 and CAD receptors in S. frugiperda larvae by RNA interference (RNAi) showed that Cry1AbS587A toxicity relied on CAD expression, in contrast to Cry1Ab. These data support the idea that the increased toxicity of Cry1AbS587A to S. frugiperda is in part due to an improved binding interaction with the CAD receptor. IMPORTANCE Spodoptera frugiperda is an important worldwide pest of maize and rice crops that has evolved resistance to Cry1Fa-expressing maize in different countries. Therefore, identification of additional toxins with different modes of action is needed to provide alternative tools to control this insect pest. Bacillus thuringiensis (Bt) Cry1Ab and Cry1Ac toxins are highly active against several important lepidopteran pests but show varying and low levels of toxicity against different S. frugiperda populations. Thus, the identification of Cry1A mutants that gain toxicity to S. frugiperda and retain toxicity to other pests could be of great value to produce transgenic crops that resist a broader spectrum of lepidopteran pests. Here, we characterized Cry1Ab domain III β-22 mutants, and we found that a Cry1AbS587A mutant displayed increased toxicity against different S. frugiperda populations. Thus, Cry1AbS587A could be a good toxin candidate to produce transgenic maize with broader efficacy against this important insect pest in the field., (Copyright © 2020 American Society for Microbiology.)

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 ( 509 STLRVN 514 ) 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. IMPORTANCE Spodoptera 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., (Copyright © 2018 American Society for Microbiology.)

An insect resistance monitoring program was developed for Mexico to accommodate the commercial introduction and stewardship of Bt cotton. Between 1998 and 2015, field-collected geographic populations of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) were evaluated against Cry1Ac and Cry2Ab proteins of Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) to establish baseline susceptibility data before the commercial use of Bollgard (Cry1Ac) and Bollgard II (Cry1Ac and Cry2Ab) cotton. An annual monitoring program was subsequently established in which a single diagnostic concentration of each Bt protein was used in a diet overlay bioassay. The diagnostic concentration represented the concentration where larvae, evaluated in baseline studies, were reduced in weight by ≥97% relative to untreated controls or failed to molt to third instar after 5 d. In the monitoring study, populations were tested against Cry1Ac from 1998 through 2015, and against Cry2Ab from 2002 through 2004 and again from 2007 through 2015. None of the Cry1Ac-exposed larvae tested during the 18-yr period reached the third larval instar after an exposure of 5 d, and weight reduction relative to untreated control larvae was uniform at about 98-99%. For the 12 yr of Cry2Ab monitoring, no larvae reached third instar, and weight reduction was uniform at >97% relative to controls. These results indicate that H. zea susceptibility to Cry1Ac and Cry2Ab has not changed during the period Bollgard and Bollgard II have been cultivated in Mexico., (© The Author 2017. Published by Oxford University Press on behalf of Entomological Society of America.)

Spiromesifen is an insecticide that inhibits the synthesis of lipids and, in Mexico, its use against the Tomato-Potato Psyllid, Bactericera cockerelli (Sulc), on chili pepper (Capsicum annum), tomato (Lycopersicon sculentum) and potato (Solanum tuberosum) began in 2005; however more information is needed to understand its toxicity on this insect pest. The aim of this research was to determine the toxicity of spiromesifen against each of the biological stages of tomato-potato psyllid, its effect on fertility and viability of eggs deposited by treated females, as well as the female preference to lay eggs on treated and non treated plants. The relative toxicity at 95% mortality (highest LC95 value /LC95 value of the respective biological stage) of spiromesifen in egg, nymph 1, nymph 2, nymph 3, nymph 4, and nymph 5 were 517.5; 31316.2; 2950.1; 315.6; 18.2 and 1-fold, respectively. There were no differences in the toxicity of spiromesifen between adult males and females. The number of laid eggs was reduced as the spiromesifen concentration used to treat female increased and egg hatch was reduced in all tested doses. In the "no choice" test, females deposited 38.6+/-2.01 eggs by leaf of non treated chili pepper type jalapeño, while in the treated with 360 mg L(-1) we observed 0.3+/-0.08 eggs by leaf. In the "choice" test, the oviposition decreased as the dose increased. There were no eggs on plants treated with 2400 mg L(-1) of spiromesifen.