The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize (Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1–2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022.

Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis ( Bt ) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

The broad adoption of transgenic crops has revolutionized agriculture. However, resistance to insecticidal proteins by agricultural pests poses a continuous challenge to maintaining crop productivity and new proteins are urgently needed to replace those utilized for existing transgenic traits. We identified an insecticidal membrane attack complex/perforin (MACPF) protein, Mpf2Ba1, with strong activity against the devastating coleopteran pest western corn rootworm (WCR) and a novel site of action. Using an integrative structural biology approach, we determined monomeric, pre-pore and pore structures, revealing changes between structural states at high resolution. We discovered an assembly inhibition mechanism, a molecular switch that activates pre-pore oligomerization upon gut fluid incubation and solved the highest resolution MACPF pore structure to-date. Our findings demonstrate not only the utility of Mpf2Ba1 in the development of biotechnology solutions for protecting maize from WCR to promote food security, but also uncover previously unknown mechanistic principles of bacterial MACPF assembly.

Lepidopteran pests have been successfully managed by the adoption of insect resistant transgenic plants expressing Cry and/or Vip insecticidal proteins derived from Bacillus thuringiensis (Bt plants). Among such pests, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is highlighted for its destructive potential in maize crops and for cases of field-evolved resistance to Bt plants. Cry insecticidal proteins expressed in Bt plants are known for their interaction with insect midgut receptors and subsequent midgut cell disruption that leads to target pest death. In the midgut of lepidopteran larval pests such as S. frugiperda, serine proteases are important in dietary protein digestion and activation or degradation of insecticidal proteins. This work was conducted to evaluate if the use of a soybean trypsin inhibitor (SBTI) could disrupt the development of a Bt-susceptible and a Bt-resistant population of S. frugiperda ingesting Bt (expressing Cry1F, Cry1A.105, and Cry2Ab2 Cry proteins) and non-Bt maize plants. The SBTI was produced and purified using recombinant expression in E. coli followed by purification in Ni-Sepharose. Bioassays using non-Bt maize leaves indicated that the development of susceptible and resistant populations of S. frugiperda was not influenced by the ingestion of SBTI. However, when the resistant population consumed Bt maize plants amended with SBTI, high mortality along with a reduction in larval weight and reduced activity of digestive trypsins were observed. Although the mode of action was not elucidated, it is possible that the consumption of SBTI increased susceptibility to Bt maize in the resistant population of S. frugiperda.

Chrysodeixis includens (Walker, 1858) and Rachiplusia nu (Guenée, 1852) are important defoliating pests belonging to the Plusiinae subfamily in the Western hemisphere. C. includens is a major lepidopteran pest of soybean in the Americas, whereas, until 2021, R. nu was more restricted to the temperate regions of South America. Recently, reports of R. nu feeding on Cry1Ac soybean and occurring in tropical regions of Brazil have raised questions regarding the distribution of this species. The morphological similarity of the larvae from the two species makes it difficult to correctly identify the species in the field, which may lead to an underestimation of R. nu in Brazilian territory. This study aimed to address these questions by using a molecular approach to identify Plusiinae caterpillars throughout three seasons in non-Bt, Cry1Ac, and Cry1Ac × Cry1F soybean fields. Here, we carried out a comprehensive spatial sampling of the primary soybean-producing regions in Brazil. The results showed that R. nu has been the main Plusiinae occurring in soybean over the last three years, and it was present in all sampled regions. For Cry1Ac and Cry1Ac × Cry1F soybeans, up to 99% of the samples collected in 2023/24 were identified as R. nu. Non-Bt soybeans had higher variations in the proportion of the two species among the regions and across seasons, indicating that populations of C. includens and R. nu are co-occurring throughout the country. This is, to our knowledge, the most robust report assessing the distribution of C. includens and R. nu in Brazil using a molecular tool. This study provides clarification of R. nu occurrence and highlights the importance of pest monitoring from an integrated pest management perspective.

Chrysodeixis includens and Rachiplusia nu are two species belonging to the Plusiinae subfamily within the Noctuidae family. Due to their morphological similarity, the identification of their larvae is difficult and time-consuming. A rapid and accurate identification of these two species is essential for their management as these species exhibit differential susceptibilities to insecticides and crops engineered to express Bacillus thuringiensis (Bt) proteins, and a molecular tool can easily provide this differentiation. Currently, molecular analysis can identify these species through genetic sequencing, an expensive and time-consuming process. In our study, after sequencing part of the mtDNA cytochrome c oxidase I (COI) gene and based on the differences found in the gene of each species, a set of species-specific primers was developed: one reverse primer common to both species and two forward primers, specific to each species, amplifying fragments of 199 base pairs (bp) for C. includens and 299 bp for R. nu. Our results indicate that the primers were specific for these species, enabling the identification of individuals directly through agarose gel. The new methodology proved to be accurate, rapid, and reliable for the correct identification of these two species of loopers.