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.