Development of a PCR-RFLP assay to identify among field-collected larvae.

The fruit fly is a model organism to study several aspects of metazoan biology. Most of the work has been conducted in adult fruit flies, including laboratory and field-derived specimens, but larvae recently became a valuable model to better understand animal physiology, development, or host-microbe interactions. While adult flies can be easily assigned to a given species based on morphological characteristics, such visual identification is more intricate at the larval stage. This could explain the limited number of studies focusing on larvae, especially field-derived samples. Here, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay that discriminates from other ecologically relevant species at the larval stage. The method, which targets the () gene, was validated using laboratory-derived larvae from seven populations originating from different geographic areas as well as six species. We further validated this PCR-RFLP assay in a natural context, by identifying wild larvae collected in two locations in France. Notably, among all PCR-RFLP profiles that matched the species, 100% were correctly identified, as confirmed by sequencing. In summary, our work provides a rapid, simple, and accurate molecular tool to identify from field-collected larvae.