Visualizing Dolichogenidea gelechiidivoris parasitism within Tuta absoluta larvae through fluorescence microscopy in...

The anatomical studies of host-parasitoid relationships are limited by tissue opacity. Traditionally, this limitation has been overcome using laborious histological dissection techniques. In this work, we present an alternative protocol based on tissue clarification techniques (1-propanol/ECi and 1-propanol/BABB) combined with 3D volumetric fluorescence microscopy (laser scanning confocal microscopy and light sheet fluorescence microscopy). These new protocols allow visualization by autofluorescence not only of the internal anatomy of Tuta absoluta (Lepidoptera: Gelechiidae), but also that of its endoparasitoid Dolichogenidea gelechiidivoris (Hymenoptera: Braconidae). Using this methodology, we were able to identify the three-dimensional position and orientation of D. gelechiidivoris parasitoid within the host as well as its size and morphology at different developmental stages. Due to its minimal invasiveness, which preserves the overall morphological context, these methods, also applicable to other species, will contribute to a deeper understanding of parasitism, providing a basis for future research on host-parasitoid interactions.