Since the larval exoskeleton is resistant to known fixative reagents, we performed an adequate fixation by injecting buffered formalin 10% with an insulin syringe in the last left proleg. The volume of solution injected, to have a turgid consistency of the larvae, was about 100 µL. Larvae were then stored at 4°C for 24 h, to fix internal organs and block melanization. Whole larvae were dissected transversally or sagittally into two halves by means of an anatomic pincers and by using a new lancet blade for each larva. The procedure was carefully performed to avoid the squeeze of the larval tissues. The two halves of larvae were placed in the same BioCassette and routinely processed in the path lab. For transversally sectioned larvae, each paraffin-embedded half was further sectioned into two/three rings, after cooling the larva at room temperature for a few minutes to harden the tissues. A crucial step to avoid the paraffin block rupture during microtome sectioning was to keep the cut larval tissues in hot paraffin for one hour to stabilize the inclusion, and to obtain a complete merge of the cut rings in the final paraffin block. Finally, four/six rings (one in the distal part, two in the middle, and one in the proximal part) were positioned in each paraffin-block. Histochemical staining on slides with serial tissue sections was then performed: haematoxylin and eosin (HE) was used to evaluate tissue morphology, periodic acid Schiff (PAS) and Grocott Methenamine staining (GMS) to highlight fungi localization and host interaction, Giemsa, Alcian blue at various pH (1, 2.5, and 3.1) to evaluate hemocytes, and Feulgen staining to evaluate DNA. All histo-chemical stainings were performed according to standard laboratory protocols.14 Sagittally sectioned larvae were routinely paraffin-embedded.13 A distance of 50 µm was maintained between serial 4-micron-thick tissue sections of the two halves, and the slides were stained with haematoxylin and eosin.
The microscopic visualization has been performed using a Leica Microscope DMLB, and the image acquisition with the NanoZoomer-XR C12000 series (Hamamatsu Photonics K.K., Tokio, Japan.).