Simplified and rapid staining of leafhopper salivary sheaths in plant tissues for electrical penetration graph waveform correlations.

Herbivorous insects in the order Hemiptera use piercing-sucking mouthparts to utilize plant sap. Among them salivary sheath feeders penetrate into the plant by their flexible stylets to reach vascular elements. Manoeuvering stylets in plant tissues is aided by the creation of salivary sheaths, which solidify from proteinous gelling saliva and remain as lasting artefacts in the plant tissues. Studying their structure reveals hidden details of the feeding behaviour and the transmission of pathogens in case of vector insects. One important aspect of studying salivary sheaths is that it can be used to confirm the biological function of electropenetrography (EPG) waveform patterns. Previously, complex and vaguely documented histological methods have been used to observe salivary sheath structure. Building on existing methodologies, we report a simplified histological procedure where each step was optimized to offer a rapid process that does not require special equipment, can be applied to many samples, has good success rate and a low cost of errors in terms of time and materials. We describe the procedure, using a Psammotettix alienus - barley model system, in three steps. (i) Clarification of entire plant parts and pre-staining salivary sheaths with aqueous fuchsin. This step allows to identify salivary sheath starting points on the surface. (ii) Knowing salivary sheath location, using hand sectioning, produce a single c. 60 μm section that contains the entire salivary sheath. (iii) Counterstain the section with methylene green and, after further clarification, study under light microscope in a glycerol - ethanol embedding solution, without fixed mounting.