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.

Consuming alternative prey does not influence the DNA detectability half-life of pest prey in spider gut contents.

BACKGROUND: Key natural enemy-pest interactions can be mapped in agricultural food webs by analysing predator gut content for the presence of a focal pest species. For this, PCR-based approaches are the most widely used methods providing the incidence of consumption of a focal pest in field sampled predators. To interpret such data the rate of prey DNA decay in the predators' gut, described by DNA detectability half-life (t 1/2), is needed. DNA decay may depend on the presence of alternative prey in the gut of generalist predators, but this effect has not been investigated in one of the major predatory arthropod groups, spiders. METHODS: In a laboratory feeding experiment, we determined t 1/2 of the key cereal pest virus vector leafhopper Psammotettix alienus in the digestive tracts of its natural enemy, the spider Tibellus oblongus. We followed the fate of prey DNA in spiders which received only the focal prey as food, or as an alternative prey treatment they also received a meal of fruit flies after leafhopper consumption. After these feeding treatments, spiders were starved for variable time intervals prior to testing for leafhopper DNA in order to establish t 1/2. RESULTS: We created a PCR protocol that detects P. alienus DNA in its spider predator. The protocol was further calibrated to the digestion speed of the spider by establishing DNA decay rate. Detectability limit was reached at 14 days, where c. 10% of the animals tested positive. The calculated t 1/2 = 5 days value of P. alienus DNA did not differ statistically between the treatment groups which received only the leafhopper prey or which also received fruit fly. The PCR protocol was validated in a field with known P. alienus infestation. In this applicability trial, we showed that 12.5% of field collected spiders were positive for the leafhopper DNA. We conclude that in our model system the presence of alternative prey did not influence the t 1/2 estimate of a pest species, which makes laboratory protocols more straightforward for the calibration of future field data.