# Simple and Rapid Detection of Salivary Sheaths at Philaenus spumarius Feeding Points

## TL;DR

This paper introduces a quick method to detect salivary sheaths left by the Meadow Spittlebug, which helps track its feeding and potential disease transmission.

## Contribution

A rapid, ethanol-based method for visualizing salivary sheaths of Philaenus spumarius is proposed for efficient vector monitoring.

## Key findings

- 75% ethanol provides a rapid and effective way to visualize salivary sheaths without harmful dyes.
- Alternative staining and diaphanization methods are less efficient and more time-consuming.
- Counting salivary sheaths can help identify resistant plant clones and model infection probabilities.

## Abstract

Philaenus spumarius (Ps) is a xylem sap-feeding hemipteran that transmits Xylella fastidiosa (Xf) subspecies and a few other plant xylem-inhabiting microorganisms, including non-phytopathogenic ones. Xylella fastidiosa is a xylem-inhabiting olive pathogenic bacterium, the causal agent of Olive Quick Decline Syndrome in southern Italy. Philaenus spumarius secretes a proteinaceous saliva that jellifies into a salivary sheath to seal the stylets in place and maintain adequate fluid uptake. The salivary sheaths remain in situ within the plant tissues and reveal the hemipteran feeding point and path of a putative transmission event. Here, we compare several rapid and quantitative procedures for detecting Ps access and feeding via salivary sheaths. The number of accesses should be a key parameter for modelling the infection probability, given the estimates of the percentage of infective vectors in the study area. Moreover, given an orchard with diverse clones, counting salivary sheaths under the same vector population pressure will indicate comparatively fewer accepted plants, thereby supporting the search for resistant accessions.

Hemipteran plant sap feeders acquire food by inserting their piercing–sucking mouthparts into the apoplast (xylem sap feeders) or symplast (phloem sap feeders). When feeding, these insects seal their stylets inside the plant with a salivary sheath, minimising embolisms. The main impact of the insect on the host or food plant consists of sucking plant sap, transmitting phytopathogenic agents, or triggering galls as hypertrophic and/or hyperplastic neoformations. This paper proposes a rapid method for locating and counting the salivary sheaths of Philaenus spumarius (the Meadow Spittlebug), the primary Italian vector of Xylella fastidiosa, at the feeding point on its host or food plants. Hand-sliced stems of herbs hosting juveniles and olive twigs or leaves feeding the adult spittlebug, preserved while mounted in 75% ethanol (v/v), show the salivary sheaths. Alternative dyeing with acid fuchsin, chlorazol black, or phloroglucinol, and diaphanisation with Essig’s fluid alone or in combination with benzyl alcohol, may help with observations. The 75% EtOH solution provides a better compromise, yielding rapid evidence. The other methods offer similar outcomes but require more time and effort and expose the operator to harmful dyes. Assessing vectors’ access to plants may enable us to quantify the potential number of pathogen transmission events or to spot rejecting clones, thereby advancing the development of robust, effective control strategies that incorporate resistance.