Defining feeding physiology through combined single-cell transcriptomics and proteomics approaches in the southern green stink bug

The southern green stink bug, Nezara viridula, is a polyphagous pest of agricultural crops and novel mechanisms to prevent plant damages from feeding are needed. Stink bugs feed by inserting their piercing-sucking mouthparts into plant tissues through physically rupturing cells and secreting ‘gel’ saliva that forms a sheath around the stylets. In addition, ‘watery’ saliva containing digestive enzymes are secreted to digest plant cell contents prior to ingestion. Products that inhibit the function of secreted proteins or reduce salivary gland activity are likely to inhibit feeding, and thus the aim of this study was to define the expression profile of individual cells of the salivary glands relevant to protein secretion to identify putative targets for technology development. Single-cell sequencing of N. viridula principle and accessory salivary glands was performed and data show 13 distinct cell clusters, with seven of these clusters identified as sub-sets of secretory cells that produce a unique profile of secreted proteins. Proteomics analysis of watery and gelling saliva was conducted to address whether the identified markers of secretory cell sub-sets could be found in the secreted saliva. Cross-analysis of datasets identified that the most abundant proteins in both gelling and watery saliva matched to markers for different secretory cell sub-sets, where one sub-set produced > 40% of matched salivary proteins in watery saliva. Understanding the origin of secreted proteins from specific sub-sets of secretory cells permits the identification of putative targets to disrupt stink bug salivary gland secretory function and prevent insect feeding and consequent damage.