Development of an LNA-Based qPCR Assay for Detecting Toumeyella parvicornis (Cockerell, 1897) (Hemiptera: Coccidae) from Insect and Honeydew DNA.

The invasive sap-feeding pest Toumeyella parvicornis (pine tortoise scale) is rapidly spreading across Europe, threatening pine ecosystems, particularly in forest-urban areas of Italy. In this scenario, early detection and monitoring strategies are critical to prevent new outbreaks and mitigate impacts in infested regions. Current surveillance is challenged by the lack of rapid, sensitive tools for indirect detection of this cryptic, canopy-dwelling pest, despite advancements in molecular diagnostics and environmental DNA (eDNA). Here, we established a highly specific qPCR assay using LNA probe chemistry for detecting T. parvicornis DNA from both adult insects and their excreted honeydew. DNA was successfully isolated/quantified from all tested matrices. We recorded average Cq values of 20.9 for insect specimens and 30.3 for collected honeydew samples. Targeting the COI barcoding region, the assay demonstrated excellent specificity in both in silico and in vitro tests, showing no cross-reactivity to other pine-associated taxa. The limit of detection for DNA isolated from insect was 64 fg/µL. This is the first diagnostic protocol to use honeydew as a matrix for indirect detection of T. parvicornis. Optimized for routine application by Plant Health Services, this eDNA-based tool offers a valuable approach for future monitoring of sap-sucking hemipterans in multiple environments.