# Evaluating Real-Time PCR to Quantify Drosophila suzukii Infestation of Fruit Crops

## TL;DR

This paper introduces a DNA-based test using real-time PCR to quickly and accurately detect and quantify infestations of spotted-wing drosophila in fruit crops.

## Contribution

The study introduces a qPCR protocol that enables rapid and accurate detection of Drosophila suzukii eggs in fruit tissue.

## Key findings

- The qPCR protocol reduces SWD identification time from weeks to about five hours.
- qPCR showed a strong negative correlation between cycle threshold and egg concentration in both fruit tissue and standard samples.
- The DNA-based test improves detection accuracy and enables quantification of infestation levels.

## Abstract

Early detection of invasive species helps growers know when to begin pest management, provides additional management options that target early life stages, and can reduce overall production losses. However, early detection of invasive insects is challenging due to their small size. Current methods for determining insect infestation in fruit rely on the time-consuming methods of either searching for eggs under a microscope or crushing fruit in a saline solution and counting the number of larvae that exit the fruit. These methods are also imprecise; definitive species identification requires rearing immature specimens to adulthood, by which time berries from the harvest may have already spoiled. To address these shortcomings, we evaluated a protocol for rapidly assessing the amount of spotted-wing drosophila (SWD) infestation in fruit using a DNA-based test, which measures the amount of SWD DNA in a fruit sample. We identified SWD with this protocol under laboratory settings and validated the protocol with fruit collected from the field. This test improves detection accuracy, enables quantification of the degree of SWD infestation, and reduces definitive species identification from several weeks to a few hours.

Common methods for detecting Drosophila suzukii (spotted-wing drosophila, SWD) in fruit, such as microscopy, physical extraction, and incubation, are time-consuming and may underrepresent egg and first instar larvae counts, the smallest life stages of SWD. To address these limitations, we evaluated a quantitative real-time PCR (qPCR) protocol to detect and quantify SWD eggs using a linear model of the log-transformed ratio of eggs to sample volume (µL) in Tris buffer and fruit tissue. Compared to traditional approaches, this method reduces identification time from several weeks to approximately five hours. We observed a negative linear correlation between qPCR cycle threshold and egg concentration in both standard and fruit tissue samples, with similar model fits (R2 = 0.7215 for field fruit tissue; R2 = 0.874 for standard samples). This DNA-based protocol improves infestation detection speed and accuracy by enabling rapid, species-specific identification of D. suzukii in fruit tissue, addressing limitations of morphological identification of eggs and larvae. Further refinement for fruit tissue could enhance real-world applicability. Rapid detection may enable timely assessment of varietal resistance to SWD and support safer control strategies targeting early life stages, helping to prevent pest development and fruit degradation.